Accepted Manuscript Routine western blot to check autophagic flux: cautions and recommendations Rubén Gómez-Sánchez, Elisa Pizarro-Estrella, Sokhna M.S. Yakhine-Diop, Mario Rodríguez-Arribas, José M. Bravo-San Pedro, José M. Fuentes, Rosa A. González-Polo PII: DOI: Reference:

S0003-2697(15)00069-X http://dx.doi.org/10.1016/j.ab.2015.02.020 YABIO 11990

To appear in:

Analytical Biochemistry

Received Date: Revised Date: Accepted Date:

19 January 2015 17 February 2015 18 February 2015

Please cite this article as: R. Gómez-Sánchez, E. Pizarro-Estrella, S.M.S. Yakhine-Diop, M. Rodríguez-Arribas, J.M. Bravo-San Pedro, J.M. Fuentes, R.A. González-Polo, Routine western blot to check autophagic flux: cautions and recommendations, Analytical Biochemistry (2015), doi: http://dx.doi.org/10.1016/j.ab.2015.02.020

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1

ROUTINE WESTERN BLOT TO CHECK AUTOPHAGIC FLUX: CAUTIONS AND

2

RECOMMENDATIONS

3

Rubén Gómez-Sánchez

4

Rodríguez-Arribas*, José M. Bravo-San Pedro, José M. Fuentes $#, Rosa A. González-Polo $#

*

, Elisa Pizarro-Estrella*, Sokhna M.S. Yakhine-Diop*, Mario

5 6

Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas

7

(CIBERNED), Departamento de Bioquímica y Biología Molecular y Genética, Universidad de

8

Extremadura, F. Enfermería y Terapia Ocupacional, 10003 Cáceres, Spain,

9 10

*

These authors contributed equally to this paper.

11 12

$ Co-senior authors

13 14

#

15

Molecular y Genética, F. Enfermería, Centro de Investigación Biomédica en Red sobre

16

Enfermedades Neurodegenerativas, Universidad de Extremadura, Avda Universidad, s/n, 10003

17

Cáceres, Spain; Fax: 34-927-257451; E-mail: [email protected] and [email protected]

To whom correspondence should be addressed, Departamento de Bioquímica y Biología

18 19

Short Title: LC3 and p62 data interpretation

20 21

Subject category: Cell Biology

22 23

Abbreviations: Atg8, autophagy-related 8; Baf. A1, bafilomycin A1; BCA, bicinchoninic acid;

24

BSA, bovine serum albumin; DMEM, Dulbecco's Modified Eagle Medium; EBSS, Earle’s

25

Balanced Salt Solution; FBS, fetal bovine serum; GAPDH, glyceraldehyde 3-phosphate

26

dehydrogenase; HF, human fibroblast; IF, immunofluorescence; LC3, microtubule-associated

27

protein

1

light

chain

3;

MEF,

mouse

embryonic

fibroblast;

NP-40,

nonyl 1

28

phenoxypolyethoxylethanol; PVDF, polyvinyl difluoride; qPCR, quantitative PCR; RIPA,

29

RadioImmunoPrecipitation Assay; SB, sample buffer; SDS, sodium dodecyl sulfate; SQSTM1,

30

sequestosome 1; TBST, Tris-buffered saline with Tween 20; WB, Western blotting

2

31

ABSTRACT

32

At present, the analysis of autophagic flux by Western blotting (WB), which measures two of

33

the most important markers of autophagy, i.e., microtubule-associated protein 1 light chain 3

34

(LC3) and p62, is widely accepted in the scientific community.

35

In this study, we addressed the possible disadvantages and limitations that this method

36

presents for a correct interpretation of the results according to the lysis buffer used for

37

extracting proteins. Here, we tested the LC3 and p62 protein levels by WB in four cell models

38

(mouse embryonic and human fibroblasts (MEFs and HFs, respectively), N27 rat mesencephalic

39

dopaminergic neurons and SH-SY5Y human neuroblastoma cells). The cells were exposed to

40

the autophagy inhibitor bafilomycin A1 (Baf. A1) in combination (or not) with nutrient

41

deprivation to induce autophagy, and they were lysed by using four different buffers (nonyl

42

phenoxypolyethoxylethanol (NP-40), RadioimmunoPrecipitation Assay (RIPA), Triton X-100

43

and sample buffer (SB) 1X).

44

Based on our observations, we want to highlight that this technique is not always

45

appropriate for analyzing and monitoring autophagy. In this report, we show conflicting data

46

that hinder the correct interpretation of the results, especially in relation to p62 protein levels, at

47

least in the models studied in this work.

48 49

Keywords: autophagy, Western blotting, lysis buffer, LC3, p62

50 51

3

52

1. INTRODUCTION

53

Macroautophagy (hereafter autophagy) is a catabolic process that is essential for recycling

54

cellular components and allows cell survival under nutrient-limited conditions. It is also

55

required to eliminate damaged organelles or other materials during homeostasis. During

56

autophagy, the cargo that will be eliminated is engulfed by a double-membrane compartment

57

called the phagophore. Upon completion, the phagophore matures into an autophagosome,

58

which transports the cargo to the lysosome, where it is degraded and recycled [1]. Autophagy is a

59

complex and dynamic process, it is important to establish appropriate methods to monitor it.

60

Two proteins have special relevance to the study and comprehension of this mechanism, LC3

61

and p62. LC3, the mammalian homolog of yeast autophagy-related 8 (Atg8), exists in two

62

forms: LC3-I (mainly cytosolic) and LC3-II (bound to the autophagy membrane structures,

63

including phagophore, autophagosome and autophagolysosome). LC3-I, is post-translationally

64

modified by an ubiquitin-like system to its lipidated form (LC3-II). This isoform is anchored in

65

the outer and inner membranes of autophagosomes, where it is sequestered into autolysosomes

66

before being degraded or recycled back into use

67

p62) acts as a bridge between LC3-II and ubiquitinated substrates. p62-bound polyubiquitinated

68

proteins are incorporated into the complete autophagosome by physical interaction between p62

69

and LC3-II. Then, they are degraded in autolysosomes, thus serving as a readout of autophagic

70

degradation, at least in certain settings [3].

[2]

. Sequestosome 1 (SQSTM1, also known as

71

Western blotting (WB) is currently used to analyze LC3-I to LC3-II conversion to

72

estimate the abundance of autophagic related structures (phagophores, autophagosomes and

73

autolysosomes) before they are degraded by lysosomal hydrolases. At this point, we want to

74

remark that there are some differences between cell lines such as the proportion between these

75

two isoforms or even transcriptional differences

76

levels serves as an indirect measurement of the efficient removal of the cargo. However, it is

77

important to note that many precautions should be taken into account to correctly analyze the

78

obtained results from this protein [5]. The guidelines published by Klionsky et al.[4] have already

[4]

.On the other hand, quantification of p62

4

79

mentioned some limitations in analyzing p62 by WB, i.e., its protein solubility, when specific

80

lysis buffers are used, without any consensus of a perfect one [4]. Moreover, it is clear that WB is

81

a complementary method for monitoring autophagy, requiring other methodologies to confirm

82

the existence of an efficient autophagic flux [4].

83

Regarding to these issues, we analyzed LC3 and p62 by WB with three different buffers

84

that are widely used (nonyl phenoxypolyethoxylethanol (NP-40), RadioImmunoPrecipitation

85

Assay (RIPA) and Triton X-100), and sample buffer (SB 1X) because of its capacity to

86

resuspend pellets obtained with the buffers previously mentioned. We took the entire pool of

87

these proteins from four cell models (mouse embryonic and human fibroblasts (MEFs and HFs,

88

respectively), N27 rat mesencephalic dopaminergic neurons and SH-SY5Y human

89

neuroblastoma cells), because of differences between cell lines, commented above. In this

90

context, we analyzed p62 and LC3 levels in both soluble and insoluble fractions upon treatment

91

with the autophagy inhibitor bafilomycin A1 (Baf. A1), upon nutrient deprivation to induce

92

autophagy or with a combination of both treatments to correctly assess autophagy flux. We

93

show possible limitations and caveats to keep in mind when the results are analyzed, observe

94

significant restrictions in some cases. These findings have led us to reconsider the possibility of

95

discarding this approach under several conditions, or at least to be cautious when interpreting

96

the findings.

97

Based on published recommendations

[4, 6]

and our own experience, we consider it

98

important to develop this work for researchers who need to know the methodological and

99

interpretation-related problems that can occur during the analysis of well-known autophagy

100

markers for monitoring autophagy flux by WB.

101 102 103 104 105 5

106

2. MATERIALS

107

2.1. Cells and culture

108

This work used the following cell lines: MEFs, HFs, SH-SY5Y human neuroblastoma cells and

109

N27 rat mesencephalic dopaminergic cells.

110

The culture media for MEFs, HFs and SH-SY5Y were as follows: Dulbecco's Modified

111

Eagle Medium (DMEM)-High Glucose (Sigma-Aldrich, D6546) supplemented with 10% fetal

112

bovine serum (FBS) (Sigma-Aldrich, F7524), 1% L-glutamine (Sigma-Aldrich, G7513) and

113

penicillin-streptomycin (Hyclone, SV30010).

114

The N27 culture medium was made of the following: RPMI 1640 medium (1X)

115

(Hyclone, SH30096.01) supplemented with 10% FBS, L-glutamine (Sigma-Aldrich, G7513)

116

and penicillin-streptomycin (Hyclone, SV30010). The starvation medium was Earle’s Balanced Salt Solution (EBSS) (Sigma-Aldrich,

117 118

E2888).

119

2.2. Plasmids

120

To improve the measurement of p62 degradation, we used the mCherry-GFP-p62 plasmid (a

121

gift from Dr. Terje Johansen)

122

autophagosomes and/or autolysosomes because GFP fluorescence is quenched by low pH, an

123

autolysosomal characteristic, and mCherry fluorescence is stable under these conditions.

124

2.3. Buffers and solutions

125

1. NP-40 lysis buffer: 0.5% (v/v) NP-40, 0.2% (v/v) Tris-HCl 0.5 M pH 6.8 and 150 mM NaCl

126

in distilled water, supplemented with protease inhibitor cocktail tablets (complete mini, EDTA-

127

free, Roche, #11836170001) and phosphatase inhibitor cocktail tablets (PhosSTOP, Roche,

128

#04906837001).

129

2. RIPA lysis buffer: 1% (v/v) Triton X-100, 1% (v/v) sodium deoxycholate, 0.1% (v/v) sodium

130

dodecyl sulfate (SDS), 20 mM Tris-HCl pH 7.4, 150 mM NaCl and 1 mM EDTA in distilled

131

water, supplemented with protease inhibitor cocktail tablets and phosphatase inhibitors, as

132

above.

[7]

. This double tag allows for the visualization of p62 in

6

133

3. Triton X-100 lysis buffer: 1% (v/v) Triton X-100 in PBS 1X, 100 mM NaF and 1 mM

134

Na3VO4, supplemented with protease inhibitor cocktail 10X (Sigma-Aldrich, P2714).

135

4. SB 1X lysis buffer: 2% (v/v) SDS, 10% (v/v) glycerol and 50 mM Tris-HCl pH 6.8 in

136

distilled water.

137

5. Sample loading buffer: 0.025% (v/v) bromophenol blue, 5% (v/v) β-mercaptoethanol, 50%

138

(v/v) glycerol, 0.01 M sodium acetate pH 5.2 and 250 mM Tris-HCl pH 6.8 in distilled water.

139

6. Electrophoresis buffer: 1X Tris/Glycine/SDS (TGS) diluted 10X TGS (Bio-Rad, 161-0772)

140

with distilled water.

141

7. Transfer buffer: 1X Tris-glycine-methanol, which was composed of 10X Tris/Glycine (Bio-

142

Rad, 161-0771) and 20% (v/v) methanol, and then diluted with distilled water.

143

8. Tris-buffered saline with Tween 20 (TBST) 1X: 0.2% Tween 20, 10 mM Tris-HCl and 50

144

mM NaCl in distilled water.

145

9. WB blocking/primary antibody solution: 10% (w/v) non-fat dried milk in TBST 1X solution.

146

10. Paraformaldehyde (PFA): 4% (w/v) PFA in PBS 1X pH 7.4.

147

11. Triton X-100 solution: ice-cold 0.1% (v/v) Triton X-100 (Sigma-Aldrich, T9284) in PBS

148

1X.

149

12. Immunofluorescence (IF) blocking solution: ice-cold 10% (v/v) FBS in PBS 1X.

150

13. IF antibody binding solution: ice-cold 0.1% (w/v) bovine serum albumin (BSA) (Sigma-

151

Aldrich, A7906) in PBS 1X.

152

14. IF mounting medium: Fluoromount-G (SouthernBiotech, 0100-01).

153

2.4. Equipment and reagents

154

1. Baf. A1: LC Laboratories, B-1080.

155

2. Protein electrophoresis apparatus (Bio-Rad Mini-PROTEAN Tetra Cell, 165-8004).

156

3. WB transfer apparatus (Bio-Rad Trans-Blot SD Semi-Dry-Transfer electrophoretic Cell, 170-

157

3940).

158

4. Polyvinyl difluoride (PVDF) membranes (Bio-Rad, 162-0177).

159

5. Phosphatase inhibitor: PhosSTOP inhibitor cocktail tablets (Roche, #04906837001). 7

160

6. Protease inhibitor: Complete mini, EDTA-free inhibitor cocktail tablets (Roche,

161

#11836170001).

162

7. Bicinchoninic acid (BCA) assay: BCA (Sigma-Aldrich, B9643) and copper (II) sulfate

163

solution (Sigma-Aldrich, C2284).

164

8. Chemiluminescent reagent: Pierce ECL WB Substrate (Thermo Scientific, 32106).

165

9. Transfection reagents: Attractene Transfection Reagent (Qiagen, 301005) and Lipofectamine

166

2000 Reagent (Invitrogen, 11668-019).

167

10. Fluorescence microscope equipment: an inverted fluorescence microscope (Olympus, IX51)

168

equipped with a camera (Olympus, DP70).

169

11. Primers for human p62: FW: 5'-GGAGAAGAGCAGCTCACAGCCA-3' (Integrated DNA

170

Technologies, 66585252) and RV: 5'-CCTTCAGCCCTGTGGGTCCCT-3' (Integrated DNA

171

Technologies, 66585253). Primers for rat p62: FW: 5’- GCTATTACAGCCAGAGTCAAGG-3’

172

(Integrated DNA Technologies, 66585254) and RV: 5’-TGGTCCCATTCCAGTCATC-3’

173

(Integrated

174

GTCGGTGTGAACGGATTTG-3' (Integrated DNA Technologies, 66585256) and RV 5'-

175

TCCCATTCTCAGCCTTGAC-3' (Integrated DNA Technologies, 66585257). Primers for

176

mouse p62: FW: 5'-TGTGCCTGTGCTGGAACTTTC-3' (Integrated DNA Technologies,

177

63351242) and RV 5'-TGTGGAACATGGAGGGAAGAG-3' (Integrated DNA Technologies,

178

63351241). Primers for mouse GAPDH: FW: 5’-AACTTTGGCATTGTGGAAG-3’ (Integrated

179

DNA Technologies, 64110744) and RV 5'-ACACATTGGGGGTAGGAAA-3' (Integrated DNA

180

Technologies, 64110744). Primers for human GAPDH: FW: 5'-AGCCACATCGCTGAGACA-

181

3' (Integrated DNA Technologies, 64065616) and RV 5'-GCCCAATACGACCAAATCC-3'

182

(Integrated DNA Technologies, 64065615).

183

12. RNeasy Mini Kit (Qiagen, 74104)

184

13. QuantiTect Reverse Transcription Kit (Qiagen, 205311).

185

14. Quantitative real-time PCR (qPCR) Master Mix: KAPA SYBR Fast Universal qPCR kit

186

(Cultek, KK4601).

DNA

Technologies,

66585255).

Primers

for

rat

GAPDH:

FW:

5'-

8

187

2.5. Antibodies

188

1. Monoclonal anti-p62 (BD Transduction Laboratories, 610498).

189

2. Polyclonal anti-LC3B (Sigma-Aldrich, L7543).

190

3. Monoclonal anti-Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (Millipore,

191

MAB374).

192

4. HRP-conjugated goat anti-rabbit IgG (Bio-Rad, 170-6515).

193

5. HRP-conjugated goat anti-mouse IgG (Bio-Rad, 170-5047).

194

6. Alexa Fluor 488 goat anti-rabbit IgG (H+L) (Molecular Probes, A11034).

195

7. Alexa Fluor 568 goat anti-mouse IgG (H+L) (Molecular Probes, A11031).

196 197 198 199 200 201 202 203 204 205 206 207 208 209 210 211 212 213 9

214

3. METHODS

215

3.1. Cell growth and treatments

216

All cell lines were incubated at 37 °C under saturating humidity in 5% CO2/95% air. The cells

217

were grown at densities of 3 x 105 (MEFs), 2 x 106 (SH-SY5Y), 4x105 (N27) and 1 x 106 (HFs)

218

in 75-cm2 tissue culture flasks. Confluent cells (80%) were trypsinized and seeded into a 6-

219

well plate at a concentration of 3 x 104 cells/ml (MEFs and HFs), 3.5 x 104 cells/ml (N27) or 1 x

220

105 cells/ml (SH-SY5Y).

221

After 24 h, the culture medium was replaced with different treatments (Control, EBSS,

222

Baf. A1 and Baf. A1 + EBSS). To block fusion between autophagosomes and lysosomes, the

223

cells were incubated with 100 nM Baf. A1. To create starvation conditions, the culture medium

224

was replaced with EBSS. For combined treatment, the cells were pre-incubated with Baf. A1

225

100 nM for 1 h and were then washed with PBS 1X and treated with Baf. A1 100 nM + EBSS.

226

All treatments lasted 4 hours.

227 228

3.2. Plasmid transfection

229

MEFs were transiently transfected by using Attractene Transfection Reagent, according to the

230

manufacturer’s protocol. HFs, SH-SY5Y and N27 cells were transiently transfected by using

231

Lipofectamine 2000 Reagent, according to the manufacturer’s protocol.

232 233

3.3. Protein extracts

234

The cells were incubated for different times depending on the lysis buffer (15 min with Triton

235

X-100, 10 min with RIPA and 5 min with NP-40) and samples were mechanically resuspended

236

by pipetting until homogenization. The incubation times with each buffer were based on

237

standard protocols that ensures complete solubilization. After that, the samples were centrifuged

238

at 13.414 g and 13 min at 4 ºC, and the resulting supernatants were quantified by BCA assay.

239

The corresponding pellets were washed 3 times with PBS 1X and resuspended in SB 1X buffer

10

240

to dilute insoluble proteins. The samples were heated in SB 1X buffer at 95 °C for 10 min

241

before their quantification.

242 243

3.4. Western blotting

244

Equal amounts of protein (25-40 µg/condition) were resolved by 12% SDS-gel electrophoresis

245

and transferred to PVDF membranes, according to a partially modified conventional protocol [8].

246

The immunodetection included the transfer (15 V during 15 min, per each membrane) and

247

blocking of the membrane with WB blocking solution (1 h at room temperature). After washing

248

the membranes 2 times with TBST 1X, the blots were incubated with the corresponding primary

249

antibodies against p62/SQSTM1 (1:5000), LC3-B (1:5000) and GAPDH (1:5000) (incubating at

250

4ºC overnight, except GAPDH (1 h at room temperature)). The membranes were washed 2

251

times with TBST 1X and subsequently incubated with their respective HRP-conjugated

252

secondary antibodies (1:10000) (1 h at room temperature). The detection of bound antibodies

253

was visualized by chemiluminescence with ECL substrate. Finally, a quantification analysis was

254

performed with ImageJ software (NIH), using GAPDH levels as a loading control.

255 256

3.5. Immunofluorescence

257

To detect endogenous p62 and LC3B, the cells were seeded on cover slips, fixed with PFA

258

solution and permeabilized for 10 min with Triton X-100 solution. To block non-specific

259

binding, the cells were incubated for 20 min with 10% FBS in PBS 1X followed by incubation

260

with primary antibodies anti-p62 (1:500) and anti-LC3B (1:500) for 1 h at room temperature.

261

After that, the cells were labeled with Alexa Fluor 488 anti-rabbit (1:1000) and 568 anti-mouse

262

(1:1000) secondary antibodies for LC3 and p62, respectively. Finally, the cover slips were

263

mounted on microscope slides, by using Fluoromount-G medium. Images were taken by using

264

an inverted fluorescence microscope, with at least 200 cells analyzed for each condition. The

265

exposition time used to measure green and red fluorescence were 1:2.5 and 1:4.5, respectively.

266 11

267

3.6 Quantitative PCR

268

An analysis of p62 mRNA expression was performed in all cell lines. RNA was extracted by

269

RNeasy Mini Kit (Qiagen, 74104); 500 ng of total RNA were reverse-transcribed into

270

complementary DNA by using a QuantiTect Reverse Transcription Kit (Qiagen, 205311), both

271

according to the manufacturer’s protocol. p62 mRNA expression was measured by qPCR with

272

KAPA SYBR Fast reagents, by using the primers described above. GAPDH gene expression

273

was used as an endogenous control, and the expression level was calculated by using the (2-∆∆Ct)

274

[9]

ratio.

275 276

3.7 Statistical analyses

277

Each experiment was repeated at least three times. The data shown here are from a

278

representative experiment. The data were evaluated by two-tailed unpaired Student's t-test and

279

ANOVA test, and all comparisons with a p value less than 0.05 (p < 0.05) were considered

280

statistically significant; ***p < 0.001, **p < 0.01 and *p < 0.05. Non-significant results are not

281

indicated in the figures. The data are expressed as the mean ± the standard error of the mean

282

(SEM).

283 284 285 286 287 288 289 290 291 292 12

293

4. RESULTS

294

1. LC3-II is quantifiable by ionic and non-ionic detergents

295

LC3 isoforms (I and II) were isolated by using the three proposed lysis buffers (NP-40, RIPA

296

and Triton X-100), and they were well resolved by 12% SDS-gel electrophoresis (Fig. 1 and 2).

297

We can observe the blockade of autophagosome/lysosome fusion by Baf. A1 (Fig. 1A, 1D, 1E,

298

1H and 2A, 2D, 2E, 2H, lanes 3, 7 and 11) through the accumulation of the LC3-II isoform.

299

Moreover, under starvation conditions (Fig. 1A, 1D, 1E, 1H and 2A, 2D, lanes 2, 6 and 10),

300

LC3-II levels increased in comparison with untreated cells (Fig. 1A, 1D, 1E, 1H and 2A, 2D,

301

lanes 1, 5 and 9), except in the N27 cells, in which we noticed lower levels (Fig. 2E and 2H),

302

becoming higher in cells treated with Baf. A1 (Fig. 1A, 1D, 1E, 1H and 2A, 2D, 2E, 2H, lanes

303

4, 8 and 12).

304 305

2. Triton-X 100 is not an appropriate detergent for the quantification of p62 levels

306

Results obtained with p62 are more questionable. In the first approach, the p62 protein

307

extraction is less efficient when using Triton X-100 lysis buffer, in comparison with NP-40 and

308

RIPA buffers (Fig. 1A, 1C, 1E, 1G and 2A, 2C, 2E, 2G), likely because of the Triton X-100

309

insoluble fraction of this protein, as previously mentioned

310

denaturing detergent, we hypothesized that the interaction between p62 and LC3 were not

311

compromised. So, it would be possible to lose the insoluble-fraction of p62 protein together

312

with the pellet.

313

Apart from this consideration, the p62 levels observed by WB do not correspond to the p62

314

autophagy-mediated degradation because, although we blocked this process with Baf. A1 under

315

starvation (Fig. 1A, 1C, 1E, 1G and 2A, 2C, 2E, 2G), these rates are lower than they are in cells

316

that were only treated with Baf. A1 in all cell lines (Fig. 1A, 1C, 1E, 1G and 2A, 2C, 2E, 2G,

317

lanes 3, 7 and 11). To improve the performance of Triton-X 100, we mixed it with SDS, a

318

denaturing anionic detergent, obtaining the RIPA buffer. Despite this, we did not resolve the

319

loss of p62.

[4]

. As Triton X-100 is a non-

13

320

3. LC3 and p62 are lost in the pellet

321

To assess how these lysis buffers extracted LC3 and p62 proteins from the pellets, we

322

resuspended the processed pellets with SB 1X. As a result, during the protein extraction with the

323

different lysis buffers, we lost more p62 than LC3 (Fig 1B, 1C, 1D, 1F, 1G, 1H and 2B, 2C,

324

2D, 2F, 2G, 2H). In focusing on the p62 protein level, we observed a considerable loss of the

325

p62 insoluble fraction with Triton X-100 lysis buffer in MEFs and N27 cells, (Fig 1B, 1C, and

326

2F, 2G, lanes 9-12), in comparison with NP-40 and RIPA buffers (Fig 1B, 1C, and 2F, 2G,

327

lanes 1-4 and 5-8). In contrast, in human cells, we observe significant loss in any used buffers

328

(Fig 1F, 1G and 2B, 2C). Thus, a critical point could be the use of Triton X-100 to check p62

329

and to verify the autophagy flux by WB in the tested cell models because we could not detect

330

real differences in the levels of this protein between treatments.

331

Furthermore, we considered checking the SB 1X in the fresh pellets, to ensure that the entire

332

p62 pool was taken. Our results show that the p62 levels were unexpected, in the same way as

333

the Baf. A1 + EBSS condition in the other studied buffers (Fig. S1).

334

Considering the results obtained by WB, we analyzed endogenous p62 by IF to monitor its

335

autophagy degradation. We didn’t observe higher differences of p62 in both untreated and

336

treated cells with Baf. A1 (Fig. S2), following the same pattern as the WB results. Moreover,

337

we immunostained endogenous LC3, and the results were quite similar to those of the WB,

338

confirming that Baf. A1 conditions present the maximum levels of LC3 (isoforms I and II) (Fig.

339

S2). Based on the results for endogenous p62, we used the mCherry-GFP-p62 plasmid to clarify

340

that point. As expected, mCherry-p62 puncta accumulates under starvation conditions, which is

341

accompanied with GFP-p62 puncta when we block the autophagy process by using Baf. A1

342

(Fig. 3).

343 344 345 346 14

347

5. DISCUSSION

348

Many studies describe p62 as an autophagic substrate and signaling adaptor

349

able to bind both LC3-II and ubiquitinated cytosolic substrates. The cargo could thereby be

350

directed to the autophagosome, and then p62 and cytosolic substrates are degraded within the

351

autolysosomes

352

degradation and autophagic efficiency.

[10, 11]

because it is

[10-12]

. For this reason, the p62 levels would be a good way to measure cargo

353

However, the use of p62 levels as a measurement of cargo degradation has already been

354

questioned [4] because of the recently discovered association between p62 and multiple proteins

355

involved in several biological processes [13]. Moreover, solubility problems have been described

356

in relation to the non-solubility of p62 aggresomes when using common non-ionic detergents,

357

such as Triton X-100

358

markers that allow us to interpret autophagic flux and establish specific criteria to prevent some

359

misconceptions, especially in certain routine techniques such as WB. WB is widely used at

360

present, among other techniques, as the first approach to identifying the autophagic status by

361

detecting both autophagic markers mentioned above (p62 and LC3). In this study, we performed

362

a deep analysis of p62 and LC3 levels by using this widespread technique, and we compared

363

three lysis buffers that are commonly used in four cell models. The purpose of this study was to

364

analyze the limitations that each buffer presents, which may affect the proper interpretation of

365

our results.

[4]

. In this sense, it is very important to find other specific autophagic

366

In this sense, we verified that the lysis buffers used here did not lead to a great

367

difference in LC3-II levels (Fig. 1, 2 and S1). We observed a large accumulation of LC3-II

368

levels with Baf. A1 treatment, when combined or not with EBSS (Fig. 1, 2 and S1). However,

369

in some cases, EBSS alone may reduce the LC3-II level depending on the model and treatment

370

time

371

autophagy

372

for use in the WB technique, independently of the lysis buffer or cell line. In addition, the loss

373

of LC3 isoforms because of solubilization issues does not modify LC3 modulation during the

[2, 4]

. This variation in the LC3-II or turnover may be caused by the dynamic process of [12]

. Taken together, these results indicate that LC3 is a reliable autophagic marker

15

374

induction and/or inhibition of autophagy process. Based on our experience, we believe that the

375

NP-40 lysis buffer efficiency is better in human lines (HFs and SH-SY5Y) because LC3

376

extraction is greater than that observed with other buffers (Fig 1F, 1H and 2B, 2D).

377

p62 seems to have very irregular modulation and does not reflect the treatments

378

administered in the soluble fraction (Fig. 1A, 1C, 1E, 1G and 2A, 2C, 2E, 2G), except when

379

cells are treated with Baf. A1 alone, in which we observed a large accumulation of this protein

380

in comparison with untreated cells in any tested buffer or cell line. In addition, we found lower

381

p62 levels when we treated with EBSS

382

protein. We first thought that dual treatment could form p62 aggregates that we would have lost

383

in the insoluble fraction [4]. Surprisingly, the analysis of the insoluble fraction did not reveal the

384

accumulation of p62 aggregates (Fig. 1B, 1C, 1F, 1G and 2B, 2C, 2F, 2G). Moreover, we

385

observed a significant loss of p62 protein when using Triton-X 100 buffer in all cell lines, and

386

with all buffers in the human lines (Fig. 1B, 1C, 1F, 1G and 2B, 2C, 2F, 2G). Additionally, we

387

believed that our treatment was not effective enough, and thus, we decided to stain for

388

endogenous p62 and LC3 levels (Fig. S2) by IF and to overexpress the mCherry-GFP-p62

389

construct (Fig. 3) for better understanding. The IF displayed a diffuse staining of p62 protein,

390

and did not provide any additional information about its degradation. Nevertheless, p62 plasmid

391

overexpression showed that dual treatment blocked autophagosome-lysosome fusion. At this

392

point, we checked the p62 mRNA levels by quantitative PCR (qPCR). The expression levels of

393

p62 mRNA under treated conditions (Baf. A1, EBSS and Baf. A1 + EBSS) were higher than the

394

control in all cell lines (data not shown). Consistent with these results, a recent study showed

395

that p62 transcription is upregulated by prolonged starvation conditions (after 4 h) [16]. Although

396

several reports have shown p62 to be an autophagic substrate and a good marker for cytosolic

397

component degradation

398

should be cautious in routinely monitoring p62 levels by WB to assess cargo elimination. WB

399

testing can involve unexpected results and variable solubility, depending on the lysis buffer and

400

cell line chosen, at least in those analyzed in this study.

[14, 15]

. Dual treatment led to a decreasing level of p62

[7, 17, 18]

, we recommend, based on our discoveries, that researchers

16

401

6. ACKNOWLEDGMENTS

402

We thank George Auburger (Experimental Neurology, Goethe University Medical School,

403

Frankfurt am Main, Germany) for the MEFs, Adolfo López de Munaín (Neurology service,

404

Instituto BioDonostia, Hospital Donostia, San Sebastian, Spain) for the HFs and Anumantha G.

405

Kanthasamy (Iowa State University, Ames, IA) for the N27 cells. We also thank Dr. Terje

406

Johansen (Molecular Cancer Research group, Institute of Medical Biology, University of

407

Tromsø, Norway) for kindly providing the mCherry-GFP-p62 construct. Rubén Gómez-Sánchez

408

was supported by an “Acción III” postdoctoral contract (Universidad de Extremadura, Spain),

409

Mario Rodríguez-Arribas was supported by a FPU predoctoral Fellowship (Ministerio de

410

Educación, Spain), Rosa-Ana González-Polo was supported by a “Contrato Reincoporación de

411

Talentos”, Gobierno de Extremadura Spain), and she received research support from the

412

Ministerio de Economía y Competitividad, Spain (PI11/00040 and PI14/00170). Dr. José M.

413

Fuentes received research support from the Ministerio de Economía y Competitividad, Spain,

414

CIBERNED (CB06/05/004 and PI12/02280) and the Consejería, Economía, Competitividad e

415

Innovación , Gobierno de Extremadura, Spain (GRU10054). This paper is supported also by

416

“Fondo Europeo de Desarrollo Regional” (FEDER) from European Union. The authors would

417

like to thank P. Delgado, R. Ronco, J. Bragado, V. Llorente-Vera and D. Ramos-Barriga for

418

invaluable and continuous technical assistance. The authors also thank FUNDESALUD for the

419

helpful assistance.

420 421 422 423 424 425 426 427 17

428

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653

8. FIGURE LEGENDS

654

Figure 1. The determination of LC3 and p62 in fibroblasts by WB. The cells were non-

655

treated or treated with 100 nM Baf. A1 and/or EBSS for 4 h, as described in the “Methods

656

section”. The cell lines were harvested by trypsinization and lysed by using NP-40, RIPA and/or

657

Triton X-100 lysis buffers. The pellets were resuspended in SB 1X lysis buffer. p62 and LC3

658

(isoforms I and II) were determined in both lysates and pellets from MEFs (A-D) and HFs (E-

659

H). GAPDH protein was used as a loading control. Representative blots from three independent

660

experiments are shown in panels A, B, E and F, and the densitometry of each band expressed in

661

arbitrary units is shown in panels C, D, G and H. The molecular mass is indicated in kDa next to

662

the blots (*p ≤ 0.05, **p ≤ 0.01).

663 664

Figure 2. The determination of LC3 and p62 in neuronal cells by WB. The cells were non-

665

treated or treated with 100 nM Baf. A1 and/or EBSS for 4 h, as described in the “Methods

666

section”. The cell lines were harvested by trypsinization and lysed by using NP-40, RIPA and/or

667

Triton X-100 lysis buffers. The resulting pellets were resuspended in SB1X lysis buffer. p62

668

and LC3 (isoforms I and II) were determined in both lysates and the pellets from SH-SY5Y

669

cells (A-D) and N27 (E-H) cells. GAPDH protein was used as a loading control. Representative

670

blots of three independent experiments are shown in panels A, B, E and F, and the densitometry

671

of each band expressed in arbitrary units is shown in panels C, D, G and H. The molecular mass

672

is indicated in kDa next to the blots (*p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001).

673 674

Figure 3. Determining mCherry-GFP-p62 by IF in fibroblasts. The cells were transfected

675

with mCherry-GFP-p62 plasmid, as described in the “Methods section”. Twenty-four hours

676

post-transfection, the cells were non-treated or treated with 100 nM Baf. A1 and/or EBSS for 4

677

h and fixed. Representative IF microphotographs and the percentages of mCherry-p62 (+)

678

puncta per cell from MEFs (A and E, respectively), HFs (B and F, respectively), SH-SY5Y (C

27

679

and G, respectively) and N27 (D and H, respectively) are shown. Scale bars: 10 μm. Data are

680

expressed as the mean ± SEM; n = 20. (*p ≤ 0.05, ***p ≤ 0.001).

681 682

Figure S1. Determining LC3 and p62 by WB using SB 1X lysis buffer. The cells were non-

683

treated or treated with 100 nM Baf. A1 and/or EBSS for 4 h, as described in the “Methods

684

section”. The cell lines were harvested by trypsinization and the cells were lysed with SB 1X

685

lysis buffer. p62 and LC3 (isoforms I and II) were determined and GAPDH protein was used as

686

a loading control. Representative p62 and LC3 (isoforms I and II) blots of three independent

687

experiments are shown from MEFs (A), HFs (C), SH-SY5Y cells (E) and N27 (G) cells. The

688

molecular mass is indicated in kDa next to the blots. The densitometry of each band as

689

expressed in arbitrary units is shown in panels B (MEFs), D (HFs), F (SH-SY5Y cells) and H

690

(N27 cells). The molecular mass is indicated in kDa next to the blots (*p ≤ 0.05, ***p ≤ 0.001).

691 692

Figure S2. Determining endogenous LC3 and p62 by IF. Cells were non-treated or treated

693

with 100 nM Baf. A1 and/or EBSS for 4 h, as described in the “Methods section”. The cell lines

694

were

695

immunofluorescence microphotographs from MEFs (A), HFs (C), SH-SY5Y (E) and N27 cells

696

(G) are shown. Scale bars: 10 μm. The quantification of fluorescence intensity per cell from

697

MEFs (B), HFs (D), SH-SY5Y (F) and N27 cells (H) are shown as histograms. The data are

698

expressed as the mean ± SEM; n = 200. (*p ≤ 0.05, **p ≤ 0.01).

fixed

and

immunostained

for

LC3

(green)

and

p62

(red). Representative

699

28