Biochemistry and Cell Biology · Biochemistry and Cell Biology. Draft 4 60 during mechanical homogenization and freezing-thawing cycles (Picard et al. 61 2011). However, the loss

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Subcellular fractionation of frozen skeletal muscle samples

Journal: Biochemistry and Cell Biology

Manuscript ID bcb-2019-0219.R1

Manuscript Type: Methods

Date Submitted by the Author: 20-Sep-2019

Complete List of Authors: Firmino Dias, Pedro; University of Campinas Institute of Biology, Departament of Biochemistry and Tissue BiologyGandra, Paulo; State University of Campinas, Institute of BiologyBrenzikofer, René; UNICAMP, FEFMacedo, Denise; University of Campinas Institute of Biology, Biochemistry and Tissue Biology

Keyword: Mitochondria, Citrate Synthase, Cytosolic Fraction, Nuclear Fraction

Is the invited manuscript for consideration in a Special

Issue? :Not applicable (regular submission)

https://mc06.manuscriptcentral.com/bcb-pubs

Biochemistry and Cell Biology

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1 SUBCELLULAR FRACTIONATION OF FROZEN SKELETAL MUSCLE

2 SAMPLES

3 Pedro Rafael Firmino Dias 1, Paulo Guimarães Gandra1, René Brenzikofer 2,

4 Denise Vaz Macedo 1*

5 1 Department of Biochemistry and Tissue Biology, Institute of Biology, University

6 of Campinas, Campinas, Brazil

7 2 School of Physical Education, University of Campinas, Campinas, Brazil

8

9 *Denise Vaz Macedo

10 Department of Biochemistry and Tissue Biology, Institute of Biology, University

11 of Campinas, 130830-862, Campinas, Brazil. E-mail: [email protected]

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mailto:[email protected]

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12 Abstract

13 Cell fractionation can be used to determine the localization and trafficking of 14 proteins between cellular compartments such as cytosol, mitochondria and 15 nuclei. Subcellular fractionation is usually performed immediately after tissue 16 dissection since freezing may fragment cell membranes and induce organellar 17 cross-contamination. Mitochondria are especially sensitive to freezing/thawing 18 and mechanical homogenization. We proposed a protocol to improve soluble 19 proteins retention in the mitochondrial fraction obtained from small amounts of 20 frozen skeletal muscle. Fifty-milligram of red portion of gastrocnemius muscle 21 from Wistar rats were immediately processed or frozen in liquid nitrogen and 22 stored at −80 °C for further processing. We compared the enrichment of 23 subcellular fractions from frozen/fresh samples obtained with the modified 24 protocol with those obtained by standard fractionation. Western blot analyses of 25 marker proteins for cytosolic (alpha-tubulin), mitochondrial (VDAC1), and nuclear 26 (histone-H3) fractions indicated that all procedures resulted in enriched 27 subcellular fractions with minimal organellar cross-contamination. Notably, the 28 activity of the soluble protein citrate synthase was higher in mitochondrial 29 fractions obtained with the modified protocol from frozen/fresh samples 30 compared to the standard protocol. Therefore, our protocol improved the 31 retention of soluble proteins in the mitochondrial fraction and may be suitable for 32 subcellular fractionation of small amounts of frozen skeletal muscle samples.33

34 Keywords: Mitochondria, Citrate Synthase, Cytosolic fraction, Nuclear fraction.

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35 Introduction

36 Alterations in skeletal muscle function under physiological and

37 pathophysiological conditions can result from the cross talk between different

38 processes, such as mitochondrial biogenesis, autophagy, and apoptosis

39 (Scarpulla et al. 2012; Siu and Alway 2005). These signaling process can mediate

40 protein traffic, activity, and abundance within subcellular components (Scarpulla

41 et al. 2012; Siu and Alway 2005). To study the changes in protein abundance in

42 different cellular compartments, one can perform the subcellular fractionation of

43 whole tissue samples.

44 Methodologies for the separation of cytosolic, nuclear, and mitochondrial

45 fractions date from the mid-1950s and are based on the use of differential

46 centrifugation (Dounce et al. 1955). Since then, many protocols improved the

47 enrichment of subcellular fractions of various tissues, including skeletal muscle

48 (Bookelman et al. 1978; Dimauro et al. 2012; Martin et al. 1983). To avoid

49 organellar cross-contamination during subcellular fractionation, it is critical to

50 minimize the damage to biological membranes that may occur during mechanical

51 homogenization and differential centrifugation of the samples (Picard et al. 2011).

52 Freezing and thawing can rupture biological membranes. Therefore, subcellular

53 fractionation is usually performed in fresh samples (Hamm 1979; Lee 1995;

54 Sherman 1972). However, since muscle dissection and cell fractionation are

55 time-consuming, only a limited number of samples can be processed at a time

56 when fresh samples are used.

57 Several studies report that a relatively pure nuclear and cytosolic fractions

58 can be obtained from frozen skeletal muscle samples (Siu et al. 2005; Siu and

59 Alway 2005). Notably, mitochondria are particularly sensitive to fragmentation

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60 during mechanical homogenization and freezing-thawing cycles (Picard et al.

61 2011). However, the loss and the cross-contamination with mitochondrial matrix

62 proteins in subcellular fractions collected from frozen muscle samples are poorly

63 characterized.

64 The possibility to obtain subcellular fractions from small aliquots of frozen

65 skeletal muscle may be advantageous for studies dealing with a limited amount

66 of samples and a large number of biological replicates. Here, we tested if a

67 modified subcellular fractionation protocol can improve the retention of soluble

68 proteins of mitochondrial fraction collected from frozen skeletal muscle. We

69 compared the purity and enrichment of fractions from frozen and fresh muscle

70 samples obtained by two distinct methods: our modified protocol and a procedure

71 that is commonly used to process fresh muscle samples (Dimauro et al. 2012).

72 Experimental Protocol

73 All experiments were approved by the Ethics Committee of the School of

74 Agricultural and Veterinary Studies, São Paulo State University (UNESP),

75 Jaboticabal, Brazil (23593/15) and certified by the Animal Experimentation Ethics

76 Committee from the State University of Campinas (CEUA/Unicamp), and are in

77 accordance with the guidelines of the Canadian Council on Animal Care.

78 Wistar rats were killed by cardiac puncture under anesthesia (Steiner et

79 al. 2004). The red portion of gastrocnemius muscle was dissected, removed, and

80 separated into small 50-mg tissue aliquots. Two 50 mg aliquots of muscle were

81 processed for subcellular fractionation immediately after dissection (referred to

82 as fresh samples). One fresh sample was processed through the subcellular

83 fractionation protocol proposed in the present study (modified protocol). On the

84 other hand, the other aliquot was processed according to a fractionation protocol

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85 that is commonly used for muscle samples (standard protocol) (Dimauro et al.

86 2012). Two additional 50 mg muscle aliquots were frozen in liquid nitrogen and

87 stored at −80 °C. One frozen sample was processed by our subcellular

88 fractionation protocol (frozen samples). The other frozen sample was used in the

89 preparation of whole tissue lysate.

90 Subcellular Fractionation

91 Frozen muscle samples were thawed in a buffer solution (referred here as

92 “intramuscular buffer solution”) containing 5 mM NaCl, 45 mM KH2PO4, 50 mM

93 K2HPO4.3H2O, 5 mM NaHPO4.H2O, 10 mM EDTA, pH 7.0 at room temperature

94 for 1 min under gentle manual shaking. After thawing, the solution was replaced

95 with cold intramuscular buffer solution. Then, frozen samples were processed

96 similarly as described for the fresh tissue samples. All procedures were

97 performed on an ice-water bath.

98 Fresh or thaw muscle samples (50 mg) were cut into small pieces with

99 scissors in a petri dish on an ice bath in cold intramuscular buffer solution and

100 then sedimented at 200 g for 1 min (microcentrifuge at 4°C). The pellet was

101 washed and centrifuged twice at 200 g for 1 min in intramuscular buffer solution.

102 After washing, the pellet was resuspended in 250 μl isolation buffer (880 mM

103 sucrose, 20 mM HEPES pH 7.4, 50 mM NaCl, 5 mM MgCl2, 5 mM EGTA and

104 protease inhibitor). The sample was homogenized manually for 4 min using a

105 loose-fitting all-glass pestle in the ice-water bath. This procedure consisted of

106 slight rotations of the pestle, with gentle upward and downward movements

107 performed without removing the whole pestle from the solution. The pestle was

108 used to gently press the tissue. The homogenate was transferred to a centrifuge

109 microtube and centrifuged for 10 min at 1000 g. The resulting pellet was named

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110 P1 and was used to prepare nuclear fraction. The supernatant was named S1

111 and was used to prepare cytosolic and mitochondrial fractions. Figure 1 shows

112 an organizational chart describing the proposed protocol.

113 To this end, P1 was resuspended in 500 μl hypotonic buffer solution (20

114 mM HEPES pH 7.4, 10 mM NaCl, 1.5 mM MgCl2 and 0.1% Triton X-100),

115 homogenized using a tight-fitting Teflon pestle (10 passes at 600 rpm under ice-

116 water bath), transferred to a microtube, and centrifuged for 10 min at 1000 g. The

117 supernatant (NS1 and NS2) was discarded, and the pellet (NP1 and NP2) was

118 resuspended in 500 μl hypotonic buffer solution followed by centrifugation for 10

119 min at 1000 g. The supernatant (NS3) was discarded, and the pellet (NP3) was

120 resuspended in 200 μl of nuclear lysis buffer solution (20 mM HEPES pH 7.4, 500

121 mM NaCl, 20% glycerol, 2 mM MgCl2, 1% Triton X-100 and protease inhibitor)

122 and kept on ice for 1h. Then, the nuclei were lysed with 20 passages through a

123 26 gauge needle, sonicated (10 pulses of 2 sec at 80% power with intervals of 20

124 sec), and centrifuged for 20 min at 20,000 g. The resulting supernatant contained

125 the nuclear fraction.

126 The S1 was centrifuged again for 10 min at 1000 g. The resulting pellet

127 (P2) was discarded, and the supernatant (S2) was centrifuged for 25 min at

128 20,000 g. The resulting supernatant (S3) and pellet (MP1) were used to obtain

129 the cytosolic and mitochondrial fractions, respectively.

130 The supernatant S3 was centrifuged again for 25 min at 20,000 g. The

131 pellet (P2) was discarded, and the supernatant (S4) was once again centrifuged

132 for 25 min at 20,000 g. The resulting supernatant (S5) contained the cytosolic

133 fraction.

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134 The MP1 pellet was carefully resuspended (without a pipette-pellet

135 contact) in 200 μl isolation buffer and centrifuged for 25 min at 20,000 g. The

136 supernatant (MS1) was discarded. The pellet (MP2) was resuspended in 200 μl

137 of isolation buffer solution and centrifuged for 25 min at 20,000 g, and the

138 supernatant (MS2) was discarded. The resulting pellet (MP3) was resuspended

139 in 20 μl lysis buffer and lysed by 3 cycles of freezing/thawing at −80°C/room

140 temperature and sonication (10 pulses of 2 sec at 50% power with intervals of 20

141 sec) and kept on ice for 20 min. The final suspension contained the mitochondrial

142 fraction.

143 Special care was taken when removing the supernatants since the

144 sediments in the pellets’ upper layers can easily be aspirated and cause

145 contamination between the fractions. Therefore, small volumes of the

146 supernatants were intentionally left on the pellets (except for the mitochondrial

147 pellets). The total protein content of each subcellular fraction was determined by

148 a modified Bradford assay (Ernst and Zor 2010). All reagents were purchased

149 from Sigma-Aldrich (St. Louis, Missouri, USA).

150 Subcellular fractionation of fresh muscle samples by standard protocol

151 Fifty milligrams of fresh muscle samples were cut into small pieces with

152 scissors into a petri dish in an ice bath, resuspended in 300 μl STM buffer (250

153 mM sucrose, 50 mM Tris-Cl pH 7.4, 5 mM MgCl2 and protease inhibitor from

154 Sigma-Aldrich), and homogenized using a Teflon pestle for 1 minute at 600 rpm

155 in the ice-water bath. The homogenate was transferred to a centrifuge microtube

156 and kept on ice for 30 min, and vortexed at full speed for 15 sec and centrifuged

157 for 15 min at 800 g. The pellet was used to obtain the nuclear fraction, and the

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158 supernatant was used to obtain the cytosolic and mitochondrial fractions as

159 described elsewhere (Dimauro et al. 2012).

160 Preparation of whole tissue lysate

161 In the preparation of whole tissue lysate, 50 mg of the red portion of

162 gastrocnemius muscle was powdered under liquid nitrogen. The powdered

163 muscle was homogenized in 500 μl lysis buffer (50 mM HEPES pH 7.4, 150 mM

164 NaCl, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS) and protease

165 inhibitor (Sigma-Aldrich) by sonication on ice-water bath (10 pulses, 2 sec each

166 with a 20 sec interval, at 80% power). The homogenates were kept in the ice-

167 water bath for 20 min, followed by a 10 min centrifugation at 1000 g. The

168 supernatant was collected, and total protein content was determined by the

169 modified Bradford method (Ernst and Zor 2010).

170 Western Blot analysis

171 Protein samples (2.5 μg protein) were resolved in 10% SDS-PAGE gel for

172 alpha-tubulin (AB176560, 1:1000) and VDAC1 (CST #4661, 1:1000), and 15%

173 SDS-PAGE gel for histone H3 (AB176842, 1:1000). After electrophoresis the

174 proteins were transferred to 0.22 μm PVDF membrane (Bio-Rad, Richmond,

175 California, USA) in CAPS buffer, blocked with 3% BSA (Sigma-Aldrich) for 1 h at

176 room temperature, and incubated overnight with the primary antibodies directed

177 for the proteins of interest. After washing, the membranes were incubated for 1 h

178 at room temperature using an HRP-conjugated secondary antibody (CST #7074,

179 1:5000). The immunoreactive bands were detected by using enhanced

180 chemiluminescence (ECL; Thermo Fisher, Rockford, Illinois, USA).

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181 To avoid differences in bands intensities due exposures of different

182 membranes and the lack of loading control present in the three fractions, we

183 loaded 10 μg of whole tissue lysate proteins in 2 wells, one at each end of the

184 gels. Then, the intensities of whole tissue lysate bands were used to equalize

185 membranes exposures. Optimal bands intensities were given by UVTech

186 software (UVTech, UK) and quantified using ImageJ analysis software.

187 Citrate Synthase Enzyme Activity

188 Citrate synthase enzyme activity was determined in a microplate

189 spectrophotometer following a well-established protocol (Srere 1969). We used

190 5 μg of protein diluted in 140 μl of 100 mM Tris-Cl pH 8.3. Twenty μl of 1 mM

191 DTNB [5.5’dithiobis (2-nitrobenzoic acid)] (Sigma-Aldrich) and 20 μl of 3 mM

192 Acetyl-CoA (Sigma-Aldrich) were added to the solution, and absorbance readings

193 were performed at 412 nm, every 15 seconds, over a 3-minutes interval. After the

194 initial reading, we added 20 μl of 5 mM Oxaloacetic acid (Sigma-Aldrich) to initiate

195 the reaction, and the absorbance was recorded using the same parameters

196 described above. Citrate synthase activity was expressed as nmol/min/μg

197 protein.

198 Statistical analysis

199 Citrate synthase activity and western blots data of the different fractions

200 obtained by the three preparation methods were subjected to the Jarque–Bera

201 test to test for normal distribution. One-way analysis of variance (ANOVA)

202 followed by Tukey’s post-hoc test were used to compare means between the

203 subcellular fractions. All analyzes and graphs for this study were executed in

204 MATLAB® 2010 (The MathWorks Inc., Massachusetts, USA). The significance

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205 level was set at p < 0.05. Data are presented as mean ± standard deviation. The

206 results presented below were collected from 5 independent experiments (n = 5).

207 Results

208 Table 1 shows the protein yield of the subcellular fractions of frozen and

209 fresh muscle tissues processed by modified protocol. There was no significant

210 difference in the protein yields within the cellular fractions obtained from either

211 frozen or fresh samples.

212 The western blotting analysis of subcellular fractions obtained from

213 modified protocol is shown in Figure 2. Alpha-tubulin (cytosolic), VDAC1

214 (mitochondrial), and histone H3 (nuclear) demonstrated intense bands in their

215 corresponding fraction in preparations from both, frozen, and fresh samples. This

216 result suggests that organellar cross-contamination was very low.

217 We then quantified the citrate synthase activity in the three subcellular

218 fractions to analyze the retention of soluble proteins in the mitochondrial fraction

219 and the cross-contamination with mitochondrial soluble proteins (Figure 3). As

220 expected, citrate synthase activity was higher in the mitochondrial fraction vs. the

221 nuclear and cytosolic fractions (this was observed for fractions obtained from

222 frozen, and fresh samples prepared through the modified protocol). This finding

223 corroborates that we obtained an enriched mitochondrial fraction. The significant

224 higher citrate synthase activity in the mitochondrial fraction from fresh vs. frozen

225 samples may result from lower retention of soluble proteins due to

226 freezing/thawing of the frozen samples.

227 Figure 4 shows the western blot and citrate synthase activity of the

228 subcellular fractions derived from the standard protocol. The fractions collected

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229 from fresh muscle samples processed by the standard protocol also presented

230 very low organellar cross-contamination when examined by western blot of

231 specific protein markers for cytosol, nuclei, and mitochondria (Figure 4). We

232 observed a lower-band density of histone H3 in the nuclear fraction of standard

233 protocol compared with the nuclear fraction from frozen and fresh samples

234 obtained by our protocol (Figure 2 and 4).

235 In the subcellular fractions collected by the standard protocol, citrate

236 synthase enzymatic activity was significantly higher in the mitochondrial fraction

237 compared with the cytosolic and nuclear fractions (Figure 4). Notably, the citrate

238 synthase activity was higher in the mitochondrial fractions obtained from fresh

239 and frozen samples by the modified protocol compared to the standard protocol

240 (431.47 ± 72.47, 295.86 ± 99.40 and 112.04 ± 72.47 nmol.min-1.ug protein-1

241 respectively; p < 0.0001). These data indicate that the retention of matrix soluble

242 proteins in the mitochondrial fraction was higher when the modified protocol was

243 used.

244 Our results confirmed that the retention of mitochondrial matrix proteins is

245 lower when subcellular fractionation is performed in frozen samples compared to

246 when fresh samples are processed (see Figure 3 and discussion). A direct

247 comparison of the subcellular fractions obtained by the modified protocol (fresh

248 and frozen samples) against subcellular fractions obtained from fresh samples

249 processed by the standard protocol already allowed us to conclude about the

250 efficiency of the modified method. Hence, we didn’t perform subcellular

251 fractionations of frozen muscle samples or any other methodological

252 modifications by using the standard protocol as a reference.

253 Discussion

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254 Our proposed protocol improved retention of matrix soluble proteins in the

255 mitochondrial fraction and represents a useful tool to investigate the changes in

256 muscle function and protein distribution under different conditions. Protein

257 analysis from subcellular fractions indicated that the three procedures (frozen,

258 fresh, and standard) presented minimal organellar cross-contamination. Citrate

259 synthase activity, used as a marker of mitochondrial matrix protein, was higher in

260 the mitochondrial fractions obtained by the proposed protocol from frozen and

261 fresh muscle samples compared with a commonly used fractionation method.

262 This observation indicates that the methodological modifications improved the

263 retention of matrix soluble proteins in the mitochondrial fraction.

264 The slightly lower enzymatic citrate synthase activity found in

265 mitochondrial fraction from modified protocol of frozen vs. fresh samples was

266 expected and is typical of the freezing process. Freezing/thawing cycles of tissue

267 samples result in the formation of ice crystals which cause the disorganization

268 and fragmentation of biological membranes (Hamm 1979; Lee 1995; Sherman

269 1972). Such changes may lead to mitochondria soluble enzymes extravasation

270 and then affect the cytosol while leaving intact the location of transmembrane

271 enzymes, such as complex II (Bookelman et al. 1978; Hamm 1979). The data

272 regarding VDAC1 amounts and citrate synthase enzyme activity, which represent

273 a transmembrane and soluble mitochondrial protein, respectively, corroborate

274 this interpretation. We observed no VDAC1 intensity differences in the

275 mitochondrial fraction among frozen and fresh sample preparations. However,

276 citrate synthase activity presented significant differences between these

277 preparations, shown as lower activity in mitochondrial fraction of frozen sample

278 compared to fresh sample.

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279 To minimize cryodamage of the muscle sample, we combined high

280 freezing rates with rapid thawing of the samples. Fast freezing rates (e.g., small

281 samples amounts frozen by immersion in liquid nitrogen) can form smaller ice

282 crystals than slow freezing rates (e.g., samples frozen at −20 and −80 °C) causing

283 less cellular damage (Hamm 1979; Meng et al. 2014; Sherman 1972). Rapid

284 thawing (e.g., thawing at room temperature solutions) is superior to slow thawing

285 (e.g., thawing at 4°C solutions) in maintaining cellular structure, possibly due to

286 the earlier inhibition of degradative enzymes released by lysosomal lysis. Such

287 enzymes can compromise cellular integrity when not inhibited by protease

288 inhibitors (Sherman 1972, Sherman 1971). We also thawed the samples in a

289 buffer solution with an osmolarity and electrolyte constitution like the

290 intramuscular medium. This possibly resulted in osmotic equilibrium between

291 sample intracellular medium and solution, reducing osmotic swelling and

292 damage. We also tested thawing the samples in PBS and isolation buffer or to

293 directly homogenize the frozen samples in the isolation buffer that was used for

294 the subcellular fractionation. However, neither approaches presented satisfactory

295 results and demonstrated low retention of citrate synthase in the mitochondrial

296 fraction (data not shown).

297 In situ, skeletal muscle mitochondria are elongated and form complex

298 branched structures making them more sensitive to fragmentation during

299 mechanical homogenization (Picard et al. 2011). This fragmentation and

300 resealing may limit the results due to the loss of the mitochondrial matrix content.

301 However, this fragmentation-resealing process may not result in the loss of

302 mitochondrial membrane proteins, such as VDAC from the mitochondrial

303 fractions. The gentle hand homogenization with all glass pestle adopted here

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304 resulted in better citrate synthase activity retention in the mitochondrial fraction

305 than when we tested a more aggressive homogenization with a Teflon pestle

306 coupled to a rotor (data not shown). Therefore, a gentle homogenization of

307 muscle samples is crucial to improve the retention of the mitochondrial matrix

308 content during subcellular fractionation. This also explains the smaller citrate

309 synthase activity observed in the mitochondrial fraction collected by the standard

310 protocol, in which a rotor-coupled Teflon pestle was used (Figure 4).

311 Mitochondrial integrity during cellular fractionation is highly affected by the

312 solution used during mitochondrial isolation. Several studies have used electron

313 microscopy to demonstrate the superiority of high sucrose concentrations in

314 maintaining the morphology of isolated mitochondria. With 250 mM sucrose, the

315 mitochondria became spherical, swollen, and with circular cristae and a less-

316 dense matrix which are irreversible changes. With 880 mM sucrose, the

317 mitochondria remained elongated with highly condensed cristae and matrix

318 (Dounce et al. 1955; Lehninger et al. 1959; Stoner and Sirak 1969; Witter et al.

319 1955). The increase in external colloidal osmotic pressure caused by the high

320 sucrose concentration and the dehydration of the organelle by osmosis likely aids

321 this organelle morphologic preservation (Hogeboom et al. 1948). Thus, it is

322 possible that the high sucrose isolation medium used in our protocol resulted in

323 increased preservation of the organelle integrity during the isolation step and,

324 consequently, in the citrate synthase retention. The standard protocol, however,

325 employs an isolation buffer containing 250 mM sucrose, which likely contributes

326 to lower retention of citrate synthase.

327 It is important to mention that contamination by organelles other than

328 nuclei and mitochondria were not accessed. For example, the mitochondrial

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329 fraction obtained by differential centrifugation is known to present endoplasmic

330 reticulum and peroxisome contamination (Deng et al. 2010). For studies in which

331 these kinds of contamination need to be controlled additional purification steps

332 are necessary. The higher-intensity histone H3 band in the western blot assay of

333 frozen and fresh samples prepared by modified protocol may have occurred due

334 to the use of 0.1% Triton X-100 in the hypotonic buffer during the second

335 homogenization (P1) and during the washing of the pellets associated with the

336 nuclear fraction. Gagnon et al. demonstrated that nuclear membrane-related

337 organelles, such as endoplasmic reticulum, can be removed by using 0.3% NP-

338 40, leading to a higher concentration of nucleus in the nuclear fraction (Gagnon

339 et al. 2014). Since both detergents are non-denaturing and non-ionic, Triton X-

340 100 may have contributed to the same phenomenon.

341 Conclusions

342 The modified protocol succeeded to improve the retention of soluble matrix

343 proteins in the mitochondrial fraction with small amounts of frozen skeletal

344 muscle. It can be advantageous in studies with a large number of samples or

345 when a limited amount of sample is available, such as in studies with humans’

346 biopsies.

347 Acknowledgments

348 This study was financed in part by the Coordenação de Aperfeiçoamento

349 de Pessoal de Nível Superior – Brazil (CAPES) finance code 001. The authors

350 also thank to Funcamp (927.7 BIO-0100) for part of the financial support.

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419 Stoner, D., Sirak, H.D., 1969. Osmotically-induced alterations in volume and

420 ultrastructure of mitochondria isolated from rat liver and bovine heart. J Cell

421 Biol 43. https://doi.org/10.1083/jcb.43.3.521

422 Witter, R.F., Watson, M.L., Cottone, M.A., 1955. Morphology and ATP-ase of

423 Isolated Mitocchondria*. J Cell Biol 1. https://doi.org/10.1083/jcb.1.2.127

424

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425 Table 1. Total protein yield of subcellular fractions obtained by the modified

426 protocol from frozen and fresh muscle samples.

Cellular Fractions Frozen samples (μg) Fresh samples (μg)

Cytosol 255.8 ± 62.1 235.4 ± 88.5

Mitochondria 38.5 ± 9.8 39.7 ± 16.9

Nucleus 353.7 ± 164.3 323.3 ± 223.6

427 Data presented as mean and standard deviation.

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428

429 Figure 1. Organizational chart of the subcellular fractionation modified protocol.

430 ITM buffer solution = intramuscular buffer solution; ISO buffer = isolation buffer;

431 HYPO buffer = hypotonic buffer; NL buffer = nuclear lysis buffer; S = supernatant;

432 P = pellet; MS = mitochondrial supernatant; MP = mitochondrial pellet; NS =

433 nuclear supernatant; NP = nuclear pellet.

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434

435 Figure 2. Representative images and graph showing the optical intensities

436 obtained by western blot assay of subcellular fractions obtained from frozen and

437 fresh muscle samples processed by the modified protocol. Data presented as

438 mean ± standard deviation. Cyt = cytosol; Mit = mitochondria; Nuc = nucleus.

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439

440 Figure 3. Citrate synthase activity (nmol/min/µg protein) in the subcellular

441 fractions obtained from frozen- and fresh-tissue processed by the modified

442 fractionation method. Data presented as mean ± standard deviation. # = p < 0.05

443 compared to all fractions collected from frozen and fresh preparations and * = p

444 < 0.05 compared to cytosol and nucleus from frozen and fresh preparations were

445 carried out ANOVA with post-hoc Tukey comparing all fractions together; & = p <

446 0.05 compared to cytosol and nucleus from frozen sample were carried out

447 ANOVA with post-hoc Tukey comparing the fractions from the same preparation.

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448

449 Figure 4. Representative image of western blots and citrate synthase enzyme

450 activity of subcellular fractions obtained from standard protocol preparations. Cyt

451 = cytosol; Mit = mitochondria; Nuc = nucleus. Data presented as mean ± standard

452 deviation. * p < 0.05 vs. Cyt and Nuc; # p < 0.05 vs. Nuc (ANOVA with post hoc

453 Tukey).

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Biochemistry and Cell Biology · Biochemistry and Cell Biology. Draft 4 60 during mechanical homogenization and freezing-thawing cycles (Picard et al. 61 2011). However, the loss