br CACCATCCCTCCC GGCTTCTGCCTCA GABARAPL ACCAGT TTTTCCAT

CACCATCCCTCCC GGCTTCTGCCTCA GABARAPL1 ACCAGT TTTTCCAT
CACATATGCAATG TCAAGCAGTGTTTA LAMP2A TTTTAAGGTCTGTC TTAATTCCAGTAAGAT
AGCTCTTTGTTGG TTGTCCTCACAGCT LC3B TGTGTAACTGTCT GACATGTATG
AGAACTGGACCC CACTTCGCTGATA MFN2 GGTTACCA CCCCTGA
CATTGTGTGACTGG TCTCTAGGCCACC MURF1 CGATTGT GAGTGAGA
TAQMAN
GAAAGGGCCAAACA GTAAATCACACGG PGC-1α GAGAGA CGCTCTT
CCCAGTGTCTTGG AGGGAAAGCAGA p62/SQSTM1 CATTCTT GGAAGCTC
Mitochondrial respiration
Oxygen consumption was measured in perme-abilized Cytochalasin D at 37 °C by high-resolution respirometry with the Oxygraph-2k (Oroboros Instru-ments). A specific experiment on male mice, distinct from the previously described ones, was performed in order to process 4 mice per day (C, n = 3; C26, n = 5 for both WT and Tg mice). EDL muscles were excised and immediately placed in ice-cold biopsy containing preservation medium [2.77 mM CaK2EGTA, 7.23 mM K2EGTA, 20 mM imidazole, 20 mM taurine,
3 mg were mechanically separated using thin twee-zers and permeabilized with 50 μg/ml saponin for 30 min at 4 °C according to the technique of Veksler et al. [41]. After rinsing of muscle bundles in respiration medium [EGTA 0.5 mM, MgCl2·6H2O 3 mM, taurine 
110 mM (pH 7.1)], the muscle fibers were transferred to the oxygraph to perform high-resolution respirom-etry corrected for wet weight. Resting respiration (state 4, absence of adenylates) was determined by the addition of 10 mM glutamate and 2 mM malate as the Complex I substrate supply, and state
3 respiration was then assessed by the addition of 2.5 mM ADP (Compl I). ADP control of coupled respiration and uncoupling control were examined
through the addition of the protonophore c a r b o n y l c y a n i d e - 4 - ( t r i f l u o r o m e t h o x y ) - phenylhydrazone (FCCP; Max R); 0.5 μM rotenone and 2.5 μM antimycin A were added to inhibit Complexes I (allowing Compl II extrapolation) and III, respectively, to observe non-mitochondrial respiration (Leak). All respiration measurements were performed in duplicate for each muscle.
Expression of results and statistical analysis
Data are presented as mean ± SEM. Statistical significance was assessed by a two-tailed paired Student's t-test or one-way ANOVA analysis follow-ed by Tukey's multiple comparisons test. A value of p b 0.05 was chosen as the limit of statistical significance.
Acknowledgments
We thank Jorge Manuel Seco and Vanessa Hernández for technological assistance, and Beth Levine and Viviana Moresi for sending the BCL-2 triple mutant plasmid. Paula Martínez-Cristóbal was the recipient of a predoctoral fellowship from the ‘la Caixa/IRB International Ph.D. Programme. Fabio Penna received a visiting fellowship under the World Wide Style program of the University of Torino. This study was supported by research grants from the MINECO (SAF2016-75246R), Grant 2014SGR48 from the Generalitat de Catalunya, CIBERDEM (“Instituto de Salud Carlos III”), INFLAMES (PIE-14/00045, Instituto de Salud Carlos III), and University of Torino (ex-60% funds). The research leading to these results has received funding from AIRC under IG 2018—ID. 21963 project (P.I. Penna Fabio). Antonio Zorzano is a recipient of an ICREA “Academia” (Generalitat de Catalunya). We gratefully acknowledge institutional funding from the MINECO through the Centres of Excellence Severo Ochoa Award, and from the CERCA Programme of the Generalitat de Catalunya. Competing Interests: The authors declare no competing financial interests.
Autophagy and mitochondria in cancer cachexia 2685
Appendix A. Suppementary data
Keywords:
autophagy;
cancer cachexia;
muscle wasting;
mitochondria;
mitophagy
†Equal contribution.
Abbreviations used:
UPS, ubiquitin–proteasome-dependent pathway; TA,
tibialis anterior; CSA, cross-sectional area; TB,
tumor-bearing; WT, wild-type.
References
2686 Autophagy and mitochondria in cancer cachexia
M.P. Wiggs, T.A. Washington, N.P. Greene, Mitochondrial degeneration precedes the development of muscle atrophy in progression of cancer cachexia in tumor-bearing mice,
Available online at www.sciencedirect.com
ScienceDirect
Original Research
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