EMBO/soda-nel huggingface dataset:
CSAG2 is a cancer-specific activator of SIRT1 (2020)
Figure 2

Figure 2. CSAG2 Is Necessary and Sufficient to Promote Tumor Cell Growth in vitro and in vivo.

(A-B) The effect of stable knockdown CSAG2 in HCT116 (A) or A375 (B) were assayed for clonogenic growth on plastic for 7 days. The number of colonies was quantified and is shown. Data are represented as the mean ± SD, n=3 biological replicates.

(C-D) The effect of stable knockdown CSAG2 in HCT116 (C) or A375 (D) were determined for anchorage-independent growth in soft agar colony formation assays for 14 days. The number of colonies was quantified and is shown. Data are represented as the mean ± SD, n=3 biological replicates.

(E-F) Knockdown of CSAG2 in A375 (F) or HCT116 (E) cells decreases xenograft tumor growth in mice. Cells with doxycycline-inducible shRNAs were implanted into NOD SCID gamma mice. Doxycycline (2 mg/mL) was administered via drinking water. Tumor growth was monitored over time. Data are represented as the mean ± SD, n=6 mice for each group.

(G-H) Stable expression of CSAG2 in HCT116 (G) and CSAG2-negative H460 (H) cells increases anchorage-independent growth in soft agar colony formation assays, n=3 biological replicates. Data are represented as the mean ± SD.

Data information: *p < 0.05, **p < 0.01, ***p < 0.001; unpaired, two-tailed student t-test or ANOVA analysis (E-F).
CSAG2 is a cancer-specific activator of SIRT1 (2020)
Figure 3

Figure 3. CSAG2 Interacts with SIRT1 and Promotes p53 Deacetylation.

(A) HEK293 cells stably expressing TAP-vector, TAP-CSAG1, or TAP-CSAG2 were subjected to pull-down followed by SDS-PAGE and LC-MS/MS. Spectral counts (SC) of the identified proteins in TAP-CSAG1 and TAP-CSAG2 pulldowns, but absence in TAP-vector control, are shown.

(B) HEK293FT cells stably expressing HA-SIRT1 were transfected with Myc-CSAG1 or Myc-CSAG2 for 48 h before IP with anti-HA followed by SDS-PAGE and immunoblotting for indicated proteins.

(C) Recombinant GST-SIRT1, but not GST, binds in vitro translated Myc-CSAG2. Myc-tagged CSAG2 was in vitro translated and GST-pulldown experiments were performed with recombinant GST or GST-SIRT1 followed by SDS-PAGE and immunoblotting for anti-Myc.

(D-G) Expression of CSAG2 decreased p53 ac-K382 levels and downregulated the expression of downstream p53 target genes in H460 cells. Cells were treated by either 1 μM doxorubicin (D-E) or 20 μM etoposide (F-G) with 0.4 μM TSA for the indicated times. Cells were then harvested and blotted for the indicated proteins. Quantitation of expression levels is shown (E and G) as the mean ± SD, n=3 biological replicates. Ac-p53 K382 levels normalized to total p53, whereas all other proteins were normalized to GAPDH.

(H-K) Knockdown of endogenous CSAG2 in HCT116 (H-I) and A375 (J-K) cells increased p53 ac-K382 levels. CSAG2-knockdown stable cells were treated by 1 μM doxorubicin/ 0.4 μM TSA for 6 hr. Cells were then harvested and blotted for the indicated proteins. Quantitation of ac-p53 K382 levels relative to total p53 is shown (I and K) as the mean ± SD, n=3 biological replicates.

(L-M) Re-expression of CSAG2 rescued p53 ac-K382 levels in CSAG2 knockdown stable cell lines. Myc-CSAG2 was transfected in HCT116 (L) or A375 (M) CSAG2 knockdown stable cell lines. Cells were treated with 1 μM doxorubicin/ 0.4 μM TSA for 6 hr and then blotted for the indicated proteins. Quantitation of ac-p53 K382 levels relative to total p53 is shown as the mean ± SD, n=3 biological replicates.

(N) CSAG2 does not enhance anchorage-independent growth of HCT116 p53-null cells. CSAG2 was stably expressed in isogenic HCT116 p53-wild-type or p53-null cells. Anchorage-independent growth in soft agar colony formation assays was determined after 14 days. The number of colonies was quantified, normalized to each vector control cell line, and is shown as the mean ± SD, n=3 biological replicates.

Data information: *p < 0.05, **p < 0.01, ***p < 0.001, unpaired, two-tailed student t-test.
CSAG2 is a cancer-specific activator of SIRT1 (2020)
Figure 4

Figure 4. CSAG2 Promotes p53 Deacetylation through SIRT1.

(A) Knockdown of SIRT1 abolished CSAG2-mediated decrease in ac-p53 K382. CSAG2 over-expressing HCT116 were transfected with SIRT1 siRNA for 72 hr before doxorubicin/TSA treatment for 6 hr. Cell lysates were blotted for the indicated proteins.

(B) Inhibition of SIRT1 enzymatic activity blocked CSAG2-induced decrease in p53 ac-K382. CSAG2 over-expressing HCT116 cells were treated with 1 μM doxorubicin/ 0.4 μM TSA with or without 1 μM EX-527 for 6 hr. Cell lysates were blotted for the indicated proteins.

(C-D) Inhibition of SIRT1 enzymatic activity prevents CSAG2-knockdown increase in p53 ac-K382. CSAG2-knockdown stable cells were treated by 1 μM doxorubicin/ 0.4 μM TSA with or without 1 μM EX-527 for 6 hr. Cells were then harvested and blotted for the indicated proteins (C) and quantitated (D; n=3 biological replicates). Data shown as mean ± SD.

(E) CSAG2 binds catalytically inactive SIRT1 H363Y. Cells were transfected with the indicated constructs before anti-Myc IP and blotting for the indicated proteins.

(F) SIRT1 H363Y blocks CSAG2 regulation of p53 ac-K382 levels. HCT116 cells were transfected with the indicated constructs and blotted for the indicated proteins.

(G) Inhibition of SIRT1 activity abolished CSAG2-induced anchorage-independent growth. Soft agar colony formation assays were performed in control or CSAG2 over-expressing HCT116 cells with or without EX-527 treatment. Data are represented as the mean ± SD, n=3 biological replicates.

(H) Summary of region on CSAG2 required for SIRT1 interaction. HEK293FT cells stably expressing HA-SIRT1 were transfected with indicated CSAG2 constructs for 48 h before IP with anti-HA followed by SDS-PAGE and immunoblotting for anti-Myc.

(I) CSAG2 Δ37 mutant fails to interact with SIRT1 in vitro. Myc-tagged CSAG2 38-127 fragment was in vitro translated and followed by in vitro binding assay with recombinant GST-SIRT1, SDS-PAGE, and immunoblotting for anti-Myc.

(J) CSAG2 Δ37 mutant does not regulate p53 ac-K382 levels. Cells were transfected with the indicated Myc-CSAG2 constructs for 48 hr before being treated with 1 μM doxorubicin/ 0.4 μM TSA. Cell lysates were blotted for the indicated proteins.

(K) CSAG2 Δ37 fails to promote anchorage-independent growth of H460 cells. Soft agar colony formation assays were performed in CSAG2-negative H460 cells stably expressing CSAG2 wild-type or CSAG2 Δ37 mutant (n=3 biological replicates). Data are represented as the mean ± SD.

Data information: **p < 0.01 unpaired, two-tailed student t-test.
CSAG2 is a cancer-specific activator of SIRT1 (2020)
Figure 5

Figure 5. CSAG2 Promotes Cellular Resistance to Genotoxic Stress through Regulation of SIRT1.

(A-B) Control or CSAG2 expressing H460 cells were treated with the indicated concentrations of doxorubicin (A) or H2O2 (B) for 24 hr before cell viability was determined by alamarBlue (n=6 biological replicates).

(C-D) shCTRL, shCSAG2-1, or shCSAG2-2 stable HCT116 cells were treated with the indicated concentrations of doxorubicin (C) or H2O2 (D) for 24 hr before cell viability was determined by alamarBlue (n=6 biological replicates).

(E-F) HCT116 cells were transiently transfected with the indicated shRNAs for 48 hr before treatment with 1 μM doxorubicin/ 0.4 μM TSA for 6 hr. Cell lysates were collected and blotted for the indicated proteins (E). Quantitation (n=3 biological replicates) of normalized cleaved PARP levels is shown (F).

(G) H460 cells were transfected with wild type, Δ37, or Δ63 CSAG2 for 48 hr. Cells were then treated with the indicated concentrations of H2O2 for 24 hr before cell viability was determined by alamarBlue (n=6 biological replicates).

(H) Inhibition of SIRT1 activity abolished CSAG2 induced cell survival under genotoxic stress. CSAG2 over-expressing H460 cells were treated with the indicated concentrations of H2O2 with or without 1 μM EX-527 for 24 hr before cell viability was determined by alamarBlue (n=6 biological replicates).

(I-J) Control or Myc-CSAG2 expressing HCT116 p53 -/- cells were treated with the indicated concentrations of H2O2 (I) or doxorubicin (J) for 24 hr before cell viability was determined by alamarBlue (n=6 biological replicates).

(K-L) shCTRL, shCSAG2-1, or shCSAG2-2 stable HCT116 p53 -/- cells were treated with the indicated concentrations of H2O2 (K) or doxorubicin (L) for 24 hr before cell viability was determined by alamarBlue (n=6 biological replicates).

(M-N) Control or Myc-CSAG2 expressing H1299 (p53 mutant) cells were treated with the indicated concentrations of H2O2 (M) or doxorubicin (N) for 24 hr before cell viability was determined by alamarBlue (n=6 biological replicates).

Data information: Data are represented as the mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001, unpaired, two-tailed student t-test.
CSAG2 is a cancer-specific activator of SIRT1 (2020)
Figure 6

Figure 6. CSAG2 Directly Enhances SIRT1 Enzymatic Activity.

(A) CSAG2 does not alter SIRT1 protein levels. HEK293FT cells were transfected with different amounts of Myc-CSAG2 for 48 hr before cells lysates were harvested and immunoblotted for indicated proteins.

(B) CSAG2 does not alter SIRT1 subcellular location. U2OS cells were transfected with or without mcherry-CSAG2 for 48 hr before fixation, SIRT1 immunostaining, and imaging. Scale bar: 10 µm.

(C) Summary of results mapping interaction region of SIRT1 recognized by CSAG2. HEK293FT cells stably expressing HA-CSAG2 were transfected with indicated Myc-SIRT1 constructs for 48 h before IP with anti-HA followed by SDS-PAGE and immunoblotting for anti-Myc.

(D) CSAG2 directly binds SIRT1 catalytic domain in vitro. HA-tagged SIRT1 fragments were in vitro translated. In vitro binding assays with recombinant GST or GST-CSAG2 were performed followed by SDS-PAGE and immunoblotting for anti-HA.

(E) CSAG2 enhances SIRT1 activity in vitro. In vitro SIRT1 activity towards p53 ac-K382 peptide was determined in the presence of GST or GST-CSAG2, n=3 biological replicates. Nicotinamide (1 mM) was added to the reaction were indicated to block SIRT1 activity.

(F) CSAG2 Δ37 mutant that does not bind SIRT1 does not enhance SIRT1 activity in vitro. In vitro SIRT1 activity towards p53 ac-K382 peptide was determined in the presence of GST, GST-CSAG2 or GST-CSAG2 38-127, n=3 biological replicates.

(G) CSAG2 enhances SIRT1 deacetylation of p53 ac-K382 peptide through increasing kcat, but not Km. not affecting the binding ability with its substrate p53. SIRT1 activity assays were performed as described above, except varying amounts of p53 ac-K382 fluorometric peptide were used in the presence of saturating NAD+ (3 mM), n=3 biological replicates.

(H-I) U2OS cells were transfected with Myc-Vector or Myc-CSAG2 for 48 hr before being treated with or without 0.4 μM TSA for 6 hr. Cell lysates were blotted for the indicated proteins (H). Quantitation of expression levels of acetylated Histone relative to total Histone is shown as the mean ± SD, n=3 biological replicates. White circles indicate Myc-Vector and black circles indicate Myc-CSAG2.

(J) HeLa, U2OS, or HCT116 cells were transfected with Myc-Vector or Myc-CSAG2 for 48 hr. Cell lysates were blotted for the indicated proteins. Quantitation of expression levels of acetylated Histone relative to total Histone is shown as the mean ± SD, n=3 biological replicates. White circles indicate Myc-Vector and black circles indicate Myc-CSAG2.

(K) Knockdown of endogenous CSAG2 in A375 cells increased ac-H4K16 levels. Cell lysates were blotted for the indicated proteins. Quantitation of expression levels of ac-H4K16 relative to total H4 is shown as the mean ± SD, n=3 biological replicates.

Data information: Data are represented as the mean ± SD (E-G). *p < 0.05, **p < 0.01, ***p < 0.001; unpaired, two-tailed Student's t-test.
Dysfunctional oxidative phosphorylation shunts branched-chain amino acid catabolism onto lipogenesis in skeletal muscle (2020)
Figure 1

Fig 1. A mouse model for the in vivo inhibition of Skm OXPHOS.

A. PCR analysis of the human H49K variant of the ATPIF1 and rtTA constructs in wild-type (wt), ACTA1-rtTA (T), ATPIF1H49K-TRE (H) or double transgenic (T/H) mice.

(B-D) (B, D) WB expression of the human (h) or human + mouse (m+h) ATPIF1 protein in Skm (B, D), brain, liver, and WAT (D) extracts. hATPIF1 is only expressed in Skm from ATPIF1H49K|T/H mice. HSP60 and α-tubulin are shown as loading controls. n= 3 mice/genotype. (C) Immunofluorescent staining of wt and ATPIF1H49K|T/H hindlimb muscle slices with hATPIF1 and βF1 antibodies. Images are representative of 3 mice/genotype, 5 fields/mouse. hATPIF1 H49K is only expressed in T/H mice.

E. Polarographic profiles of isolated mitochondria from wt (lower trace) and ATPIF1H49K|T/H (upper trace) animals. Histograms show a reduction in state 3 respiration consistent with the inhibition of ATP synthase in ATPIF1H49K|T/H mice. Bars are the mean ± s.e.m. of n= 3 mice/genotype, 3 traces/mouse; OL, oligomycin; Ant A, antimycin A.

(F, G) Quantitative proteomic analysis (iTRAQ) of hindlimb Skm from wt and LowOXPHOS mice. The pie charts (F) and volcano plot (G) present the upregulated (log2[LowOXPHOS/wt]≥1, red) or downregulated (log2[LowOXPHOS/wt]≤-1, blue) proteins. A (-)log p value of >4 was considered statistically significant.

H. Cytoscape representation of the most upregulated proteins in Skm from LowOXPHOS mice.

I. Cytoscape representation of GSEA analysis. Fourteen significantly altered pathways and their normalized enrichment score (NES) are shown. 1. Hallmarks of adipogenesis; 2. Go_Mitocondrial signal; 3. Kegg_TCA Cycle 4; Pyruvate Metabolism Reactome; 5. Hallmarks of OXPHOS; 6. Go_ETC; 7. Go_NADPH metabolism; 8. Kegg_Propionate metabolism; 9. Redox system; 10. Kegg_BCAA catabolism; 11. Go_Lipid metabolism; 12. Kegg_Lipid oxidation; 13. NEFA, Tg and ketone bodies; 14. Go_FAD binding proteins. See also Fig EV1.

J. Enrichment score (ES) graphs from GSEA analysis.

K. Skm iTRAQ ratio of proteins from BCAA catabolism. Higher intensities of red or blue colors represent higher or lower LowOXPHOS/wt expression ratios, respectively.

Data information: (F-K) Data are the mean ± s.e.m. of n= 12 animals/ genotype measured in 2 different iTRAQ analysis *p <0.05 when compared to wt by ANOVA and Student's t-test. See also Figures EV1 and EV2.
Dysfunctional oxidative phosphorylation shunts branched-chain amino acid catabolism onto lipogenesis in skeletal muscle (2020)
Figure 3

Fig 3. OXPHOS-mediated alterations in lipid and amino acid metabolism contribute to IR. Data on wild type (wt, black bars and traces) and LowOXPHOS (orange bars and traces) mice are shown.

(A-E) (A, C). Plasma (A) and Skm (C) BCAA levels in wt and LowOXPHOS mice (wt, n= 4; LowOXPHOS, n= 4). (B, D, E). Scheme of the BCAA catabolism pathway (B, left panel) and 14C(u)-leucine and 14C(u)-isoleucine uptake (B, right panels), oxidation (D) and incorporation into lipids (E) in myocytes expressing or not ATPIF1H49K. Bars are the mean ± s.e.m. of n= 3 experiments, 6 replicas/condition.

F. Longitudinal (left panels) and transversal (right panels) slices of soleus stained with hematoxylin/eosin. im-WAT infiltrations and fiber shrinkage (arrows) in HFD-fed LowOXPHOS mice (wt + HFD, n= 4; LowOXPHOS + HFD, n= 4; 10 fields/mouse).

(G-I). Motor function tests. Rotarod (G; wt, n= 7; LowOXPHOS, n= 6), Open Field (H; wt, n= 4; LowOXPHOS, n= 4) and gripforce (after fatigue, I; wt, n= 7; LowOXPHOS, n= 6) tests. In H, lines represent the movement of the mice in the cage. The histogram represents the relative time that wt and and LowOXPHOS mice expended in the corners.

(J, K) Quantitative Skm lipidomics at day 80 of HFD (J). The color scale (brown to blue) in the heat-map represents LowOXPHOS/wt amounts of detailed lipid species. A p-value ≤ 0.05 was considered statistically significant (red). In the lower heat-map (K), the color scale (yellow to blue) highlights an increase in DAG in HFD-fed LowOXPHOS mice. Each sample is a pool from 4 mouse extracts. Histograms show the total amounts of Skm DAG (wt, n= 8; LowOXPHOS, n= 8).

L. Blood triglycerides on the 80th day of HFD (wt + HFD, n= 10; LowOXPHOS + HFD, n= 10).

M. Skm relative expression of the GLUT1, GLUT3, PPARβ/δ and PGC1α by qPCR (wt, n= 5; LowOXPHOS, n= 5).

N. qPCR relative expression of myokines (wt, n= 5; LowOXPHOS, n= 5). Red or blue color represents higher or lower % expression, respectively, compared to that in wt. SPARC, osteonectin; OSTN, musclin; LIF, interleukin 6 family cytokine; FKN, fractalkine; FSTL1, follistatin-like protein 1; FNDC5, irisin; ADIPOQ, adiponectin.

O. Blood glucose following the administration of HFD (wt + HFD, n= 10; LowOXPHOS + HFD, n= 10). T2D onset occurred on day 80 in wt and day 60 in LowOXPHOS mice.

P. Insulin (ITT) and glucose (GTT) tolerance tests on day 60 of HFD (wt + HFD, n= 10; LowOXPHOS + HFD, n= 10). LowOXPHOS but not wt mice appeared diabetic.

Data information: Bars are the mean ± s.e.m. of the indicated (n) mice/genotype *p <0.05 when compared to wt by ANOVA or Student's t-test. See also Fig EV3, 4 and Table EV3.
Dysfunctional oxidative phosphorylation shunts branched-chain amino acid catabolism onto lipogenesis in skeletal muscle (2020)
Figure 4

Fig 4. The lipogenic switch alters the redox system and lipid-related OXPHOS components. Data on wild type (wt, black bars) and LowOXPHOS (orange bars) mice are shown.

A. v-WAT iTRAQ ratio of proteins from BCAA catabolism (wt, n= 4; LowOXPHOS, n= 4). Higher intensities of blue colors represent lower LowOXPHOS/wt expression ratios.

B. Odd chain FAs in Skm and WAT from wt (n= 8) or LowOXPHOS (n= 8) mice.

(C, D). Representative WB expression (C) and iTRAQ analysis (D) of v-WAT proteins from lipid metabolism. Each sample contains extracts from 3 mice. The histogram shows the quantifications (wt, n= 6; LowOXPHOS, n= 6).

E. v-WAT weight expressed as a percentage of the wt (wt, n= 12; LowOXPHOS, n= 12).

F. Quantitative lipidomics in v-WAT. The color scale (brown to blue) in the heat map represents LowOXPHOS/wt amounts of specific lipid species. A p-value ≤ 0.05 was considered statistically significant (red). Each sample is a pool from 4 mouse extracts, n= 8/genotype.

G. v-WAT amounts of triacylglycerides (TAGs) and diacylglycerides (DAGs) (wt, n= 8; LowOXPHOS, n= 8).

H. Higher saturated lipid species in LowOXPHOS mice compared to wt (wt, n= 8; LowOXPHOS, n= 8).

I. MitoSox staining in myocytes expressing or not ATPIF1H49K. The right histogram shows the quantification of mitochondrial ROS. Bars are the mean ± s.e.m. of n= 3 experiments, 12 replicas/condition.

J. Representative WB of Skm lipid peroxidation and redox system proteins. Two samples per condition are shown. Each sample contains extracts from 3 mice. Histograms represent quantification (wt, n= 6; LowOXPHOS, n= 6). 4-Hydroxynonenal (4HN), peroxiredoxin 2, 3 and 6 (PRX), superoxide dismutase 1 and 2 (SOD), catalase (CATA) and glutathione reductase (GSR) immunoblots are shown. βF1 is presented as a loading control.

K. Representative WB of Skm OXPHOS and related system proteins. Two samples per condition are shown. Each sample contains extracts from 3 mice. Histograms represent quantification (wt, n= 6; LowOXPHOS, n= 6). NDUFA9 (CI), SDHA and B (CII), ETF subunits A and B, ETFDH, CoreII (CIII), subunit 1 (CIV), βF1(CV) and hATPIF1 immunoblots are shown. Tubulin is presented as a loading control. See also Fig EV5.

L. Skm iTRAQ ratio of FADH2-binding proteins (wt, n= 12; LowOXPHOS, n= 12). A higher intensity of red color represents a higher LowOXPHOS/wt expression ratio.

M. Oxidized FAD levels and the reduced/oxidized FAD ratio in Skm extracts (wt, n= 8; LowOXPHOS n= 8).

Data information: Bars are the mean ± s.e.m. of the indicated (n) mice/genotype. *p <0.05 when compared to wt by Student's t-test. See also Fig EV4, 5 and Table EV3.
Dysfunctional oxidative phosphorylation shunts branched-chain amino acid catabolism onto lipogenesis in skeletal muscle (2020)
Figure 5

Fig 5. Hindering the ATP synthase activity feeds back to OXPHOS dysfunction by inhibiting CII and generating a SDHA-dependent burst in ROS.

A. Enzymatic activity of respiratory complex CII in Skm isolated mitochondria from wt and LowOXPHOS mice fed with chow or HFD (wt, n= 4; LowOXPHOS, n= 4; wt + HFD, n= 4; LowOXPHOS + HFD, n= 4).

B. Polarographic profiles of isolated mitochondria from wt (lower trace) and LowOXPHOS (upper trace) animals using succinate (left graph) or palmitoyl-carnitine (right graph) as a substrate. Quantification of maximal respiration in the right histogram. Bars are the mean ± s.e.m. of n= 3 mice/genotype, 3 traces/mouse; OL, oligomycin; Ant A, antimycin A.

(C-E) 2D-PAGE of Skm extracts (C, D) and acetylated proteins (E) from wt (n= 4) and LowOXPHOS (n= 3) mice. The isoelectric point (pI) of the CII subunits SDHB (C) and SDHA (D) was calculated by protein migration in pH 3-10 NL strips.

F. Immunocapture (IP) of SDHA blotted with anti-acetyl-K antibody (upper panel) and of acetylated proteins blotted with anti-SDHA antibody (middle panel) in Skm isolated mitochondria from wt (n= 3) and LowOXPHOS (n= 3) mice.

(G, H). PyMOL cartoon representations of acetylated lysines in SDHA related to CII activity. 6 acetylated-K are shown.

I. MitoSox staining in myocytes expressing or not ATPIF1H49K. The left scheme illustrates where each ETC inhibitor works. The right histogram shows the quantification of mitochondrial ROS. Bars are the mean ± s.e.m. of n= 3 experiments, 12 replicas/condition.

J. MitoSox staining in myocytes expressing or not ATPIF1H49K. The left scheme illustrates where each CII inhibitor works. The right histogram shows the quantification of mitochondrial ROS. Bars are the mean ± s.e.m. of n= 3 experiments, 12 replicas/condition.

K. Representative blue native immunoblots (BN) and clear native in-gel activity (CN) of Skm mitochondrial membrane proteins from wt (n= 3) and LowOXPHOS (n= 3) 2-month-old mice. The migration of the respiratory complexes/supercomplexes CI-V is indicated. Subassembly of CI (with no activity, suggesting degradation) was observed in LowOXPHOS mice. VDAC is shown as a loading control.

Data information: *, # p <0.05 when compared to wt or LowOXPHOS, respectively, by ANOVA and Student's t-test. See also Fig EV5.
Dysfunctional oxidative phosphorylation shunts branched-chain amino acid catabolism onto lipogenesis in skeletal muscle (2020)
Figure 6

Fig 6. Edaravone as a mitochondrial metabolism enhancer.

(A, B) The schematic illustrates the screening process of 702 drugs related to the mitochondrial respiratory capacity assessed by Seahorse XFe96 in C2C12 myocytes. Palmitate is used as a substrate. A total of 41 hits were identified. Maximum respiration (Max R, % of untreated cells) of compounds categorized by their therapeutic use (B).

(C, D) The schematic illustrates the screening process of the 41 hits on FFA β-oxidation using 9,103H(N) palmitic acid as a substrate in C2C12 myocytes. Ten enhancers were identified. D. Correlation between the maximum respiration (x) and palmitate β-oxidation (y). Colors identify classes of pharmaceuticals as in B. Edaravone was selected as a hit.

E. Representative respiratory profile of C2C12 myocytes treated (green trace) or not treated (black trace) with 2 µM edaravone. The CPT1 inhibitor etomoxir was used as a negative control. OCR, oxygen consumption rate; OL, oligomycin; DNP, 2,4-dinitrophenol; Rot, rotenone; Ant A, antimycin A. Quantification in right histogram. Bars are the mean ± s.e.m. of 12 replicas/condition.

(F, G) FFA β-oxidation in C2C12 myocytes transfected with CRL or ATPIF1H49K plasmids (F) and in primary myotubes from wt and LowOXPHOS mice (G), treated (green bars) or not with 2 µM edaravone. Bars are the mean ± s.e.m. of n= 3 experiments, 9 replicas/condition.

Data information: (E-G) *p <0.05 when compared to wt by ANOVA and Student's t-test.