Fig 2: Effect of the upregulated level of p-AMPK caused by shWTAP on HCC autophagy. (A) Western blot analysis of some autophagy-related proteins expressed in BEL-7404 and SMMC-7721 cells. (B) Western blot analysis of LC3-I and LC3-II in shWTAP BEL-7404 cells treated with or without Compound C. (C) Western blot analysis of LC3-I and LC3-II in shWTAP BEL-7721 cells treated with or without Compound C. (D) Statistical analysis of LC3 II/LC3 I ratio between shCON and shWTAP–treated 7404 cell lines. (E) Statistical analysis of LC3 II/LC3 I ratio between shCON and shWTAP-treated 7721 cell lines. (F) Confocal fluorescence image of shWTAP cells treated with or without Compound C. (G) Statistical analysis of the numbers of LC3 puncta/cell. The cells indicated with red arrows were enlarged on the right. Error bars indicate the mean ± SD from three independent experiments (n = 3). * indicate a statistically significant difference (p < 0.05), ** indicate a statistically significant difference (p < 0.01) and *** indicate a statistically significant difference (p < 0.001).

Fig 3: Fibre type dependence of glycogen-related proteins in control and T2D individuals. Amount of glycogen phosphorylase (GP), glycogen debranching enzyme (GDE), glycogen synthase (GS), glycogen branching enzyme (GBE) and AMPK-ß2 in Type I and Type II fibres isolated from control (CON) and T2D individuals (before (pre) and after (post) hyperinsulinaemic, euglycaemic clamp). A Western blot showing detection of GP, MHC I, MHCII protein in single fibres segments isolated from CON and T2D pre and post clamp and a calibration curve of mixed human muscle homogenates loaded at 4–40 µg wet weight muscle. B Mean data expressed relative to the average GP amount in Type II fibres from CON on the given gel (mean + S.D). C, D As for A and B, but showing GDE. E and F. As for A and B, but showing GS. G, H As for A and B, but showing GBE. I, J As for A and B, but showing AMPK-ß2. Note this Western blot was also probed with GBE as seen in panel G. Two-way within subject ANOVA. #indicates significant difference (P < 0.05) compared to Type I fibres from the same group. * indicates significant difference (P < 0.05) compared to the same type of control fibres. $ indicates significant difference (P < 0.05) compared to the pre fibres. A–D, N = 11/12–2 individuals/fibres; E–H, N = 4–11/12–2 individuals/fibres; I–J, N = 7–11/12–2 individuals/fibres. ? indicates fibre type not ascertained and through a process of elimination, likely a MHC IIx fibre. Contiguous lanes on a given western blot are shown, with vertical lines included to separate the samples from the different groups as indicated

Fig 4: Proposed model of 2-AA’s action in skeletal muscle. 2-AA triggers ROS generation via activating XO activity and NOX2 expression and by impairing the activity of antioxidant enzymes as well as by downregulation of SIRT1 protein. Reduction of PGC-1ß protein level impairs ROS detoxification, whereas upregulation of UCP3 gene (25) and protein levels impacts the mitochondrial functions (26), resulting in energy depletion. Energetic exhaustion and oxidative stress increases lead to muscle atrophy by activating AMPK, which controls autophagy and proteasomal degradation as assessed by the activation of autophagy markers ATG5, LC3B, and SQSTM1 and autophagy markers MuRF1 and MAFbx, respectively. NAC can ameliorate 2-AA-induced oxidative stress and thus rescue skeletal muscle protein degradation by scavenging the ROS.

Fig 5: The functional effects of NBR2 are partially mediated by AMPK(A and B) UMRC2 cells stably expressing EV or NBR2 expression vectors were transfected with AMPK siRNA (AMPK si1 or si2) or control siRNA (Ctrl si). Protein lysates were prepared and analyzed by Western blotting (a), or cells were cultured in 25 mM glucose-containing medium for different days as indicated, and then subjected to cell proliferation analysis (b) (Mean ± s.d., n=3 biologically independent extracts, two-tailed paired Student’s t-test). (c–g) MDA-MB-231 cells with stable expression of control shRNA (Ctrl sh) or NBR2 shRNA (NBR2 sh) were infected with empty vector (EV) or constitutively active AMPK (AMPK CA). These cells were cultured in 25 or 0 mM glucose-containing medium for 24 hours, and protein lysates were prepared and analyzed by Western blotting (c); The cells were cultured in 0 mM glucose-containing medium for different days as indicated, and then subjected to crystal violet staining to measure cell number (d) (Mean ± s.d., n=3 biologically independent extracts, two-tailed paired Student’s t-test); The cells were cultured in 25 or 0 mM glucose-containing medium for 24 hours, then subjected to Annexin V/PI staining followed by FACS analysis to measure the percentages of Annexin V positive/PI negative cells cells (e) (Mean ± s.d., n=5 fields per group, each field was assessed from an independent experiment, two-tailed paired Student’s t-test), or to Western blotting analysis to measure PARP cleavage (f); The cells were seeded in soft agar containing high or low concentrations of glucose as indicated. Bar graph showing the mean colony numbers from the soft agar assay (g) (Mean ± s.d., n=5 fields per group, each field was assessed from an independent experiment, two-tailed paired Student’s t-test). (h) Relative tumor volumes of MDA-MB-231 xenograft tumors of different genotypes at different weeks (Mean ± s.d., n = 5 xenograft tumors, *: P < 0.05, **: P < 0.01, two-tailed paired Student’s t-test). Source data for b, d, e can be found in Supplementary Table 1. Unprocessed original scans of blots are shown in Supplemental Fig. 8.