Fig 2: AMPK and AKT regulate Api5 stability. MCF7 cells were treated with (a) 20 μM AMPKi, and (c) 5 μM AKTi for 4 h and 24 h, respectively. Immunoblotting analysis was performed using Api5 specific antibody to check for Api5 expression levels. GAPDH was used as loading control. (b,d) Quantification showing the fold change in Api5 levels after normalisation to GAPDH. (a) p21 and (c) pAkt S473 were used as positive control to confirm AMPK and Akt inhibition, respectively. (e) pAkt S473 activation upon AMPK inhibition. pAkt, Akt and GAPDH blots were cropped and the full blot is provided in Supplementary figure S12. (f) Quantification showing the fold change in pAkt S473 activation after normalisation to GAPDH. (e) E2F1 levels upon AMPK inhibition and (g) Quantification of E2F1 levels after normalising to GAPDH. (h,i) E2F1 protein levels were analysed following Akt inhibition using immunoblotting.

Fig 3: CD147 regulates IGFBP2 expression via the PTEN/PI3K/AKT signaling pathway. (A) Cell lysates of sh-CD147 and sh-Mock A375 and SK-MEL-28 cells were subjected to western blotting to detect p-AKT, p-mTOR and PTEN expression. Quantitation of (B) p-AKT, (C) p-mTOR and (D) PTEN expression was performed using ImageJ software. (E) Sh-Mock A375 and SK-MEL-28 cells were cultured with 50 μg/ml LY294002, a PI3K signaling pathway inhibitor, for 24 h. IGFBP2 and CD147 expression was detected by western blotting and (F) quantitative analysis of IGFBP2 expression was performed using ImageJ software. n=3 for each experiment. *P<0.05, **P<0.01 vs. sh-Mock or 0 μg/ml LY294002. AKT, protein kinase B; CD147, cluster of differentiation 147; m-TOR; p, phosphorylated; PI3K, phosphoinositide 3-kinase; PTEN, phosphatase and tensin homolog; sh, short hairpin RNA.

Fig 4: Exercise increases (A) FGF21 protein, (B) FGF21 mRNA, and (C) p‐Akt/Akt protein of skeletal muscle in mice (CON, n = 8; RUN, n = 10). Values are means ± SD. *P < 0.05 versus CON.

Fig 5: Role of SENP2 for basal and insulin-stimulated glycogen synthesis and Akt phosphorylation in human myotubes. (A) Myotubes were starved for 1.5 ​h before incubated for 3 ​h with 5.5 ​mM D-[14C(U)]glucose (37 kBq/ml) with no insulin (basal), 20 ​nM or 100 ​nM insulin. Results are shown as mean ​± ​SEM in absolute values, nmol/mg protein, from four individual experiments (n ​= ​4). (B-D) Akt phosphorylation and total Akt protein expression by immunoblotting. Myotubes were incubated for 15 ​min with 100 ​nM insulin before protein was isolated and the amount of total Akt and pAkt were assessed by immunoblotting. B, a representative immunoblot. C and D, quantified immunoblots relative to SCR. Results are shown as mean ​± ​SEM from six individual experiments (n ​= ​6). (E) mRNA expression of selected genes. RNA was harvested and expression of glucose transporter (GLUT) 1, pyruvate dehydrogenase kinase (PDK) 4 and insulin receptor substrate (IRS) 1 were analyzed by qPCR. Results are shown as mean ​± ​SEM relative to the housekeeping gene ribosomal protein lateral stalk subunit P0 (RPLP0) and normalized to SCR cells, from six individual experiments (n ​= ​6). ∗Statistically significant versus SCR cells (p ​≤ ​0.05, paired t-test). #Statistically significant versus to SCR basal (p ​≤ ​0.05, paired t-test). KD, cells with SENP2 knockdown; SCR, control cells i.e., with scrambled vector.