Fig 2: The effect of Pkm2 depletion on differentiation, mitochondria OXPHOS and glycolysis.a Representative western blotting results of PKM2 and PKM1 in control (shCtrl) and Pkm2-knockdown (shPkm2) podocytes (n = 3). b Bar charts show means of optical density (O.D.), and normalized to cells transfected with shCtr-RNAi lentivirus. c Real-time PCR analysis of Pkm2 and Pkm1 mRNA levels in the absence or presence of Pkm2-RNAi lentivirus (n = 3–6). d PK activity in different podocytes as indicated (n = 3). e ATP level was normalized by protein content, and used shCtrl podocyte as control. f Representative western blotting results of nephrin and synaptopodin expression level in cultured podocytes as indicates (n = 3). g Representative western blotting results of PGC-1a and TFAM expression level (n = 3). h Representative blot images of OPA-1 and DRP-1 (n = 3). i Representative blot images of F6PK and LDHA (n = 3). j Immunofluorescence staining for nephrin (red), phalloidine for F-actin (green) and DAPI for nuclear (blue). Scale bar=5 µm. k Representative immunofluorescence and electron microscopy (EM) images showing alterations in mitochondrial morphologies between different podocytes as indicated. In the immunofluorescence images, cells are labeled with MitoTracker Red (red) for mitochondria and DAPI (blue) for nuclear. Left scale bar = 2 µm. Right scale bar = 500 nm. Pictures show representative fields of over 10 cells photographed. l–n Average mitochondrial area, mitochondrial mass and MMP. o, p Relative lactate production and glucose consumption in culture medium derived from podocytes, and shCtrl podocyte was used as control (n = 3). q Effects of PKM2 on OCR in podocytes (n = 4). OCR traces were obtained using a Seahorse XF96 Analyzer. r Statistical analyses of baseline respiratory capacity, ATP-coupled respiratory capacity, maximum respiratory capacity and reserve respiratory capacity in OCR. s Effects of PKM2 on ECAR in podocytes (n = 4). t Statistical analyses of glycolysis, glycolytic capacity and glycolytic reserve in ECAR.

Fig 3: Expression of LDHA, PKM2 and HK2 in ovarian tissues. (A) Relative Ldha mRNA level of ovaries (n = 4 rats per group). (B) Relative Pkm2 mRNA level of ovaries (n = 4 rats per group). (C) Relative Hk2 mRNA level of ovaries (n = 4 rats per group). (D) Western blotting assays of LDHA, PKM2 and HK2 in ovarian tissues, with ß-tubulin served as a loading control (n = 4 rats per group). (E) Relative LDHA protein level of ovaries (n = 4 rats per group). (F) Relative PKM2 protein level of ovaries (n = 4 rats per group). (G) Relative HK2 protein level of ovaries (n = 4 rats per group). (H) Immunohistochemistry assays of LDHA, PKM2 and HK2 in ovarian tissues (n = 4 rats per group), the lowest panel shows the negative control. Data were presented as mean ± SEM. Significant differences were respectively presented as *P < 0.05 and **P < 0.01.

Fig 4: GFHPD application induces a metabolic shift from glycolysis towards amino acid metabolism and reduces colonic energy supply in AOM/DSS-treated mice. (a–f) Serum samples collected from indicated treatment groups at day 126 were analyzed with regard to (a) L-lactate, (b) D-glucose, (c) insulin, (d) C-peptide, (e) branched-chain amino acids (BCAA), or (f) L-alanine. ctrl. Ab (n = 10 CD; n = 7–10 GFHPD), anti-EGFR Ab (n = 8–10 CD; n = 9–11 GFHPD). (g) Representative images of HE-stained kidney tissue collected at day 126 from indicated treatment groups of AOM/DSS tumor mice. Original magnification, 2.5×. (h–n) Protein expression of (h) LDHA/C, (i) p32, (j) TOM20, (k,l) OXPHOS complexes I–V, (m) phosphorylated AMPKα, (n) phosphorylated AKT, or α-Tubulin was determined by Western blot experiments utilizing biopsy samples collected from colorectal tumors (n = 6 per group) of indicated treatment groups at day 126. Densitometric analysis was performed using ImageJ software and data were normalized to α-Tubulin values. (o) Schematic overview of systemic amino acid metabolism. * p ≤ 0.05, ** p ≤ 0.01. *** p ≤ 0.001. AU = arbitrary units.

Fig 5: HSF1 and c-Myc mRNA levels positively correlate in human HCC specimensA. Relationship between HSF1 and c-Myc expression levels; data were extracted from previous publications of our group and subjected to linear regression analysis [12, 15]. B., C. A statistically significant, positive correlation was also found between mRNA levels of HSF1 and those of two canonical c-Myc targets, namely ODC and LDHA. D. Example of c-Myc amplification patterns in HCC specimens as detected by fluorescence in situ hybridization (FISH). Upper panel: disomy (normal pattern); lower panel: c-Myc amplification.