Fig 2: miR-135 knockdown leads to increased glutamine dependence. a Schematic representation of 13C- glucose metabolism. b Heatmap of significantly changed labeled metabolites in glycolysis, amino acids and tricarboxylic acid cycle in control and miR-135 knockdown MIA PaCa-2 cells analyzed by LC-MS. c Labeled pyruvate, lactate, serine and glycine were analyzed in control (anti-NC), miR-135 knockdown (anti-miR-135) and miR-135/PFK1 double knockdown MIA PaCa-2. Fold change is calculated by the labeled metabolites normalized to control MIA PaCa-2 cells. Each value represents mean ± SD in three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001, Student’s t test. d Labeled and unlabeled a-ketoglutarate, succinate, fumarate, and glutamate were analyzed in control (anti-NC) and miR-135 knockdown (anti-miR-135) MIA PaCa-2 cells by using LC-MS. Fold change is calculated by the labeled or unlabeled metabolites divided by pool and normalized to control MIA PaCa-2 cells. Metabolite data are shown as mean ± SD in three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001, one-tailed Student’s t test. e Control (anti-NC), miR-135 knockdown (anti-miR-135) and miR-135/PFK1 double knockdown MIA PaCa-2 cells were cultured in complete medium for 24 h. Glutamine uptake was measured using the Nova Bioprofile 100 analyzer. f Control (anti-NC), miR-135 knockdown (anti-miR-135), shPFK1 and miR-135/PFK1 double knockdown MIA PaCa-2 cells were cultured in glutamine-free medium for indicated time points. Cell viability was assessed by Trypan blue exclusion. Each value represents mean ± SD in three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001, Student’s t test. Cells were lysed and immunoblotting was performed with indicated antibodies

Fig 3: DOX treatment inhibited PFKM expression in H9c2 cells. (A–C) PFKM expression in DOX-treated H9c2 cells. (D–F) PFKM expression in DOX-treated H9c2 cells for different time points. *P < 0.05, ***P < 0.001 vs 0 μM or 0 h. (see Supplementary Fig. S1–S4)

Fig 4: HDAC1-induced PFKM transcriptional repression regulated DOX-mediated OXPHOS and glycolysis in H9c2 cells. H9c2 cells were transfected with PFKM siRNA or nonspecific siRNA (siNC) and treated with 2 μM DOX in the absence or presence of 0.1 μM MGCD for 24 h, and (A) cell viability, (B) OCR and (C) ECAR, (D) ATP and (E) lactate level were measured. (F) Schematic representation of the regulation of DOX-induced cardiotoxicity via OXPHOS and glycolysis through HDAC1-induced PFKM transcriptional repression. ***P < 0.001 vs Control; ###P < 0.001 vs DOX + siNC + vehicle; ΔΔP < 0.01 vs DOX + siPFKM-1 + vehicle.

Fig 5: TRAP1 regulates Warburg metabolism through modulation of PFK1 activity/expression. (A) Representative fluorescence images showing proximity ligation assay signals (red), detected in HCT116 cells stained with TRAP1 and PFK1 (left panel). The right panel represents the negative control stained with not related antibodies. Nuclei are DAPI‐labeled (blue). Scale bar, 10 µm. (B, C) HKII, PFK1, and TRAP1 immunoblot analysis (B) and relative PFK1 and HKII activity (C) in transient TRAP1‐silenced HCT116 cells. (D, E) PFK1 and TRAP1 immunoblot analysis (D), and relative PFK1 activity and lactate production (E) in transient TRAP1‐silenced SA54 CRC spheres. (F, G) PFK1 and TRAP1 immunoblot analysis (F), and relative lactate production and PFK1 activity (G) in PKF1‐silenced HCT116 cells transfected with TRAP1 cDNA. (H, I) PFK1 and TRAP1 immunoblot analysis (H) and relative lactate production and PFK1 activity (I) in shTRAP1 HCT116 cells transfected with PFK1 cDNA. Graphs represent mean ± SD of three experiments. The Mann–Whitney test was used to establish the statistical significance between two groups (P < 0.05). (G) ANOVA test: P = 0.0001; Bonferroni post hoc test: siNEG pmock vs siPFK1, lactate production: P < 0.01, PFK1 activity: P < 0.01, siNEG pmock vs siNEG pTRAP1, lactate production: P < 0.001, PFK1 activity P < 0.001; siNEG pTRAP1 vs siPFK1 pTRAP1, lactate production P < 0.05, PFK1 activity: P < 0.001. (I) ANOVA test: P = 0.004; Bonferroni post hoc test: scramble pmock vs shTRAP1 pmock, lactate production: P < 0.01, PFK1 activity: P < 0.01; scramble pPFK1 vs shTRAP1 pPFK1 lactate production: P < 0.05, PFK1 activity: P < 0.01.