Fig 2: Kinase activity of ULK1/2 is instrumental for hypoxic glycolysis mediated by PKM2 independent of BNIP3. (A) Western-blotting comparing the levels of ULK1 and ULK2 expression in SW-1990 cells transfected with ULK1, ULK2 and ULK1/2 shRNA in the presence or absence of Flag-tagged wild-type ULK1/2 expression under hypoxia, respectively. GAPDH was used as internal control of cytoplasmic lysates. Scr, scrambled shRNA; WB, western-blotting. (B and C) Glucose consumption (B) or lactate production (C) of SW-1990 cells transfected with ULK1, ULK2 and ULK1/2 shRNA in the presence or absence of Flag-tagged wild-type ULK1/2 expression under hypoxia. Data are expressed as mean ± s.d. of three independent experiments. *P<0.05 and **P<0.01. Two-sided ANOVA with Bonferroni post hoc t test correction was used to calculate the P value. (D and E) Glucose consumption (D) or lactate production (E) of SW-1990 cells treated with SBI-0206965 (SBI) at the indicated concentrations under hypoxia. Data are expressed as mean ± s.d. of three independent experiments. *P<0.05 and **P<0.01 versus D. D, DMSO. Two-sided ANOVA with Bonferroni post hoc t test correction was used to calculate the P value. (F) Western-blotting detecting the amount of ULK1, ULK2 and BNIP3 expression in SW-1990 cells transfected with BNIP3 shRNA (sh.BNIP3) in the presence or absence of ULK1/2 shRNA expression or ULK1/2 shRNA plus Flag-tagged wild-type ULK1/2 coexpression under hypoxia. (G and H) Glucose consumption (G) or lactate production (H) of SW-1990 cells transfected with BNIP3 shRNA (sh.BNIP3) in the presence or absence of ULK1/2 shRNA expression or ULK1/2 shRNA plus Flag-tagged wild-type ULK1/2 coexpression under hypoxia. Data are expressed as mean ± s.d. of three independent experiments. *P<0.05 and **P<0.01. Two-sided ANOVA with Bonferroni post hoc t test correction was used to calculate the P value. (I) Western-blotting assessing the amount of BNIP3 expression in SW-1990 cells with BNIP3 shRNA (sh.BNIP3) transfection. (J and K) Glucose consumption (J) or lactate production (K) of SW-1990 cells transfected with BNIP3 shRNA (sh.BNIP3) in the presence or absence of 50 μmol/L SBI-0206965 (SBI) treatment under hypoxia. Data are expressed as mean ± s.d. of three independent experiments. *P<0.05 and **P<0.01. Two-sided Student's t test was used to calculate the P value. (L) Western-blotting detecting the amount of ULK1, ULK2 and PKM2 expression in SW-1990 cells transfected with PKM2 shRNA (sh.PKM2) in the presence or absence of ULK1/2 shRNA expression or ULK1/2 shRNA plus Flag-tagged wild-type ULK1/2 coexpression under hypoxia. (M and N) Glucose consumption (M) or lactate production (N) of SW-1990 cells transfected with PKM2 shRNA (sh.PKM2) in the presence or absence of ULK1/2 shRNA expression or ULK1/2 shRNA plus Flag-tagged wild-type ULK1/2 coexpression under hypoxia. Data are expressed as mean ± s.d. of three independent experiments. *P<0.05 and **P<0.01. Two-sided ANOVA with Bonferroni post hoc t test correction was used to calculate the P value. N.S., no significant. (O) Western-blotting examining the amount of PKM2 expression in SW-1990 cells with PKM2 shRNA (sh.PKM2) transfection. (P and Q) Glucose consumption (P) or lactate production (Q) of SW-1990 cells transfected with PKM2 shRNA (sh.PKM2) in the presence or absence of 50 μmol/L SBI-0206965 (SBI) treatment under hypoxia. Data are expressed as mean ± s.d. of three independent experiments. *P<0.05. Two-sided Student's t test was used to calculate the P value.

Fig 3: Effect of PKM2 in the maintenance of the Warburg effect in HNC cells.(a-b) Percentage of decreased lactate production and glucose uptake in curcumin-PEC treated versus PEC control H157 cells after 48 h of treatment. Decreased lactate production and glucose uptake were rescued after PKM2 over-expression (PKM2-OE) in curcumin-PEC treated cells. (c) Measurement of apoptotic death by caspase 3/7 activity in curcumin-PEC treated versus control-PEC H157 cells after 48 h. The apoptotic death was partially rescued after PKM2 over-expression (PKM2-OE) in curcumin-PEC treated cells. (d) Reduced cell proliferation in curcumin-PEC treated H157 cell rescued partially by PKM2 overexpression (PKM2-OE) in curcumin-PEC treated H157 cells. (e) Representative image of invasion assay in curcumin-PEC treated versus control-PEC H157 cells after 24 h. Over-expression of PKM2 (PKM2-OE) partially rescues the invasion property of curcumin-PEC treated cells and the quantitative evaluation of invasive property of H157 cells. Three independent experiments were conducted with mean values ± SD. P-value calculated using two-tailed Student’s t-test, * P < 0.05, ** P < 0.01,*** P < 0.001, ns = non-significant

Fig 4: OA suppression of aerobic glycolysis contributes to its anti-tumor activity.(A) PC-3 cells was counted 12, 24, 36 and 48 hr after the treatment of OA (100 µg/ml) or/and MHY1485 (2 µM) using hemacytometry. The average values of three independent experiments were shown as Mean ± SD. **, P<0.01. The representative figure was shown for each group. (B) Cells were treated with OA (100 µg/ml) or/and MHY1485 (2 µM) for 10 days, the number of colonies of PC-3 cells were counted and analyzed using ImageJ software. The representative figure was shown for each group. (C) OA suppresses the activation of mTOR in cancer cells. This inhibitory effect on mTOR signaling, in turn, abrogated c-Myc/hnRNPA1/hnRNPA2-dependent PKM2 expression. Consequently, the aerobic glycolysis was inhibited in cancer cells treated with OA.

Fig 5: SMAR1-mediated PKM alternative splicing is HDAC6 dependent. a Expression of PKM isoforms upon shRNA-mediated knockdown of HDAC6 in MCF7 by western blot. b Expression of PKM isoforms upon SMAR1 overexpression followed by Tubacin (5 µM) treatment in MCF7 by western blot. c Confocal microscopy to check the expression of eGFP and mCherry in MCF7 cells transfected with PKM minigene along with Flag-SMAR1. Relative fluorescence intensity of eGFP and mCherry was quantified with the help of ImageJ and fold change in eGFP/mCherry ratio was calculated for control, control + Tubacin (5 µM), Flag-SMAR1 and Flag-SMAR1 + Tubacin (5 µM). Fold change in eGFP/mCherry ratio for SMAR1 overexpression and Tubacin treatment validates the role of HDAC6 in SMAR1-mediated regulation of PKM alternative splicing (n = 3). Error bars show mean values ± SD. Differences were considered statistically significant with *p < 0.05, **p < 0.01, and ***p < 0.001; ns non-significant difference (p > 0.05)