Fig. 5: Modulation of HK2 contributes to the role of MCL1 in promoting bioenergetics and tumorigenesis.

Mitochondria- (A) and glycolysis- derived (B) ATP production rate measured by Seahorse XF Real-Time ATP Rate Assay of CHL-1 cells overexpressing glycolysis regulators HK2, PKM2, ALDOA or vector and transduced with either scrambled shRNA or shRNA against MCL1 for 72 h. (n = 3 biologically independent samples). Data is presented as mean +/- SD and significance is determined by paired two-tailed t-test. C Immunoblotting analysis of lysates derived from CHL-1 cells overexpressing HK2 or vector and transduced with either scrambled shRNA or shRNA against MCL1 for 72 h. The samples derived from the same experiment but different gels for HK2, pS6K1, MCL1, Tubulin, another for S6K1, pS6 and another for S6 were processed in parallel. D Percentage of cell viability of CHL-1 cells transduced as in (A) after 96 h in culture. (n = 3 biologically independent samples). Growth rate (E), weight (F) and images (G) of subcutaneous xenografts established in NSG mice from CHL-1 cells overexpressing HK2 or vector and transduced with either scrambled shRNA or doxycycline-inducible shRNA against MCL1. After establishment of xenografts, mice were kept on 1 mg/ml doxycycline supplemented in the drinking water to induce MCL1 shRNA. (n = 5 mice per group). Data is presented as mean +/- SEM and significance is determined by unpaired two-tailed t-test. Correlation between the mRNA levels of HK2 and MCL1 (H), BCL-2 (I) and BCL2L1 (J) in The Cancer Genome Atlas (TCGA TCGA-SKCM (n = 471 patients) analyzed using TIMER2.0 (http://timer.cistrome.org/). Spearman’s rho value was used to evaluate the degree of their correlation.