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. 2015 Jul 28;25(9):1025–1042. doi: 10.1038/cr.2015.90

Figure 6.

Figure 6

RHOBTB3 is a negative regulator of the Warburg effect. (A) Knockout of RHOBTB3 elevates the expression of HK2, LDHA GLUT1 and PDK1. RHOBTB3−/− MEFs and WT MEFs were maintained in normoxia or exposed to hypoxia for 16 h. Cells were then lysed and the protein extracts were analyzed by immunoblotting with antibodies indicated. (B) RHOBTB3 deficiency leads to increased mRNA levels of GLUT1 and LDHA. Total RNAs from RHOBTB3−/− MEFs and WT MEFs, maintained in normoxia or exposed to hypoxia for 16 h, were purified, and analyzed by real-time PCR analysis for the expression levels of GLUT1 and LDHA. Values are presented as mean ± SEM, n = 3 for each group, three replicate experiments. ***P< 0.001 (ANOVA followed by Tukey). (C, D) RHOBTB3 decreases rates of glucose consumption (C) and lactate production (D). RHOBTB3−/− and WT MEFs were maintained in normoxia or exposed to hypoxia for 8 h, and glucose consumption rates (C) and lactate production rates (D) were measured. Values are presented as mean ± SEM, n = 3 for each group, ***P< 0.001 (ANOVA followed by Tukey). (E, F) RHOBTB3 and LIMD1 cooperatively decrease glucose consumption (E) and lactate production (F). HEK293T cells were infected with lentiviruses expressing siRNAs targeting GFP, RHOBTB3 and/or LIMD1. At 16 h post-infection, cells were maintained in normoxia or exposed to hypoxia for 8 h and glucose consumption rates (E) and lactate production rates (F) were measured. Values are presented as mean ± SEM, n = 3 for each group, *P< 0.05, **P< 0.01, ***P< 0.001 (ANOVA followed by Tukey).