Fig. 4
TXNIP regulation by 1,25(OH)2D3 is independent of metabolic reprogramming. a Schematic overview of treatment-induced metabolic changes that may reduce levels of glycolytic intermediates capable of inducing the nuclear translocation of MondoA/MLX and hence TXNIP expression. Genetic and/or pharmacological strategies for inhibiting key metabolic players are depicted. b Time-dependent increase in G6PD mRNA expression in MCF-7 cells with 1,25(OH)2D3 (100 nM) is associated with an attenuation of the initial increase in TXNIP mRNA levels with treatment. Upper and lower ends of floating bars represent maximum and minimum values, respectively, whereas lines in the middle represent the means. The ∆∆Ct method was used to calculate the relative expression with vinculin as the housekeeping gene. The data are obtained from two biological replicates. c Similar to the trend observed on the mRNA level, the induction in G6PD protein level in response to 1,25(OH)2D3 first occurs after 12 h, which coincides with the first reduction in TXNIP protein expression. d Co-treatment with DHEA, but not knocking-down G6PD levels, elevates TXNIP mRNA levels in 1,25(OH)2D3-treated MCF-7 cells. Cells transfected with either anti-G6PD siRNA or a NC were treated with 1,25(OH)2D3 for 72 h, whereas increasing concentrations of DHEA were added to the conditioned medium of MCF-7 cells treated for 48 h with either DMSO or 1,25(OH)2D3, for an additional 24 h. e G6PD inhibition using siRNA or DHEA reduces TXNIP protein expression independent of 1,25(OH)2D3. f–h Western blot analysis of TXNIP expression in MCF-7 cells treated with a combination of either DMSO or 1,25(OH)2D3 and various metabolic inhibitors. Cells were initially treated with DMSO or 1,25(OH)2D3 for 48 h, after which increasing concentrations of the different inhibitors were added to the conditioned medium for an additional 24 h. All inhibitors were found to reduce TXNIP protein levels in 1,25(OH)2D3-treated cells. In the absence of 1,25(OH)2D3, AZD 3965 and CBR-5884 were found to mildly induce TXNIP levels