Figure 2.

Time-dependent regulation of TXNIP expression and associated cellular processes in HL-60 cells by 1,25(OH)₂D₃. (A) 1,25(OH)₂D₃ (100 nM) induced a transient increase in TXNIP mRNA levels that was diminished at later time points. The dashed red line indicates the baseline TXNIP mRNA expression in DMSO-treated cells set to 1; (B,C) Immunoblot showing TXNIP protein levels in response to 1,25(OH)₂D₃; induction is observed after 24 h and also after 96 h of treatment. Basal TXNIP levels exhibit temporal fluctuations, independent of treatment. Treatment with 2-deoxyglucose (10 mM), a potent inducer of TXNIP expression, and MG-132 (5 µM), a proteasomal inhibitor to prevent degradation, were used as positive controls. The compounds were added into the conditioned medium of DMSO- and 1,25(OH)₂D₃-treated HL-60 cells either 24 h (2-deoxyglucose) or 6 h (MG-132) before the end of the initial treatment period. “+” and “−” denote the presence or absence of the indicated molecule/treatment, respectively; (D) Immunoblot and (E) densitometric quantification of TXNIP protein levels after 24 h of treatment of HL-60 cells with 1,25(OH)₂D₃ indicate a mild increase in TXNIP level, which lacks statistical significance; (F) Immunoblot and (G) densitometric quantification after 96 h of treatment of HL-60 cells with 1,25(OH)₂D₃ showing a statistically significant induction of TXNIP levels; (H) Intracellular ROS levels, presented as fold change of treated vs. control cells, were found to be significantly induced by 1,25(OH)₂D₃ only at the latest time point (96 h). The dashed red line indicates the baseline ROS level in DMSO-treated cells set to 1; (I) Glucose uptake, which is known to be inhibited by TXNIP, was found to be significantly induced by 1,25(OH)₂D₃ at an early time point (6 h), and reduced at a later one (72 h). Baseline glucose uptake level in DMSO-treated cells is indicated by the dashed red line set to 1. Statistical significance was calculated using a two-tailed Student’s t-test. p-Values less than or equal to 0.05 and 0.01, are depicted by * and **, respectively. Error bars ± SD; n = 3.