Effect of ectopic TXNIP overexpression in TXNIP-KD HEK293t cells. (A) TXNIP-KD HEK293t cells were transfected with 1 or 5 μg of a TXNIP-GFP expression vector (or empty plasmid) for 24 or 48 h. Cells were then harvested and levels of E2F1, BCL2, SOD2, P21 and tubulin were measured by Western blots. (B) Scanning densitometry analysis of TXNIP, SOD2 and E2F1 levels, as shown in panel A. (C) Western blot analysis of IGF1R/PI3K/AKT signaling mediators in TXNIP-GFP transfected cells. (D) RT-QPCR analysis of TXNIP mRNA levels in TXNIP-GFP-transfected cells. Values are normalized to the GAPDH housekeeping gene. Bars represent mean ± SEM of three independent experiments. A value of 1 was given to the mRNA levels in untreated cells. * p-value ≤ 0.05; ** p-value ≤ 0.01.