The effect of HPB242 on specificity protein 1 (Sp1) protein expression in HN22 and HSC4 cells. HN22 and HSC4 cells were incubated with different concentrations of HPB242 for 48 hours. The cells were harvested and prepared for western blots as described under Methods. Protein expression levels of Sp1 were detected using a specific antibody against Sp1, and its levels were quantified after actin normalization. (A) The HPB242-treated cells were compared with untreated cells, and data are shown as the means ± SD of three independent experiments. The asterisk indicates a significant difference compared with the negative control (untreated cells) (*p < 0.05). (B) Time-dependent effects of HPB242 on Sp1 and Cleaved-caspase3 expression were performed in HN22 and HSC4 cells for 48 hours with 6 hours intervals. (C) The effect of HPB242 (0–20 μg/ml) for 48 hours on Sp1 mRNA expression was determined by RT-PCR. The graphs indicate the ratio of Sp1 to β-actin expression. (D) The effect of HPB242 on Sp1 protein turnover in HN22 and HSC4 cells. The protein lysates were obtained from cells pretreated with protein synthesis inhibitor such as cycloheximide (CHX) for 2 hour and then exposed to HPB242 for 48 hours. The protein expression of Sp1 was analyzed by western blot analysis. (E) Immunocytochemistry analysis was performed in HPB242 treated HN22 and HSC4 cells. HN22 and HSC4 cells were treated with different concentrations of HPB242 for 48 hours, and cells were immunostained with Sp1 specific antibody, Cleaved-caspase3 specific antibody, and then signals were detected with Jackson 488- and 647-conjugated anti-mouse secondary antibody. DAPI was used for nucleus staining. 254×190 mm (96 × 96 DPI).