The induction of HSP40 by TChal contributes to the stabilization of p53 activity.

A. U2OS cells were treated with TChal at the indicated dose and times. The p53 and HSP40 expressions were measured by western blot analysis. B. U2OS cells were treated with 50 μM of TChal for 24 h, and cell lysates were isolated with a modified RIPA buffer. The cell lysates were immunoprecipitated with 2 μg of p53 or normal IgG antibodies using A/G PLUS-agarose, as described in the Materials and Methods section, and then subjected to western blot analysis using Hsp40 antibody. C. U2OS cells were transfected with 30 nM of Hsp40 or control siRNAs for 24 h, followed by treatment with 50 μM of TChal for 24 h. Then, cells lysates were subjected to western blot analysis. D. Vectors containing the MDM2-Luc promoter were co-transfected with Hsp40 or control siRNAs into U2OS cells, and cells were treated with 50 μM of TChal for 24 h. Luciferase activity was measured; y-axis represents RLU (relative luciferase unit). E. HSP40 silencing suppressed the effect of TChal on p53-dependent transcription. U2OS cells were transfected with Hsp40 or control siRNAs, followed by treatment with 50 μM of TChal for 24 h. Total RNA was isolated, and RT-PCR was performed as described in the Materials and Methods section. A representative gel from 3 independent experiments are shown.