Supplementary Figure 2: Statins induce ubiquitination and degradation of mutp53 via an E3 ubiquitin ligase CHIP, but not MDM2.
From: DNAJA1 controls the fate of misfolded mutant p53 through the mevalonate pathway
(a) Quantitative RT-PCR for human p53 and GAPDH using total RNAs from SK-Br-3 and U2OS cells treated with DMSO (D) or lovastatin (L) at 4 μM for 24 h. Data are shown as relative p53 mRNA expression to that of GAPDH. Error bars, means ± s.d. (n = 3 independent experiments), NS: not significant; Student’s t test (two-tailed). (b) WB for p53 and vinculin using KHOS/NP (p53R156P) cells treated with cyclohexamide (CHX, 50 nM) for indicated time period following pre-incubation with D or 4 μM of L for 12 h (top). Graph showing relative p53R175H levels with time compared to those without CHX treatment (bottom). (c) Ubiquitination assays for p53 using KHOS/NP (left) or CAL33 (p53R175H, right) cells treated with D or 4 μM of L for 22 h. High molecular bands indicate ubiquitinated mutp53. (d) WB for indicated proteins using KHOS/NP cells infected with non-silencing control or MDM2 (M1, 2, 3) shRNA-encoding lentiviral vectors and treated with L at 4 μM for 24 h. (e) WB for indicated proteins using KHOS/NP cells infected with non-silencing control or CHIP (CH1, 2) shRNA-encoding lentiviral vectors and treated with L at 4 μM for 24 h. (f) WB for p53 and vinculin using SK-Br-3 (p53R175H) cells infected with non-silencing control or CHIP shRNA-encoding lentiviral vectors and treated with cyclohexamide (CHX, 50 nM) for indicated time period following pre-incubation with D or 4 μM of L for 12 h (left). Graph showing relative p53R175H levels with time, compared to those without CHX treatment (right). Error bars, means ± s.d. (n = 3 independent experiments). Statistics source data for a and f are provided in Supplementary Table 1.
