Fig 1: HDAC inhibition restores PUMA expression and DNA damage–induced apoptosis in Caki-2 cells. (A) Brightfield microscopic analysis of Caki-2 cells treated with 1 μM TSA for the indicated time intervals. Note appearance of cells with apoptotic morphology at 24 hours. Scale bar = 20 μm. (B) qRT-PCR analysis of PUMA mRNA expression following treatment with 1 μM TSA or DMSO used as control for 24 hours. (C) Immunoblot analysis of Caki-2 cells treated with 1 μM TSA for 24 hours or left untreated. Note the reexpression of PUMA and induction of PARP cleavage following TSA treatment. (D) Immunoblot analysis of Caki-2 cells for p53, cyclin A, PUMA, PARP cleavage (arrow), and GAPDH after transfection with siRNA duplexes targeting PUMA or control siRNA (24 hours) followed by treatment with 1 μM TSA for 36 hours or left untreated. Note the suppression of PUMA upregulation and apoptosis by TSA in cells transfected with PUMA siRNA. (E) Relative cell viability (MTT) analysis of Caki-2 cells after transfection with siRNA duplexes targeting PUMA (24 hours) followed by treatment with 1 μM TSA for 36 hours. (F) Trypan blue dye exclusion assay of Caki-2 cells treated with 200 ng/ml NCS and/or a sublethal concentration of TSA (0.5 μM) in comparison to DMSO-treated controls. (G) Brightfield microscopic analysis of Caki-2 cells treated for 24 hours with either DMSO, 200 ng/ml NCS, 0.5 μM TSA, or a combination of 200 ng/ml NCS and 0.5 μM TSA. Scale bar = 20 μm. (H) Immunoblot analysis of Caki-2 cells treated with 200 ng/ml NCS and/or 0.5 μM TSA for 24 hours or left untreated. Note reexpression of PUMA and induction of apoptosis as evidenced by PARP cleavage and expression of cleaved caspase-3.
Fig 2: PUMA and p53 expression modulates sensitivity of RCC cells to various DNA-damaging agents.Relative cell viability (MTT) analysis of Caki-2, Caki-1, and A498 RCC cells following treatment with either NCS (200 ng/ml), cisplatin (10 μM), gemcitabine (10 μM), or daunorubicin (0.5 μM) for 24 hours after transient transfection with PUMA (48 hours) or control plasmid, or two shRNAs to knockdown p53 or control shRNA (48 hours). Nontransfected cells are shown as control. Asterisks indicate statistically significant differences (*P ≤ .05, **P ≤ .005, ***P ≤ .0005; Student's t test for independent samples, two-tailed).
Fig 3: Resistance to DNA damage–induced apoptosis is caused by defective PUMA upregulation. (A) qRT-PCR analysis of HEK293 and Caki-2 cells for p21Cip1 and PUMA mRNA expression after NCS treatment with 200 ng/ml for 72 hours or left untreated (control) and normalization to the housekeeping gene GAPDH. Mean and standard errors of three independent experiments are shown. (B) Immunofluorescence microscopic analysis of PUMA expression in cells after treatment with 200 ng/ml NCS or left untreated. Scale bar = 50 μm. (C) Immunoblot analysis of HEK293 and Caki-2 cells treated with NCS 72 hours or left untreated. (D) Cell viability of Caki-2 cells normalized to controls following transient overexpression (48 hours) of p53 or PUMA. Each bar represents mean and standard error of three experiments. (E) qRT-PCR analysis of HEK293 and Caki-2 cells for CTCF mRNA expression after NCS treatment with 200 ng/ml for 72 hours or left untreated (control) and normalization to the housekeeping gene GAPDH. Mean and standard errors of three independent experiments are shown. (F) Immunoblot analysis of Caki-2 cells transfected with siRNA duplexes targeting CTCF or control duplexes for 24 hours and treated with 200 ng/ml NCS for 72 hours or left untreated.
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