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. 2018 Oct 11;7(10):82. doi: 10.1038/s41389-018-0092-0

Fig. 1. DNA hypomethylating agents cause ZBTB38 down-regulation at protein level in solid and hematologic cancer cells.

Fig. 1

a Schematic representation of the experimental protocol. Cancer cells were treated twice with 10 µM 5-Azacytidine (Aza) or DMSO and harvested 24 h later for analysis. b Dot blot analysis of CpG methylation on preparation of genomic DNA from cells treated or not with 5-azacytidine (n = 2). c Gene expression analysis of hyper-methylated silenced genes CDH13 and DAPK1 by real-time PCR in HeLa cells treated with 5-azacytidine (Aza) compared to control (Ctrl) cells (n = 3). Expression level is expressed as the fold change between treated and control cells. d Western blot analysis of ZBTB38, MCM3, and GAPDH protein expression in HeLa cells treated with 5-azacytidine (Aza) compared to control (Ctrl) cells. e Gene expression analysis of ZBTB38 by real-time PCR in HeLa cells treated with 5-azacytidine (Aza) compared to control (Ctrl) cells (n = 3). f Western blot analysis of ZBTB38 and GAPDH protein expression in U2OS, HepG2, and HCT116 cancer cells treated with 5-azacytidine (Aza) compared to control (Ctrl) cells. g Western blot analysis of ZBTB38 and GAPDH protein expression in THP-1 and MOLM-14 AML cells treated with 5-azacytidine (Aza) compared to control (Ctrl) cells. h Western blot analysis of ZBTB38 and GAPDH protein expression in HeLa and HCT116 cancer cells treated with 5-azacytidine (Aza), 5-azacytidine plus proteasome-inhibitor MG132 (4 h) compared to control cells. i Western blot analysis of ZBTB38-ubiquitination in HCT116 cells treated with 5-azacytidine (Aza) compared to control (Ctrl). Co-immuno-precipitates of ZBTB38 were run on a SDS-page and ubiquitin-moieties detected using a specific antibody. j Western blot analysis of ZBTB38, ZBTB4, ZBTB33, GAPDH, phospho-CHK2 (P-CHK2), phospho-H2AX (P-H2AX) and phospho-ATM (P-ATM) in HeLa and HCT116 cells treated with 10 µM of either 5-azacytidine, decitabine or zebularine and in control mock-treated cells. k Western blot analysis of ZBTB38, DNMT1, phospho-H2AX (P-H2AX), H2AX, and GAPDH protein levels in different human cell lines (HeLa, U2OS, HCT116) treated with a specific siRNA against DNMT1 or a control siRNA. l Western blot analysis of ZBTB38 and GAPDH in HeLa cells treated with 10 µM of 5-azacytidine alone or co-treated with caffeine (1 mM) for 24 h