Fig. 5. Depletion of ZBTB38 induces CDKN1C up-regulation at the mRNA level in solid and hematologic cancers.

a Western blot analysis of cell death, cell cycle, and DNA replication factors expression in THP-1 cells treated with siRNAs (control and ZBTB38) and with decitabine (1 µM for 24 h) or not. The experiment was performed on day 1. Levels of 5 mC and 5 hmC evaluated by dot blot are indicated. TP53 expression in MOLM-14 cells is also presented as control. b Analysis by RT-qPCR of CDKN1C expression in U2OS, HCT116, HeLa, THP-1, and MOLM-14 cells treated with an siRNA against ZBTB38 and control siRNA (n = 3). c RT-qPCR analysis of the expression of genes located in the vicinity of CDKN1C at the ICR1 locus in HeLa, HCT116, and THP-1 cells (n = 3). d Analysis by RT-qPCR of CDKN1A, CDKN1B, and CDKN1C expression levels in HeLa, HCT116, and THP-1 cells treated with an siRNA against ZBTB38 and control siRNA (n = 3). e Analysis of CDKN1C expression by RT-qPCR in THP-1 and MOLM-14 cells transfected with siRNA against USP9X or control siRNA (n = 3). f Western blot analysis of CDKN1C protein expression in U2OS, HeLa, and HCT116 cells challenged with 5-azacytidine (4 µM for 24 h) and further treated with siRNAs against ZBTB38. g Depletion of CDKN1C rescues the cytotoxicity caused by DNMTi and ZBTB38 silencing in THP-1 and MOLM-14 cells (n = 3). Cell viability was analyzed 2 days post-DNMTi exposure (azacytidine: 1 µM for 24 h and decitabine: 2 µM for 24 h) by scoring trypan blue-positive cells and by reporting the ratio between the number of viable cells at Day 2 compared to Day 0. ***P < 0.05. A representative western blot of CDKN1C and ZBTB38 protein expression in each condition is shown for one of the three replicate