Figure 4.
Downregulation of MYC targets in ML-DS cells expressing WT ZBTB7A. (A) Growth of CMK11-5 cells constitutively expressing WT ZBTB7A or its mutants. CMK11-5 cells were infected with various ZBTB7A-ZsGreen bicistronic retroviruses, and cell fate was monitored by flow cytometry. The combined results from 3 independent experiments are shown. Data are presented as mean ± SD. ∗P < .05; ∗∗P < .01 by Welch t test. (B) Western blot analysis of ZBTB7A expression. The analyzed cells include WT KPAM1 (lane 1), KPAM1-expressing WT ZBTB7A under the control of a Dox-inducible promoter before (lane 2) and after (lane 3) Dox treatment, and TAM (lanes 4, 5, and 6). The red arrow indicates ZBTB7A expression. (C) Cell cycle analysis of ZBTB7A-inducible KPAM1 cells. Combined results from 3 independent experiments are shown. WT ZBTB7A induction led to an increase in the G0/G1-phase population and a decrease in the S-phase population; ∗P < .05, ∗∗P < .01, Welch t test. Error bars indicate SD. (D) RNA-seq analysis of KPAM1 cells. GSEA of transcriptomes in WT ZBTB7A-inducible KPAM1 cells after vs before ZBTB7A induction. (E) GSEA using a gene set of MYC targets shows that MYC was significantly downregulated upon WT ZBTB7A expression. The red arrow indicates MYC expression. (F) Quantification of ZBTB7A binding to the P1 promoter of MYC by ChIP-qPCR in WT ZBTB7A-inducible KPAM1 cells before and after ZBTB7A induction. ZBTB7A, anti-ZBTB7A antibody (13E9); IgG, IgG isotype control. (G) Western blot analysis of MYC before and after treatment of the ML-DS cell lines (CMK11-5, MGS, KPAM1, and CHYNA) with 500 nM JQ1 for 24 hours (top) or 50 nM ARV-825 for 12 hours (bottom). β-actin served as the loading control. (H) ML-DS cells were treated with various concentrations of JQ1 or (I) ARV-825 for 72 h. Viable cell numbers were determined using the Cell Counting Kit-8. Combined results from at least 3 independent experiments are shown. Data are presented as mean ± SD. (J) ML-DS cell lines were treated for 72 hours with palbociclib at a wide range of concentrations, and viable cell numbers were determined using Cell Counting Kit-8. Representative cell viability curves are shown from 1 of 3 independent experiments. Data are presented as mean ± SD. FDR, false discovery rate; NES, normalized enrichment score; qPCR, quantitative PCR.

Downregulation of MYC targets in ML-DS cells expressing WT ZBTB7A. (A) Growth of CMK11-5 cells constitutively expressing WT ZBTB7A or its mutants. CMK11-5 cells were infected with various ZBTB7A-ZsGreen bicistronic retroviruses, and cell fate was monitored by flow cytometry. The combined results from 3 independent experiments are shown. Data are presented as mean ± SD. ∗P < .05; ∗∗P < .01 by Welch t test. (B) Western blot analysis of ZBTB7A expression. The analyzed cells include WT KPAM1 (lane 1), KPAM1-expressing WT ZBTB7A under the control of a Dox-inducible promoter before (lane 2) and after (lane 3) Dox treatment, and TAM (lanes 4, 5, and 6). The red arrow indicates ZBTB7A expression. (C) Cell cycle analysis of ZBTB7A-inducible KPAM1 cells. Combined results from 3 independent experiments are shown. WT ZBTB7A induction led to an increase in the G0/G1-phase population and a decrease in the S-phase population; ∗P < .05, ∗∗P < .01, Welch t test. Error bars indicate SD. (D) RNA-seq analysis of KPAM1 cells. GSEA of transcriptomes in WT ZBTB7A-inducible KPAM1 cells after vs before ZBTB7A induction. (E) GSEA using a gene set of MYC targets shows that MYC was significantly downregulated upon WT ZBTB7A expression. The red arrow indicates MYC expression. (F) Quantification of ZBTB7A binding to the P1 promoter of MYC by ChIP-qPCR in WT ZBTB7A-inducible KPAM1 cells before and after ZBTB7A induction. ZBTB7A, anti-ZBTB7A antibody (13E9); IgG, IgG isotype control. (G) Western blot analysis of MYC before and after treatment of the ML-DS cell lines (CMK11-5, MGS, KPAM1, and CHYNA) with 500 nM JQ1 for 24 hours (top) or 50 nM ARV-825 for 12 hours (bottom). β-actin served as the loading control. (H) ML-DS cells were treated with various concentrations of JQ1 or (I) ARV-825 for 72 h. Viable cell numbers were determined using the Cell Counting Kit-8. Combined results from at least 3 independent experiments are shown. Data are presented as mean ± SD. (J) ML-DS cell lines were treated for 72 hours with palbociclib at a wide range of concentrations, and viable cell numbers were determined using Cell Counting Kit-8. Representative cell viability curves are shown from 1 of 3 independent experiments. Data are presented as mean ± SD. FDR, false discovery rate; NES, normalized enrichment score; qPCR, quantitative PCR.

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