Figure 4
Figure 4. Direct Hes1 downregulation of Bbc3 expression inhibits apoptosis in T-ALL cells. (A) Quantitative RT-PCR of Bbc3 expression in Hes1 conditional knockout T-ALL (ΔE-NOTCH1 Rosa26 Cre-ERT2 Hes1flox/flox) after treatment with vehicle only (ETOH) or tamoxifen (4-OH TMX) in vitro. (B) Western blot analysis of Bbc3 expression in Hes1 conditional knockout leukemia cells and on tamoxifen-induced Hes1 deletion. Bar graph indicates the corresponding quantification of protein expression. (C) Western blot analysis of BBC3 expression DND41 and MOLT4 cells on HES1 shRNA knockdown. (D) Schematic representation of the BBC3 locus indicating the potential HES1 N-box and TP53 binding sites. (E) Relative enrichment of the BBC3 promoter sequences in control (immunoglobulin G) and HES1 ChIPs from CUTLL1 T-ALL cells. (F) Luciferase reporter activity in HEK 293T cells of a BBC3 promoter construct (Wild Type), a BBC3 promoter containing a scramble sequence in the N-box bound by HES1 (Scramble), and a BBC3 promoter with the deletion of the N-box bound by HES1 (Deletion). (G) Relative enrichment of the BBC3 promoter sequences in control (immunoglobulin G) and TP53 ChIPs from TP53 competent MOLT4 T-ALL cells in basal conditions and on etoposide-induced DNA damage. (H) Luciferase reporter activity of the BBC3 wild-type reporter construct in response to increasing doses of TP53, increasing doses of HES1, and the combination of TP53 and HES1. (I) Quantitative RT-PCR analysis of Bbc3 expression in Hes1 conditional knockout T-ALL (ΔE-NOTCH1 Rosa26 Cre-ERT2 Hes1flox/flox) cells infected with lentiviruses expressing a control shRNA (shLUC) and 2 independent shRNAs targeting Bbc3 (shBbc3 1 and shBbc3 2). (J) Quantization of cell death induced by Hes1 deletion in control (shLUC) and Bbc3 knockdown (shBbc3) in Hes1 conditional knockout T-ALL cells treated with 4-OH tamoxifen. (K) Western blot analysis of Bbc3 knockdown in Hes1 conditional knockout leukemia cells grown in low-serum conditions (3% fetal bovine serum) and on tamoxifen-induced Hes1 deletion. Bar graphs indicate mean values of triplicate measurements; error bars represent standard deviation.

Direct Hes1 downregulation of Bbc3 expression inhibits apoptosis in T-ALL cells. (A) Quantitative RT-PCR of Bbc3 expression in Hes1 conditional knockout T-ALL (ΔE-NOTCH1 Rosa26 Cre-ERT2 Hes1flox/flox) after treatment with vehicle only (ETOH) or tamoxifen (4-OH TMX) in vitro. (B) Western blot analysis of Bbc3 expression in Hes1 conditional knockout leukemia cells and on tamoxifen-induced Hes1 deletion. Bar graph indicates the corresponding quantification of protein expression. (C) Western blot analysis of BBC3 expression DND41 and MOLT4 cells on HES1 shRNA knockdown. (D) Schematic representation of the BBC3 locus indicating the potential HES1 N-box and TP53 binding sites. (E) Relative enrichment of the BBC3 promoter sequences in control (immunoglobulin G) and HES1 ChIPs from CUTLL1 T-ALL cells. (F) Luciferase reporter activity in HEK 293T cells of a BBC3 promoter construct (Wild Type), a BBC3 promoter containing a scramble sequence in the N-box bound by HES1 (Scramble), and a BBC3 promoter with the deletion of the N-box bound by HES1 (Deletion). (G) Relative enrichment of the BBC3 promoter sequences in control (immunoglobulin G) and TP53 ChIPs from TP53 competent MOLT4 T-ALL cells in basal conditions and on etoposide-induced DNA damage. (H) Luciferase reporter activity of the BBC3 wild-type reporter construct in response to increasing doses of TP53, increasing doses of HES1, and the combination of TP53 and HES1. (I) Quantitative RT-PCR analysis of Bbc3 expression in Hes1 conditional knockout T-ALL (ΔE-NOTCH1 Rosa26 Cre-ERT2 Hes1flox/flox) cells infected with lentiviruses expressing a control shRNA (shLUC) and 2 independent shRNAs targeting Bbc3 (shBbc3 1 and shBbc3 2). (J) Quantization of cell death induced by Hes1 deletion in control (shLUC) and Bbc3 knockdown (shBbc3) in Hes1 conditional knockout T-ALL cells treated with 4-OH tamoxifen. (K) Western blot analysis of Bbc3 knockdown in Hes1 conditional knockout leukemia cells grown in low-serum conditions (3% fetal bovine serum) and on tamoxifen-induced Hes1 deletion. Bar graphs indicate mean values of triplicate measurements; error bars represent standard deviation.

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