The Δ133p53 isoform is an antiapoptotic protein that promotes the survival of human T-ALL cells through induction of Bcl-xL expression. (A) The abundance of the Cherry+ mTagBFP2+ cell fraction, tracked over time in culture by flow cytometry. The CUTLL-1 cell line was independently transduced with 2 different clones of shRNA/mCherry lentiviral constructs against the HES4 gene or with the scramble control, in combination with the Δ133p53/ mTagBFP2 construct or the EV as indicated. Cherry+ mTagBFP2+ cells were sorted using FACS, cultured in vitro, and measured at the indicated time points by flow cytometry. The graphs report the result of 3 independent experiments performed in triplicate. ∗∗∗P < .001 (2-way ANOVA). (B) Abundance of the Δ133p53-transduced mTagBFP2+ cell fraction, tracked over time in culture by flow cytometry. The DND-41 cell line was independently transduced with the Δ133p53/mTagBFP2 lentiviral construct or EVs as control. mTagBFP2+ alive cells were measured at the indicated time points by flow cytometry for propidium iodide (PI) exclusion. The graphs report the result of 3 independent experiments performed in triplicate. ∗∗∗P < .001 (2-way ANOVA). (C) Abundance of the Δ133p53-transduced mTagBFP2+ cell fraction in 2 independent clones of PDXs. Two days after transduction the cells were sorted using FACS and treated with the GSI Compound E (1 μM) for 3 days and measured at the indicated time points by flow cytometry for PI exclusion. The graphs report the result of 3 independent experiments performed in triplicate. ∗∗P < .01; ∗∗∗P < .001 (Student t test). (D) Correlation analysis of messenger RNA (mRNA) expression among HES4 and selected p53 target genes in the human CUTLL-1 cell line after transduction with HES4 or the EV as reported in Figure 5. (E) Correlation analysis of mRNA expression between HES4 and selected p53 target genes across 4 different gene expression data sets of human T-ALLs. The mRNA expression levels of different genes among 262 patients with T-ALL from the COG TARGET study were normalized using rLog. The Affymetrix microarray (HG-U133 Plus 2.0) data were downloaded from the Gene Expression Omnibus (GEO) database (accession numbers: GSE13204 [n = 174], GSE32215 [n = 228], and GSE26713 [n = 117]) and normalized using RMA (Robust Multiarray Averaging). (F) The BCL2L1 mRNA expression level in the ALL-SIL and DND-41 cell lines and in the D115-1 and H3255-1 PDXs after transduction with the lentivectors that encoded HES4, Δ133p53, or EVs. Cells were collected 4 days after the transduction and sorted using FACS before RNA isolation and performing the TaqMan reverse transcriptase-digital droplet polymerase chain reaction assay. The values in the plot indicate the ratio of the BCL2L1 mRNA expression level normalized to the B2M gene expression as control. In each data set, 3 biologic replicates for each condition are indicated. Error bars indicate the SD. ∗P < .05; ∗∗∗P < .001 (Student t test). (G) Protein expression level of Bcl-xL determined by flow cytometry in the ALL-SIL and DND-41 cell lines and in the D115-1 and H3255-1 PDXs transduced as reported in panel F. (H) Flow cytometric analysis of the early apoptotic level using annexin V binding and 7-aminoactinomycin D (7AAD) exclusion in the ALL-SIL and DND-41 cell lines and the D115-1 and H3255-1 PDXs transduced as reported in panel F. Two days after the transduction, the cells were sorted using FACS and treated with GSI for 3 days before flow cytometry analysis. The graphs report the results of 2 independent experiments performed in triplicate. Each reported statistical value was compared with EV as the control. ∗∗P < .01; ∗∗∗P < .001 (Student t test). (I) Flow cytometric analysis of early apoptotic level by annexin V binding and 7AAD exclusion in the D115-1 and H3255-1 PDXs after transduction with lentivectors encoding wt intracellular NOTCH1 domain (ICN1) or ICN1-R1984A dimer mutant alone and in combination with the HES4 or Δ133p53 constructs. Cells transduced with EV were also included as control. Two days after the transduction, the cells were sorted using FACS and treated with GSI for 3 days before flow cytometry analysis. The graphs report the result of 2 independent experiments performed in triplicate. ∗P < .05; ∗∗P < .01; ∗∗∗P < .001 (Student t test).

The Δ133p53 isoform is an antiapoptotic protein that promotes the survival of human T-ALL cells through induction of Bcl-xL expression. (A) The abundance of the Cherry+ mTagBFP2+ cell fraction, tracked over time in culture by flow cytometry. The CUTLL-1 cell line was independently transduced with 2 different clones of shRNA/mCherry lentiviral constructs against the HES4 gene or with the scramble control, in combination with the Δ133p53/ mTagBFP2 construct or the EV as indicated. Cherry+ mTagBFP2+ cells were sorted using FACS, cultured in vitro, and measured at the indicated time points by flow cytometry. The graphs report the result of 3 independent experiments performed in triplicate. ∗∗∗P < .001 (2-way ANOVA). (B) Abundance of the Δ133p53-transduced mTagBFP2+ cell fraction, tracked over time in culture by flow cytometry. The DND-41 cell line was independently transduced with the Δ133p53/mTagBFP2 lentiviral construct or EVs as control. mTagBFP2+ alive cells were measured at the indicated time points by flow cytometry for propidium iodide (PI) exclusion. The graphs report the result of 3 independent experiments performed in triplicate. ∗∗∗P < .001 (2-way ANOVA). (C) Abundance of the Δ133p53-transduced mTagBFP2+ cell fraction in 2 independent clones of PDXs. Two days after transduction the cells were sorted using FACS and treated with the GSI Compound E (1 μM) for 3 days and measured at the indicated time points by flow cytometry for PI exclusion. The graphs report the result of 3 independent experiments performed in triplicate. ∗∗P < .01; ∗∗∗P < .001 (Student t test). (D) Correlation analysis of messenger RNA (mRNA) expression among HES4 and selected p53 target genes in the human CUTLL-1 cell line after transduction with HES4 or the EV as reported in Figure 5. (E) Correlation analysis of mRNA expression between HES4 and selected p53 target genes across 4 different gene expression data sets of human T-ALLs. The mRNA expression levels of different genes among 262 patients with T-ALL from the COG TARGET study were normalized using rLog. The Affymetrix microarray (HG-U133 Plus 2.0) data were downloaded from the Gene Expression Omnibus (GEO) database (accession numbers: GSE13204 [n = 174], GSE32215 [n = 228], and GSE26713 [n = 117]) and normalized using RMA (Robust Multiarray Averaging). (F) The BCL2L1 mRNA expression level in the ALL-SIL and DND-41 cell lines and in the D115-1 and H3255-1 PDXs after transduction with the lentivectors that encoded HES4, Δ133p53, or EVs. Cells were collected 4 days after the transduction and sorted using FACS before RNA isolation and performing the TaqMan reverse transcriptase-digital droplet polymerase chain reaction assay. The values in the plot indicate the ratio of the BCL2L1 mRNA expression level normalized to the B2M gene expression as control. In each data set, 3 biologic replicates for each condition are indicated. Error bars indicate the SD. ∗P < .05; ∗∗∗P < .001 (Student t test). (G) Protein expression level of Bcl-xL determined by flow cytometry in the ALL-SIL and DND-41 cell lines and in the D115-1 and H3255-1 PDXs transduced as reported in panel F. (H) Flow cytometric analysis of the early apoptotic level using annexin V binding and 7-aminoactinomycin D (7AAD) exclusion in the ALL-SIL and DND-41 cell lines and the D115-1 and H3255-1 PDXs transduced as reported in panel F. Two days after the transduction, the cells were sorted using FACS and treated with GSI for 3 days before flow cytometry analysis. The graphs report the results of 2 independent experiments performed in triplicate. Each reported statistical value was compared with EV as the control. ∗∗P < .01; ∗∗∗P < .001 (Student t test). (I) Flow cytometric analysis of early apoptotic level by annexin V binding and 7AAD exclusion in the D115-1 and H3255-1 PDXs after transduction with lentivectors encoding wt intracellular NOTCH1 domain (ICN1) or ICN1-R1984A dimer mutant alone and in combination with the HES4 or Δ133p53 constructs. Cells transduced with EV were also included as control. Two days after the transduction, the cells were sorted using FACS and treated with GSI for 3 days before flow cytometry analysis. The graphs report the result of 2 independent experiments performed in triplicate. ∗P < .05; ∗∗P < .01; ∗∗∗P < .001 (Student t test).

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