tim somervaille rrid cvcl 1632 skno1 m dsmz rrid cvcl 2196 tf1  (DSMZ)

Journal: Cell Reports Medicine

Article Title: ΔNp73 isoform defines a TP53 -mutant-like poor-risk subgroup of acute myeloid leukemia

doi: 10.1016/j.xcrm.2025.102540

Figure Lengend Snippet: ΔNp73 overexpression is associated with downregulation of the TP53 signaling pathway in TP53 wt AMLs (A) Western blot analysis for ΔNp73 and total TP73 in total cell extracts from MOLM13 cells transduced with lentivirus containing the EV (pMEG) or the ΔNp73α or ΔNp73β cDNA. (B) Volcano plot displaying the differentially expressed genes in MOLM13 cells with ΔNp73-OE versus EV control ( n = 2). (C) Expression of CD14 and CD117 in MOLM13 EV (pMEG) and ΔNp73α-OE cells ( n = 3). (D) GSEA analysis using the fold change values from the analysis depicted in (A). False discovery rate (FDR)-q values are indicated. (E) ChIP-seq data on MOLM13 cells used in (A) using antibodies against TP53 or GFP (for the GFP-ΔNp73 fusion), and TAp73. Heatmaps with signals ± 5 kb from the transcription start site (TSS) are shown. (F) Representative screenshots of TP53, TAp73, and ΔNp73 antibody binding at four TP53 target loci. (G) Venn diagram depicting overlapping peaks detected for the TP53 ChIP-seq in MOLM13 EV control cells and the GFP-ΔNp73 in MOLM13-ΔNp73 OE cells. Lower: GO analysis for the overlapping peaks (51 targets). (H and I) Cumulative cell count of MOLM13 ( TP53 wt, H) and TF1 ( TP53 mut, I) cells transduced with ΔNp73α, ΔNp73β, and EV control, cultured for 9 days ( n = 4). (J) Western blot analysis for TP53 and total TP73 in total cell extracts from MOLM13 cells transduced with EV (pMEG) or the shRNA targeting the TP53 gene (shTP53). Cumulative cell count of MOLM13 TP53 KD cells (sh TP53 ) transduced with ΔNp73α, ΔNp73β, and EV control, cultured for 9 days, is shown in the right ( n = 4). Data are reported as mean ± SEM for (H) and (I). The p values and cell lines are indicated in the graphs; ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001; ANOVA and Bonferroni post-test.

Article Snippet: TF1 (male origin) , DSMZ , ACC 334 RRID:CVCL_0559.

Techniques: Over Expression, Western Blot, Transduction, Control, Expressing, ChIP-sequencing, Binding Assay, Cell Characterization, Cell Culture, shRNA

Journal: Cell Reports Medicine

Article Title: ΔNp73 isoform defines a TP53 -mutant-like poor-risk subgroup of acute myeloid leukemia

doi: 10.1016/j.xcrm.2025.102540

Figure Lengend Snippet: ΔNp73 expression is associated with drug resistance and is regulated by an intragenic region in the TP73 gene (A) MOLM13 cells (ΔNp73-OE and EV control) were treated with FLT3 inhibitors quizartinib (AC220) and midostaurin (PKC) and AML-related drugs venetoclax (VEN) and cytarabine (AraC) for 72 h. Apoptosis and viable cell numbers were assessed by flow cytometry. Experiments were performed in quadruplicates. Results are expressed as the mean ± standard error of the mean (SEM). ED 50 , half maximal effective concentration ( n = 4). (B and C) Drug-induced apoptosis in TF1 cells (ΔNp73-OE and EV control) (B) and MOLM13 cells (transduced with shTP53 and ΔNp73-OE, as depicted in the figure) (C) treated with AML-related drugs (AraC and VEN; concentrations indicated in the plots, 72 h) detected by flow cytometry ( n = 4). (D) DNAseI gene tracks in six AML samples from the BLUEPRINT consortium. The red arrows denote highly accessible sites (+24 kb from the TSS) in the TP73 gene. The blue arrow denotes the TA promoter, and the green arrow denotes the ΔN promoter of the TP73 gene locus. (E) Relative mRNA expression levels of Δ Np73 after Cas9-mediated TP73 enhancer excision in MOLM13 cells (MOLM13-KO) at baseline and upon AraC treatment (1 μM, 48 h) ( n = 4). (F) GSEA analysis using the fold change values from the RNA-seq analysis comparing MOLM13-KO versus MOLM13-SCR cells ( n = 2). (G) TP53 (+0.2) and CDKN1A (−0.8) ChIP-qPCRs with error bars representing SEM based on three independent experiments. (H) Cumulative cell count of Cas9-mediated excision of TP73 intragenic enhancer region in MOLM13 and HL60 cells (KO versus SCR control) cultured for 9 days ( n = 4). (I and J) Drug-induced apoptosis (I) and viable cell counts (J) in MOLM13-KO cells treated with AML-related drugs (drugs and concentrations indicated in the plots, 72 h) detected by flow cytometry ( n = 4). (K) Genome browser screenshots of DNA hypersensitivity sites (DHSs) and digital footprints of the TP73 intragenic enhancer region in the TP73 loci, revealing the two regions of the intragenic enhancer. Results from motif analysis are displayed at the bottom. (L) Relative mRNA expression levels of Δ Np73 after Cas9-mediated TP73 enhancer excision of the separate regions 1 and 2 in MOLM13 cells (MOLM13-KO included as a control) at baseline and upon AraC treatment (1 μM, 48 h) ( n = 4). (M) Drug-induced apoptosis in region 2 KO MOLM13 cells treated with AML-related drugs (drugs and concentrations indicated in the plots, 72 h) detected by flow cytometry ( n = 4). (N) Representative screenshots of CEBPA antibody binding at the TP73 enhancer region in primary AML samples. Data are reported as mean ± SEM for (A)–(C), (E), (H)–(J), (L), and (M). The p values and cell lines are indicated in the graphs; ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001; ANOVA and Bonferroni post-test.

Article Snippet: TF1 (male origin) , DSMZ , ACC 334 RRID:CVCL_0559.

Techniques: Expressing, Control, Flow Cytometry, Concentration Assay, Transduction, RNA Sequencing, Cell Characterization, Cell Culture, Binding Assay