bh3 mimetics abt 737  (Mimetics)

Journal: bioRxiv

Article Title: Inhibition of V-ATPase function drives apoptosis via GCN1/GCN2 kinase signaling

doi: 10.64898/2026.03.27.714872

Figure Lengend Snippet: a. Gene ontology analysis of differentially expressed genes of HAP-1 cells after 24 h treatment with 50 nM of NoA or DMSO vehicle control. b. Schematic of mitochondrial apoptosis regulation by BCL-2 family members. Chronic cellular stress BH3-only proteins upregulation antagonize pro-survival BCL-2 members and activate BAX/BAK, resulting in mitochondrial outer membrane permeabilization and apoptosis. c. Heatmap showing regulation of canonical ATF4 target genes extracted from the RNA-seq data. Values show row scaled vst-normalized expression of differentially expressed genes. Values of n=4 technical replicates are shown. d. Heatmap showing regulation of canonical JUN target genes extracted from the RNA-seq data. Values show row scaled vst-normalized expression of differentially expressed genes. Values of n=4 technical replicates are shown. e. Heatmap showing regulation of pro-apoptotic BH-3 proteins extracted from the RNA-seq data. Values show row scaled vst-normalized expression of differentially expressed genes. Values of n=4 technical replicates are shown. f. Western blot analysis of HAP-1 WT cells treated with 50 nM NoA. Cells were harvested at 0, 3, 6, 12 and 24 h after treatment, and protein lysates were probed with the indicated antibodies. g. Nalm-6 WT and KO clones were treated with 12, 25 and 50 nM of NoA. Annexin V surface binding and Propidium Iodide uptake was measured after 72 h to assess viability of the cells. Bar plots show mean ± SD of three biological replicates. Legend: AV−/PI− = live cells, AV+/PI− = early apoptosis, AV+/PI+ and AV−/PI+ = late apoptosis. Statistical significance was determined using a two-way ANOVA with Bonferroni correction for multiple comparisons, comparing percentages of live cells of corresponding concentrations between WT and KO clones. * P < 0.05, ** P < 0.005 h. Western blot analysis of Nalm-6 and HCT-116 WT cells treated with 50 nM NoA. Cells were harvested at 0, 12, 24 and 36 h after NoA treatment and protein lysates probed with indicated antibodies. MCL-1 OMM refers to the full-length, anti-apoptotic isoform MCL-1.

Article Snippet: Loss of MCL-1 creates a vulnerability that renders cells dependent on co-expressed BCL-2 family proteins, enabling potent synergy with the BH3 mimetics ABT-737 or venetoclax.

Techniques: Control, Membrane, RNA Sequencing, Expressing, Western Blot, Clone Assay, Binding Assay

Journal: bioRxiv

Article Title: Inhibition of V-ATPase function drives apoptosis via GCN1/GCN2 kinase signaling

doi: 10.64898/2026.03.27.714872

Figure Lengend Snippet: a. Bottom: Representative cell cycle profiles of HAP-1 WT and BB dKO cells after exposure to 12.5, 25 and 50 nM of NoA. Right: Ǫuantification of the SubG1 population represented as bar graphs, showing means and SD of N=3 biological replicates. Statistical significance was determined using a two-way ANOVA with Bonferroni correction for multiple comparisons, comparing SubG1 percentages of corresponding concentrations of WT with BBdKO cells at every time point. **** P < 0.0001 b. HAP-1 WT and HAP-1 BB dKO KO cells were treated with 12.5, 25 and 50 nM of BafA1. Annexin V surface binding and Propidium Iodide uptake was measured after 48 and 72 h to assess viability of the cells. Bar plots show mean ± SD of three biological replicates. Legend: AV−/PI− = live cells, AV+/PI− = early apoptosis, AV+/PI+ and AV−/PI+ = late apoptosis. Statistical significance was determined using a two-way ANOVA with Bonferroni correction for multiple comparisons, comparing percentages of live cells of corresponding concentrations between WT and BB dKO cells at every time point. **** P < 0.0001. c. WB analysis of HAP-1 WT, GCN1 KO, and GCN2 KO cells treated with NoA or BafA1 (25 or 50 nM) for 24 h. Cell lysates were probed for the indicated proteins. d. HAP-1 WT and GCN1 KO cells were treated with BafA1 (6.25, 12.5, 25, or 50 nM). HAP-1 WT cells were treated either with BafA1 alone or in combination with the GCN2 inhibitor GCN2iB (10 μM). Annexin V surface binding and propidium iodide (PI) uptake were measured after 48 and 72 h to assess cell viability. Bar plots represent mean ± SD of three biological replicates. Legend: AV−/PI−, live cells; AV+/PI−, early apoptosis; AV+/PI+ and AV−/PI+, late apoptosis. Statistical significance was determined by two-way ANOVA with Bonferroni correction for multiple comparisons, comparing percentages of live cells at corresponding concentrations between WT and GCN1 KO cells or WT cells co-treated with GCN2iB at each time point. ** P < 0.01, *** P < 0.001, ** P < 0.0001. e. HCT-116 WT and Octa KO clones were treated with 12.5, 25 and 5 0nM of BafA1. Annexin V surface binding and Propidium Iodide uptake was measured after 24, 48 and 72 h to assess cell viability. Bar plots show mean ± SD of three biological replicates. Legend: AV−/PI− = live cells, AV+/PI− = early apoptosis, AV+/PI+ and AV−/PI+ = late apoptosis. Statistical significance was determined using a two-way ANOVA with Bonferroni correction for multiple comparisons, comparing percentages of live cells of corresponding concentrations between WT and Octa KO clones. * P < 0.015, ** P < 0.004, *** P < 0.001, **** P < 0.0001 f. HAP-1 WT cells were treated with indicated V-ATPase inhibitors (25 nM) or ABT-737 (1 μM) alone or in combination for 24 h. Viability was assessed by Annexin V surface binding and Propidium Iodide uptake. Viable fraction was defined as double negative cells and is depicted as mean ± SD of three biological replicates. g. Dot plot showing synergy scores for combinations of V-ATPase inhibitors with BH3 mimetics (ABT-737 and ABT-199). Synergy scores were calculated using the SynergyFinder web tool based on concentration matrices of two biological replicates. Cell viability was defined as the fraction of Annexin V−/PI− (double-negative) cells, representing live cells. h. Proposed mechanism for V-ATPase inhibition induced cell death

Article Snippet: Loss of MCL-1 creates a vulnerability that renders cells dependent on co-expressed BCL-2 family proteins, enabling potent synergy with the BH3 mimetics ABT-737 or venetoclax.

Techniques: Binding Assay, Clone Assay, Concentration Assay, Inhibition