Fig 1: CTSD knockdown in AML cells enhances the TRIM21-mediated ubiquitination and degradation of BCL2, BCL-XL, and MCL1.A Numbers of upregulated and downregulated differentially expressed proteins (DEPs). B Volcano plot of the distribution of DEPs in the proteomic analysis. C The protein levels of BCL2, BCL-XL, and MCL1 in U937 cells with or without CTSD knockdown were measured using western blotting. D Effects of CTSD knockdown on the degradation of BCL2, BCL-XL, and MCL1. CON- or CTSD-shRNA U937 cells were incubated with CHX (20 μg/mL) or CHX plus MG132 (10 μM) for the indicated times, and proteins were detected using western blotting. E Effects of CTSD knockdown on the ubiquitination of BCL2, BCL-XL, and MCL1 in U937 cells. Protein lysates were immunoprecipitated (IP) with anti-BCL2, anti-BCL-XL, or anti-MCL1 Abs. Ubiquitinated BCL2, BCL-XL, or MCL1 were detected using immunoblotting. F Venn diagram showing the common proteins containing TRIM21 identified in the mass spectrometry analysis of BCL2, BCL-XL, and MCL1. G The protein levels of TRIM21, FBXW7, PELI1, and ITCH in U937 cells with or without CTSD knockdown were detected using western blotting. H Ubiquitination of BCL2 after co-transfection with or without E3 ligase plasmid TRIM21 was detected using immunoblotting. I Ubiquitination of BCL-XL and MCL1 after co-transfection with or without TRIM21 were detected using immunoblotting. J The protein levels of CTSD and TRIM21 in U937 cells transfected with indicated CTSD-shRNA2 or TRIM21-shRNAs were detected by western blotting. K The protein levels of BCL2, BCL-XL, and MCL1 in U937 cells from the indicated shRNA transfection groups were measured using western blotting. L Flow cytometric analysis of apoptotic cell proportions in U937 cells from the indicated shRNA transfection groups. Annexin V+ cells were quantified using FlowJo software. Data are presented as the mean ± S.E.M. Statistical significance was calculated using a one-way ANOVA. M Growth curves of U937 cells from the indicated shRNA transfection groups. Data are presented as the mean ± S.E.M. Statistical significance was calculated using a two-way ANOVA.
Fig 2: N-8 attenuates the progression of AML in vivo.A Schematic strategy for investigating the effect of N-8 on the progression of AML in mice xenografted with U937 AML cells (AML mice). Ara-C, cytarabine. po, Per Os. ip, Intraperitoneal. QD, Quaque Die. QOD, Quaque Other Die. B Flow cytometry analysis of the percentage of leukemia cells in the PB, BM, and spleen of the indicated groups (n = 5 mice per group). hCD45+ cells were quantified using FlowJo software. Data are presented as the mean ± S.E.M. Statistical significance was calculated using a one-way ANOVA. C Flow cytometry analysis of the percentage of apoptotic cells in the PB, BM, and spleen of the indicated groups (n = 5 mice per group). Annexin V+ cells were quantified using FlowJo software. Data are presented as the mean ± S.E.M. Statistical significance was calculated using a one-way ANOVA. D The protein levels of BCL2, BCL-XL, and MCL1 in the spleen of AML mice treated with or without N-8 were detected using western blotting. E Kaplan-Meier survival curves for the indicated mice (n = 5 mice per group). Statistical significance was calculated using a two-sided log-rank test. F The body weight curves of AML mice treated with the indicated agents (n = 5 mice per group). Data are presented as the mean ± S.E.M. Statistical significance was calculated using a two-way ANOVA. G Schematic of the strategy used to investigate the effect of N-8 on the progression of AML in the MLL-AF9 leukemia model. VEN, venetoclax. po, Per Os, QD, Quaque Die. H Representative spleen images (left) and statistical analysis of spleen weights (right) obtained from mice with the indicated treatment. Scale bar, 1 cm. I Flow cytometry analysis of the percentage of leukemia cells in the PB, BM, and spleen of the indicated groups (n = 5 mice per group). GFP+ cells were quantified using FlowJo software. Data are presented as the mean ± S.E.M. Statistical significance was calculated using a one-way ANOVA. J Flow cytometry analysis of the percentage of apoptotic cells in the PB, BM, and spleen of the indicated groups (n = 5 mice per group). Annexin V+ cells were quantified using FlowJo software. Data are presented as the mean ± S.E.M. Statistical significance was calculated using a one-way ANOVA. K The protein levels of BCL2, BCL-XL, and MCL1 in the spleen and BM of AML mice treated with or without N-8 was detected using western blotting. L Kaplan-Meier survival curves for the indicated mice (n = 5 mice per group). Statistical significance was calculated using a two-sided log-rank test. M The body weight curves of leukemia mice treated with the indicated agents (n = 5 mice per group). Data are presented as the mean ± S.E.M. Statistical significance was calculated using a two-way ANOVA. N Schematic diagram illustrating the mechanism by which CTSD inhibition induces cell apoptosis.
Fig 3: N-8 destabilizes BCL2, BCL-XL, and MCL1 to inhibit AML. Effect of N-8 on apoptosis of U937, MV4-11, MOLM-13 (A), MV4-11-VEN-R, MOLM-13-VEN-R (B), and primary AML cells (C). Cells were treated with the indicated concentrations of N-8, evaluated after 48 h and stained with Annexin V/PI. The percentage of Annexin V+ cells was calculated using FlowJo software. Data are presented as the mean ± S.E.M. Statistical significance was calculated using a one-way ANOVA. D Effect of N-8 on proliferation of U937, MOLM-13, and MOLM-13-VEN-R cells. Data are presented as the mean ± S.E.M. Statistical significance was calculated using a two-way ANOVA. E Effect of N-8 on apoptosis of U937 cells with or without CTSD knockdown. U937 cells with or without CTSD knockdown were treated with the indicated concentrations of N-8, evaluated after 12 or 24 h and stained with Annexin V/PI. The percentage of Annexin V+ cells was calculated using FlowJo software. Data are presented as the mean ± S.E.M. Statistical significance was calculated using a two-way ANOVA. Effect of N-8 on the protein levels of BCL2, BCL-XL, and MCL1 in U937, MV4-11, MOLM-13 (F), MV4-11-VEN-R, and MOLM-13-VEN-R cells (G). Cells were treated with the indicated concentrations of N-8 for 24 h, and proteins were detected using western blotting. H Effect of N-8 on the degradation of BCL2, BCL-XL, and MCL1 proteins. U937 and MV4-11 cells were incubated with CHX (20 μg/mL) or CHX plus MG132 (10 μM) for the indicated times, and proteins were detected using western blotting. I Effect of N-8 on the ubiquitination of BCL2, BCL-XL, and MCL1. Lenti-X 293 T cells were transfected with the indicated plasmids for 24 h and treated with the indicated concentrations of N-8. After 24 h, ubiquitinated BCL2, BCL-XL, or MCL1 was detected using immunoblotting.
Supplier Page from Sino Biological, Inc. for Human BCL2L1/Bcl-XL Gene ORF cDNA clone in cloning vector