Figure 1.
Knockout and knockdown of MTCH2 reduces the growth and viability of AML cells. (A) A CRISPR screen in Cas9-OCI-AML2 cells identified depleted gRNAs that target the 1050 nuclear-encoded mitochondrial proteins. Positive hits were identified at a FDR of less than 5%. Genes were ranked based on MAGeCK scores (log2 of P values) as calculated by the MAGeCK algorithm. (B) OCI-AML2-Cas9-expressing cells were transduced with gRNA targeting MTCH2. Seven days after transduction, MTCH2 protein levels were measured by immunoblotting. Viability and proliferation of cells were measured over time by trypan blue exclusion staining starting 4 days after transduction. Data represent mean viable cell counts ± standard deviation (SD) from representative experiments. (C–E) OCI-AML2 (C), TEX (D), and U937 (E) cells were transduced with shRNA targeting MTCH2 or control sequences. Seven days after transduction, levels of MTCH2 were detected by immunoblotting. Viability and proliferation of cells were measured from 4 days after the transduction of MTCH2 or control shRNA, using trypan blue exclusion staining. Data represent the mean ± SD of 3 independent experiments. Colony formation from control and MTCH2 knockdown clones. Mean ± SD colony counts are shown. *P < .05; **P < .01; ***P < .001, using 1-way ANOVA. (F) MLL-AF9 was transduced into MTCH2 F/F Vav1-Cre+ or MTCH2 F/F murine hematopoietic cells in vitro and then injected into secondary recipients. The survival of the mice was measured over time (MTCH2 F/F n = 10; MTCH2 F/F Vav1-Cre+, n = 7). (G) Human CD34-enriched cord blood was transduced with GFP-expressing plasmids containing control or MTCH2 shRNA2 sequences in lentiviral vectors. Forty-eight hours after transduction, 60 000 cells were injected intrafemorally into NSG mice (5 mice/group). Eight weeks after transplant, bone marrow aspirates were analyzed for human engraftment (anti-human CD45+) and GFP+ expression. The transduction efficiencies of the control and MTCH2 lentiviral vectors were 20.9% and 19.8%, respectively. No statistically significant differences were detected.

Knockout and knockdown of MTCH2 reduces the growth and viability of AML cells. (A) A CRISPR screen in Cas9-OCI-AML2 cells identified depleted gRNAs that target the 1050 nuclear-encoded mitochondrial proteins. Positive hits were identified at a FDR of less than 5%. Genes were ranked based on MAGeCK scores (log2 of P values) as calculated by the MAGeCK algorithm. (B) OCI-AML2-Cas9-expressing cells were transduced with gRNA targeting MTCH2. Seven days after transduction, MTCH2 protein levels were measured by immunoblotting. Viability and proliferation of cells were measured over time by trypan blue exclusion staining starting 4 days after transduction. Data represent mean viable cell counts ± standard deviation (SD) from representative experiments. (C–E) OCI-AML2 (C), TEX (D), and U937 (E) cells were transduced with shRNA targeting MTCH2 or control sequences. Seven days after transduction, levels of MTCH2 were detected by immunoblotting. Viability and proliferation of cells were measured from 4 days after the transduction of MTCH2 or control shRNA, using trypan blue exclusion staining. Data represent the mean ± SD of 3 independent experiments. Colony formation from control and MTCH2 knockdown clones. Mean ± SD colony counts are shown. *P < .05; **P < .01; ***P < .001, using 1-way ANOVA. (F) MLL-AF9 was transduced into MTCH2 F/F Vav1-Cre+ or MTCH2 F/F murine hematopoietic cells in vitro and then injected into secondary recipients. The survival of the mice was measured over time (MTCH2 F/F n = 10; MTCH2 F/F Vav1-Cre+, n = 7). (G) Human CD34-enriched cord blood was transduced with GFP-expressing plasmids containing control or MTCH2 shRNA2 sequences in lentiviral vectors. Forty-eight hours after transduction, 60 000 cells were injected intrafemorally into NSG mice (5 mice/group). Eight weeks after transplant, bone marrow aspirates were analyzed for human engraftment (anti-human CD45+) and GFP+ expression. The transduction efficiencies of the control and MTCH2 lentiviral vectors were 20.9% and 19.8%, respectively. No statistically significant differences were detected.

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