Figure 2
Figure 2. GAC protein is prominently expressed in AML and modulates the OCR. (A) Analysis of human leukemic cell lines. AML cells from 8 patients and normal CD34+ HPCs from 4 healthy donors were analyzed by western blotting using anti-GLS1, anti-GLS2, and anti-ACTIN antibodies. (B) MOLM-14 (left) and OCI-AML2 (right) cells were transfected with a lentiviral vector expressing a doxycycline-inducible GLS1 shRNA (#5) construct. Stably infected cell lines were established by puromycin selection. After 2 days of doxycycline exposure, the OCR was measured using a Seahorse XF96 extracellular flux analyzer under both basal conditions and after the addition of CCCP and antimycin, as indicated. The inhibition of GAC expression was controlled in western blots using anti-GAC antibody. Histograms show data that are representative of 3 independent experiments. (C) OCI-AML2 cells were cultured with or without CB-839 (1 µM), BPTES (10 µM), or compound 968 (10 µM) for 6 hours, and the OCR was measured using a Seahorse XF96 extracellular flux analyzer. Histograms show data that are representative of 3 independent experiments. (D) OCI-AML2 cells were cultured with or without CB-839 (1 µM), BPTES (10 µM), or compound 968 (10 µM) for 4 hours, and 5 × 106 cells were washed twice in cold PBS; the pellet was frozen and each indicted metabolite was measured. (E) OCI-AML2 cells were cultured with or without CB-839 (1 µM) and αKG (5 mM) for 6 h, and the OCR was measured. Histograms show data that are representative of 3 independent experiments. (F) OCI-AML2 cells were transfected with a lentiviral vector expressing a doxycycline-inducible V5-tagged GACWT or GACK320A construct. Stably infected cell lines were established by puromycin selection. After 6-hour doxycycline exposure with or without CB-839, the OCR was measured under both basal conditions and after the addition of CCCP and antimycin, as indicated. Histograms show data that are representative of 3 independent experiments (G) OCI-AML2 cells stably infected with GACWT or GACK320A were cultured for 6 hours with or without doxycycline or CB-839 (1 µM) and were analyzed by western blotting using anti-GAC and anti-ACTIN antibodies. *P < .05, **P < .01, ***P < .001.

GAC protein is prominently expressed in AML and modulates the OCR. (A) Analysis of human leukemic cell lines. AML cells from 8 patients and normal CD34+ HPCs from 4 healthy donors were analyzed by western blotting using anti-GLS1, anti-GLS2, and anti-ACTIN antibodies. (B) MOLM-14 (left) and OCI-AML2 (right) cells were transfected with a lentiviral vector expressing a doxycycline-inducible GLS1 shRNA (#5) construct. Stably infected cell lines were established by puromycin selection. After 2 days of doxycycline exposure, the OCR was measured using a Seahorse XF96 extracellular flux analyzer under both basal conditions and after the addition of CCCP and antimycin, as indicated. The inhibition of GAC expression was controlled in western blots using anti-GAC antibody. Histograms show data that are representative of 3 independent experiments. (C) OCI-AML2 cells were cultured with or without CB-839 (1 µM), BPTES (10 µM), or compound 968 (10 µM) for 6 hours, and the OCR was measured using a Seahorse XF96 extracellular flux analyzer. Histograms show data that are representative of 3 independent experiments. (D) OCI-AML2 cells were cultured with or without CB-839 (1 µM), BPTES (10 µM), or compound 968 (10 µM) for 4 hours, and 5 × 106 cells were washed twice in cold PBS; the pellet was frozen and each indicted metabolite was measured. (E) OCI-AML2 cells were cultured with or without CB-839 (1 µM) and αKG (5 mM) for 6 h, and the OCR was measured. Histograms show data that are representative of 3 independent experiments. (F) OCI-AML2 cells were transfected with a lentiviral vector expressing a doxycycline-inducible V5-tagged GACWT or GACK320A construct. Stably infected cell lines were established by puromycin selection. After 6-hour doxycycline exposure with or without CB-839, the OCR was measured under both basal conditions and after the addition of CCCP and antimycin, as indicated. Histograms show data that are representative of 3 independent experiments (G) OCI-AML2 cells stably infected with GACWT or GACK320A were cultured for 6 hours with or without doxycycline or CB-839 (1 µM) and were analyzed by western blotting using anti-GAC and anti-ACTIN antibodies. *P < .05, **P < .01, ***P < .001.

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