non-template negative control (ntc  (Millipore)

Journal: Cancers

Article Title: Cotargeting of Mitochondrial Complex I and Bcl-2 Shows Antileukemic Activity against Acute Myeloid Leukemia Cells Reliant on Oxidative Phosphorylation

doi: 10.3390/cancers12092400

Figure Lengend Snippet: IACS-010759 and venetoclax cooperatively induce intrinsic apoptosis. ( a , b ) MV4-11 cells cultured in media supplemented with glucose were treated with vehicle control, IACS-010759, venetoclax, or in combination for 4 or 8 h. Cellular fractionation was performed. Mitochondrial and cytosolic fractions were subjected to western blot analysis. This experiment was performed 2 independent times in triplicate. One representative image is shown. Relative densitometry measurements were determined using Odyssey Software V3.0, normalized to β-actin or VDAC1, and compared to the vehicle control. Results from one representative experiment are graphed as mean ± SEM in panel b. * indicates p < 0.05, ** indicates p < 0.01, and *** indicates p < 0.001 compared to vehicle control; ### indicates p < 0.001 compared to single drug treatments. ( c ) MV4-11 cells were cultured and treated as described in panel a. Whole cell lysates were subjected to western blot analysis. Relative densitometry measurements were determined using Odyssey Software V3.0, normalized to β-actin, and compared to the vehicle control. ( d , e ) Lentiviral shRNA double knockdown of Bak and Bax (designated Bak/Bax KD) was performed in MV4-11 cells. Non-template control (NTC)-shRNA was used as the control for the Bak/Bax double knockdown. Western blots probed with anti-Bak, -Bax, or -β-actin antibody are shown in panel d. shRNA knockdown cells cultured in media supplemented with glucose were treated with IACS-010759 and venetoclax, alone or combined, for 24 h. Cells were then stained with Annexin V-FITC/PI and analyzed by flow cytometry. *** indicates p < 0.001 compared to the NTC-shRNA cells. ( f ) MV4-11 cells cultured in media supplemented with glucose were treated with combined IACS-010759 and venetoclax in the absence or presence of the pan-caspase inhibitor Z-VAD-FMK for 24 h. Cells were then stained with Annexin V-FITC/PI and analyzed by flow cytometry. *** indicates p < 0.001 compared to the combined venetoclax and IACS-010759 treatment. ( g , h ) MOLM-13 cells cultured in media supplemented with glucose were treated with vehicle control, IACS-010759, venetoclax, or in combination for 8 h. ( i ) Cellular fractionation was performed. Mitochondrial and cytosolic fractions were subjected to western blot analysis. This experiment was performed 2 independent times in triplicate. One representative image is shown. Relative densitometry measurements were determined using Odyssey Software V3.0, normalized to β-actin or VDAC1, and compared to the vehicle control. Results from one representative experiment are graphed as mean ± SEM in panel h. *** indicates p < 0.001 compared to vehicle control; ### indicates p < 0.001 compared to single drug treatments. ( j ) MOLM-13 cells cultured in media supplemented with glucose were treated with IACS-010759 and venetoclax, alone or combined, for 8 h. Cells were then stained with Annexin V-FITC/PI and analyzed by flow cytometry. *** indicates p < 0.001 compared to the vehicle control and single drug treatments. ( k ) MOLM-13 cells cultured in media supplemented with glucose were treated with combined IACS-010759 and venetoclax in the absence or presence of the pan-caspase inhibitor Z-VAD-FMK for 24 h. Cells were then stained with Annexin V-FITC/PI and analyzed by flow cytometry. *** indicates p < 0.001 compared to the combined venetoclax and IACS-010759 treatment.

Article Snippet: Non-template negative control (NTC)-, Bax-, and Bak-shRNA lentiviral constructs were purchased from Sigma-Aldrich (St. Louis, MO, USA).

Techniques: Cell Culture, Cell Fractionation, Western Blot, Software, shRNA, Staining, Flow Cytometry

Journal: Cancers

Article Title: Cotargeting of Mitochondrial Complex I and Bcl-2 Shows Antileukemic Activity against Acute Myeloid Leukemia Cells Reliant on Oxidative Phosphorylation

doi: 10.3390/cancers12092400

Figure Lengend Snippet: Forcing glycolysis-reliant AML cells to utilize OXPHOS renders them susceptibility to IACS-010759 and venetoclax induced intrinsic apoptosis mediated by cytochrome c release. ( a , b ) THP-1 cells, cultured in media supplemented with glucose, were treated with vehicle control, IACS-010759, venetoclax, or in combination for 24 h. Cellular fractionation was performed. Mitochondrial and cytosolic fractions were subjected to Western blot analysis. This experiment was performed 2 independent times in triplicate. One representative image is shown. Relative densitometry measurements were determined using Odyssey Software V3.0, normalized to β-actin or VDAC1, and compared to the vehicle control. Results from one representative experiment are graphed as mean ± SEM in panel b. * indicates p < 0.05, ** indicates p < 0.01, and *** indicates p < 0.001 compared to vehicle control. ( c ) THP-1 cells were cultured and treated as described in panel a. Whole cell lysates were subjected to Western blot analysis. Relative densitometry measurements were determined using Odyssey Software V3.0, normalized to β-actin, and compared to the vehicle control. ( d , e ) Lentiviral shRNA double knockdown of Bak and Bax (designated Bak/Bax KD) was performed in THP-1 cells. Non-template control (NTC)-shRNA was used as the control for the Bak/Bax double knockdown. Western blots probed with anti-Bak, -Bax, or -β-actin antibody are shown in panel d. shRNA knockdown cells cultured in media supplemented with glucose were treated with IACS-010759 and venetoclax, alone or combined, for 24 h. Cells were then stained with Annexin V-FITC/PI and analyzed by flow cytometry. *** indicates p < 0.001 compared to the NTC-shRNA cells. ( f ) THP-1 cells cultured in media supplemented with glucose were treated with combined IACS-010759 and venetoclax in the absence or presence of the pan-caspase inhibitor Z-VAD-FMK for 24 h. Cells were then stained with Annexin V-FITC/PI and analyzed by flow cytometry. *** indicates p < 0.001 compared to the combined venetoclax and IACS-010759 treatment. ( g – i ) THP-1 cells cultured in media supplemented with galactose were treated with vehicle control, IACS-010759, venetoclax, or in combination for 8 h. In panel g, cells were then stained with Annexin V-FITC/PI and analyzed by flow cytometry. *** indicates p < 0.001 compared to the vehicle control and single drug treatments. For panels ( h , i ), cellular fractionation was performed. Mitochondrial and cytosolic fractions were subjected to Western blot analysis. This experiment was performed 2 independent times in triplicate. One representative image is shown. Relative densitometry measurements were determined using Odyssey Software V3.0, normalized to β-actin or VDAC1, and compared to the vehicle control. Results from one representative experiment are graphed as mean ± SEM in panel i . * indicates p < 0.05, ** indicates p < 0.01, and *** indicates p < 0.001 compared to vehicle control, ### indicates p < 0.001. ( j ) THP-1 cells were cultured and treated as described in panel ( g ). Whole cell lysates were subjected to Western blot analysis. Relative densitometry measurements were determined using Odyssey Software V3.0, normalized to β-actin, and compared to the vehicle control. ( k ) THP-1 NTC and Bak/Bax double knockdown cells cultured in media supplemented with galactose were treated with IACS-010759 and venetoclax, alone or combined, for 24 h. Cells were then stained with Annexin V-FITC/PI and analyzed by flow cytometry. *** indicates p < 0.001 compared to the NTC-shRNA cells.

Article Snippet: Non-template negative control (NTC)-, Bax-, and Bak-shRNA lentiviral constructs were purchased from Sigma-Aldrich (St. Louis, MO, USA).

Techniques: Cell Culture, Cell Fractionation, Western Blot, Software, shRNA, Staining, Flow Cytometry