Figure 4.
Underlying mechanisms of action of G9a/GLP targeting in MM. (A-B) Effect of G9a/GLP targeting on H3K9me1-3 levels. (A) Western blot analysis of H3K9me1, H3K9me2, and H3K9me3 levels in OPM-2 cells (left panel) and XG-7 cells (right panel) after 96 hours of treatment with or without BIX01294 (BIX; 2 µM). (B) XG-7 cells transduced with 3 shG9a constructs were cultured or not with doxycycline (Dox; 1 mg/mL) for 7 days, after which H3K9me2 and H3K9me3 levels were analyzed by western blot. Histone 3 was used as loading control; 1 experiment representative of 3 experiments is shown. Quantification and normalization were performed with ImageJ, and quantification relative to the control condition is shown. (C) Effect of G9a/GLP targeting on autophagy. Immunofluorescence staining for DAPI, LC3B, and α-tubulin on OPM-2 and XG-20 cells, treated or not with 5 µM BIX01294 or UNC0638 for 24 hours. Scale bars, 10 µm. Photographs shown are representative of 1 experiment; quantification data are based on 3 independent experiments. ****P < .001. (D-E) Western blot analysis of involved signaling pathways and downstream targets. OPM-2 cells (D) and XG-7 cells (E) were treated with 1.25 µM, 2.5 µM, or 5 µM BIX01294 for 24 hours, after which whole-cell lysates were analyzed for the indicated proteins. Tubulin was used as loading control; 1 experiment representative of 3 experiments is shown. (F) mRNA expression levels of MYC and eIF4E in OPM-2 cells (upper panel) and XG-7 cells (lower panel) treated or not with 5 µM BIX01294 (BIX) for 24 hours. Data are mean ± standard deviation (SD) of 3 independent experiments. *P < .05. (G) Effect of the autophagy inhibitor 3-ME on BIX01294 (BIX)-induced cell death. OPM-2 cells were treated for 4 hours with 3-ME (1 and 2 mM), after which BIX (2.5 µM) was added for an additional 48 hours. The effect on apoptosis was assessed using Annexin V-APC/7-aminoactinomycin D (7-AAD), followed by flow cytometric analysis. The percentage of apoptotic cells is the sum of the percentage of Annexin V+ and Annexin V+/7-AAD + cells. Data are mean ± SD of 3 independent experiments. *P < .05 vs Bix alone.

Underlying mechanisms of action of G9a/GLP targeting in MM. (A-B) Effect of G9a/GLP targeting on H3K9me1-3 levels. (A) Western blot analysis of H3K9me1, H3K9me2, and H3K9me3 levels in OPM-2 cells (left panel) and XG-7 cells (right panel) after 96 hours of treatment with or without BIX01294 (BIX; 2 µM). (B) XG-7 cells transduced with 3 shG9a constructs were cultured or not with doxycycline (Dox; 1 mg/mL) for 7 days, after which H3K9me2 and H3K9me3 levels were analyzed by western blot. Histone 3 was used as loading control; 1 experiment representative of 3 experiments is shown. Quantification and normalization were performed with ImageJ, and quantification relative to the control condition is shown. (C) Effect of G9a/GLP targeting on autophagy. Immunofluorescence staining for DAPI, LC3B, and α-tubulin on OPM-2 and XG-20 cells, treated or not with 5 µM BIX01294 or UNC0638 for 24 hours. Scale bars, 10 µm. Photographs shown are representative of 1 experiment; quantification data are based on 3 independent experiments. ****P < .001. (D-E) Western blot analysis of involved signaling pathways and downstream targets. OPM-2 cells (D) and XG-7 cells (E) were treated with 1.25 µM, 2.5 µM, or 5 µM BIX01294 for 24 hours, after which whole-cell lysates were analyzed for the indicated proteins. Tubulin was used as loading control; 1 experiment representative of 3 experiments is shown. (F) mRNA expression levels of MYC and eIF4E in OPM-2 cells (upper panel) and XG-7 cells (lower panel) treated or not with 5 µM BIX01294 (BIX) for 24 hours. Data are mean ± standard deviation (SD) of 3 independent experiments. *P < .05. (G) Effect of the autophagy inhibitor 3-ME on BIX01294 (BIX)-induced cell death. OPM-2 cells were treated for 4 hours with 3-ME (1 and 2 mM), after which BIX (2.5 µM) was added for an additional 48 hours. The effect on apoptosis was assessed using Annexin V-APC/7-aminoactinomycin D (7-AAD), followed by flow cytometric analysis. The percentage of apoptotic cells is the sum of the percentage of Annexin V+ and Annexin V+/7-AAD + cells. Data are mean ± SD of 3 independent experiments. *P < .05 vs Bix alone.

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