CBX7 inhibitors block proliferation, induce terminal differentiation, and promote apoptosis of OCI-AML3 cells. Phenotype of OCI-AML3 cells after treatment with MS452, EC-134, or BDA-41 for 4 days. (A-B) Cell cycle analysis. (A) Representative flow plots of Ki67-AF488 and DAPI (4′,6-diamidino-2-phenylindole) signal indicating the cell cycle phases G0, G1, S, G2, and M, and fragmented DNA. (B) Quantification of the percentage of the unfragmented cells in G0 + G1 and S + G2 + M phase of ≥2 replicates with SEM as error bar. Student t test was used to calculate the P value between DMSO and treatment, ∗P < .05. (C-D) Proliferation rate. (C) Representative histograms of the MFI of the CellTrace Violet signal in cells at the start of treatment (black), and after 4 days without treatment (gray), or MS452 (red), EC-134 (purple), or BDA-41 (blue) treatment. (D) Quantification of the proliferation rate was calculated by dividing the CellTrace Violet MFI at day 4 from the MFI at day 0. Lines go through the means of the individual data points of ≥2 replicates. (E-I) Differentiation potential. (E-F) Representative histograms of the MFI of (E) CD11b-BV421 or (F) CD14-AF700 obtained with flow cytometry in untreated DMSO-treated control cells (black), and MS452- (red), EC-134– (purple) or BDA-41–treated (blue) cells. (G-H) Normalized expression of (G) CD11b and (H) CD14 calculated by normalizing the MFI of the treated samples to the MFI of their experimental DMSO control. Data points are plotted as the mean of ≥3 replicates with SEM as error bar. (I) May-Grünwald Giemsa staining of untreated DMSO control cells or cells treated with CBX7 inhibitors. (J-K) Apoptosis induction. (J) Representative flow plots of 7-aminoactinomycin D (7-AAD) and annexin-V–BV421 signal indicating the gates for live, apoptotic, or dead cell populations. (K) Quantification of the percentage of cells in each gate. Bars represent the mean of n = 8 for DMSO, n = 3 for MS452, n = 2 for EC-134, and n = 3 for BDA-41 with SEM as error bar. Student t test was used to calculate the P value between treatment and their experimental DMSO control, ∗P < .05, with green stars indicating differences in live cells, orange stars indicating differences apoptotic cells, and red stars indicating differences dead cells.

CBX7 inhibitors block proliferation, induce terminal differentiation, and promote apoptosis of OCI-AML3 cells. Phenotype of OCI-AML3 cells after treatment with MS452, EC-134, or BDA-41 for 4 days. (A-B) Cell cycle analysis. (A) Representative flow plots of Ki67-AF488 and DAPI (4′,6-diamidino-2-phenylindole) signal indicating the cell cycle phases G0, G1, S, G2, and M, and fragmented DNA. (B) Quantification of the percentage of the unfragmented cells in G0 + G1 and S + G2 + M phase of ≥2 replicates with SEM as error bar. Student t test was used to calculate the P value between DMSO and treatment, ∗P < .05. (C-D) Proliferation rate. (C) Representative histograms of the MFI of the CellTrace Violet signal in cells at the start of treatment (black), and after 4 days without treatment (gray), or MS452 (red), EC-134 (purple), or BDA-41 (blue) treatment. (D) Quantification of the proliferation rate was calculated by dividing the CellTrace Violet MFI at day 4 from the MFI at day 0. Lines go through the means of the individual data points of ≥2 replicates. (E-I) Differentiation potential. (E-F) Representative histograms of the MFI of (E) CD11b-BV421 or (F) CD14-AF700 obtained with flow cytometry in untreated DMSO-treated control cells (black), and MS452- (red), EC-134– (purple) or BDA-41–treated (blue) cells. (G-H) Normalized expression of (G) CD11b and (H) CD14 calculated by normalizing the MFI of the treated samples to the MFI of their experimental DMSO control. Data points are plotted as the mean of ≥3 replicates with SEM as error bar. (I) May-Grünwald Giemsa staining of untreated DMSO control cells or cells treated with CBX7 inhibitors. (J-K) Apoptosis induction. (J) Representative flow plots of 7-aminoactinomycin D (7-AAD) and annexin-V–BV421 signal indicating the gates for live, apoptotic, or dead cell populations. (K) Quantification of the percentage of cells in each gate. Bars represent the mean of n = 8 for DMSO, n = 3 for MS452, n = 2 for EC-134, and n = 3 for BDA-41 with SEM as error bar. Student t test was used to calculate the P value between treatment and their experimental DMSO control, ∗P < .05, with green stars indicating differences in live cells, orange stars indicating differences apoptotic cells, and red stars indicating differences dead cells.

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