OR-2100 downregulates mitosis-related molecules via an E2F-mediated pathway, leading to mitotic perturbation and chromosomal aberration. (A) Schematic image of the cell cycle and multipolar spindles. (B) Representative images of OCI-Ly18 cells (left, shCtr; middle, shCDCA8; right, OR-2100-treated). Immunofluorescence images were captured at the original magnification ×100; α-tubulin (green) and DAPI (blue). (C) The quantification of aberrant spindles was conducted using IHC; the percentage of aberrant spindles were significantly increased among TP53 wt cells. (D) Flow cytometry analysis was performed to detect the percentage of cells with a >4N karyotype. The percentage of abnormal OCI-Ly18 cells that harbored a >4N karyotype was significantly increased by OR-2100 treatment when compared with AZA treatment, whereas that of SU-DHL-6 cells was increased modestly (gray, Ctr; red, AZA; purple, OR-2100). (E) The expression of β-tubulin, borealin (CDCA8), and survivin (BIRC5) in OCI-Ly18 cells and SU-DHL-6 cells treated with HLM006474 (an E2F inhibitor), as measured by western blotting. The expression of borealin and survivin protein decreased in OCI-Ly18 cells but not in SU-DHL-6 cells. (F) The cell viability assays conducted for RC cells and OCI-Ly18 cells treated with HLM006474. HLM006474 inhibited the growth of RC cells and OCI-Ly18 cells. (G) The phosphorylation of Rb (S807/811), E2F1, β-actin, borealin (CDCA8), and survivin (BIRC5) in RC cells and OCI-Ly18 cells treated with HMAs (AZA, DAC, and OR-2100) was measured by western blotting. Representative images show that the expression of phosphorylated Rb (S807/811), E2F1, borealin, and survivin decreased in the presence of DAC and OR-2100 to a greater extent than in the presence of AZA. (H) Chromosomal analysis of the OCI-Ly18 cells. (gray, Ctr; purple, OR-2100 treated). Cell and chromosome numbers are shown with the images of G-banding analysis (left, Ctr; right, OR-2100 treated). E2Fi, E2F1 inhibitor; Mut, mutation; ns, not significant; Phospho-Rb, phosphorylated retinablastoma protein; wt, wild type. Panel A created with BioRender.com.

OR-2100 downregulates mitosis-related molecules via an E2F-mediated pathway, leading to mitotic perturbation and chromosomal aberration. (A) Schematic image of the cell cycle and multipolar spindles. (B) Representative images of OCI-Ly18 cells (left, shCtr; middle, shCDCA8; right, OR-2100-treated). Immunofluorescence images were captured at the original magnification ×100; α-tubulin (green) and DAPI (blue). (C) The quantification of aberrant spindles was conducted using IHC; the percentage of aberrant spindles were significantly increased among TP53 wt cells. (D) Flow cytometry analysis was performed to detect the percentage of cells with a >4N karyotype. The percentage of abnormal OCI-Ly18 cells that harbored a >4N karyotype was significantly increased by OR-2100 treatment when compared with AZA treatment, whereas that of SU-DHL-6 cells was increased modestly (gray, Ctr; red, AZA; purple, OR-2100). (E) The expression of β-tubulin, borealin (CDCA8), and survivin (BIRC5) in OCI-Ly18 cells and SU-DHL-6 cells treated with HLM006474 (an E2F inhibitor), as measured by western blotting. The expression of borealin and survivin protein decreased in OCI-Ly18 cells but not in SU-DHL-6 cells. (F) The cell viability assays conducted for RC cells and OCI-Ly18 cells treated with HLM006474. HLM006474 inhibited the growth of RC cells and OCI-Ly18 cells. (G) The phosphorylation of Rb (S807/811), E2F1, β-actin, borealin (CDCA8), and survivin (BIRC5) in RC cells and OCI-Ly18 cells treated with HMAs (AZA, DAC, and OR-2100) was measured by western blotting. Representative images show that the expression of phosphorylated Rb (S807/811), E2F1, borealin, and survivin decreased in the presence of DAC and OR-2100 to a greater extent than in the presence of AZA. (H) Chromosomal analysis of the OCI-Ly18 cells. (gray, Ctr; purple, OR-2100 treated). Cell and chromosome numbers are shown with the images of G-banding analysis (left, Ctr; right, OR-2100 treated). E2Fi, E2F1 inhibitor; Mut, mutation; ns, not significant; Phospho-Rb, phosphorylated retinablastoma protein; wt, wild type. Panel A created with BioRender.com.

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