Skip to main content
. 2023 Aug 21;5(1):vdad102. doi: 10.1093/noajnl/vdad102

Figure 4.

Figure 4.

Synergistic LMP400/Niraparib combination leads to cell growth arrest and apoptosis in PTEN-deficient cells through enhanced DNA damage and attenuated DNA repair. (A) Western blot demonstrating the PTEN expression in TR, but not TRP cells. Increased p-AKT (S473) was observed upon PTEN deletion in TRP cells. (B) The dose–response curves of TR and TRP cells treated with increasing concentrations of LMP400, Niraparib, or both for 72 hours. (C) The dose–response curves of TR and TRP cells treated with increasing concentrations of LMP400 with or without addition of 1 μM Niraparib. (D) Colony formation assay in TR and TRP cells treated with 15 nM LMP400 or 0.5 μM Niraparib or both for 72 hours before releasing to drug-free media. (E) Synergism plots in TR and TRP cells generated by Compusyn software. (F) Cell cycle analysis performed by flow cytometry in TR and TRP cells after treatment with 50 nM LMP400 or 2 μM Niraparib or both for 48 hours. (G) Western blot of cell cycle-related proteins in TR and TRP cells treated with 30 nM LMP400 or 1 μM Niraparib or both for 72 hours. (H) Immunocytochemical staining of γ-H2AX in TR and TRP cells treated with 50 nM LMP400 or 2 μM Niraparib or both for 48 hours. (I) Single-cell electrophoresis (comet) assay performed in TR and TRP cells treated with 30 nM LMP400 or 1 μM Niraparib or both for 72 hours. Tail moment was measured in at least 50 cells per each experimental group. (J) Western blot analysis of DNA damage and repair proteins in TR and TRP cells treated with 30 nM LMP400 or 1 μM Niraparib or both for 72 hours. (K) Caspase 3/7 activity assay in TR and TRP cells treated with 30 nM LMP400 or 1 μM Niraparib or both for 72 hours. The data is expressed as mean ±SEM normalized to the average value of the control group. (L) Flow cytometry analysis of TR and TRP cells by double staining with Annexin V and PI after treatment with 30 nM LMP400 or 1 μM Niraparib or both for 72 hours. Apoptosis fraction includes both early (Annexin V+, PI−) and late apoptosis (Annexin V+, PI+). Necrosis was measured as Annexin V−, PI+.