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. Author manuscript; available in PMC: 2019 Aug 1.
Published in final edited form as: Mol Cancer Res. 2018 May 8;16(8):1241–1254. doi: 10.1158/1541-7786.MCR-17-0581

Figure 1. PTEN loss suppresses NHEJ and PTEN complementation rescues the NHEJ defect in PTEN-null melanomas.

Figure 1

(A) Schematic representation of the NHEJ luciferase based reporter assay. (B) Relative NHEJ efficiency of short-term patient derived melanoma cultures. (C) Western blot analysis of PTEN expression in YUGASP, YUSAC2 and 501MEL cells 96 h after transfection of siRNA targeting PTEN. (D) Relative NHEJ efficiency of short-term patient derived melanoma culture YUGASP after siRNA suppression of PTEN, XRCC4, XLF, XRCC5, BRCA2, and RAD51. (E) Relative NHEJ efficiency of YUSAC2 and 501 melanoma cell lines after PTEN suppression by siRNA compared to scrambled control siRNA. (F) EJ5 end-joining reporter assay in U2OS cells performed after PTEN suppression by siRNA compared to scrambled control siRNA. (G) Neutral comet assay performed 6 h after IR in YUGASP, YUSAC2 and 501MEL cells 96 h transfection with siPTEN and 24 h after exposure to 10 μM DNA-PKi NU7441 or DMSO control. (H) Radiation survival with or without PTEN knockdown in YUGASP, YUSAC2 and 501MEL melanoma cells using siPTEN compared to siSCR. (I) Western blot analysis of PTEN expression in YUGEN8 and YUROL cultures complemented with either pBABE-PURO PTEN or pBABE-PURO empty vector. Vinculin is used as a loading control. (J) NHEJ reporter assay performed in YUGEN8 and YUROL with and without PTEN complementation. Reporter activity is normalized to empty vector control. (K) Quantification of γH2AX foci per nucleus 0 through 24 h post 5 Gy IR in YUGEN8 cultures complemented with either pBABE-PURO PTEN or pBABE-PURO empty vector. (L) Quantification of neutral comet assay performed in 6 h after 5 Gy IR in YUGEN8 cells complemented with either pBABE-PURO PTEN or pBABE-PURO empty vector, 96 h after knockdown of indicated factors with siRNA or 24 h after 10 μM DNA-PKi, NU7441. (M) Radiation survival, quantified by colony formation, of clonal PTEN complemented cell lines compared to empty vector control for YUGEN8 and YUROL. For all panels, error bars represent SEM (n=3) and statistical analysis by t-test.