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Fig. 6 | Journal of Experimental & Clinical Cancer Research

Fig. 6

From: The disruption of the CCDC6 – PP4 axis induces a BRCAness like phenotype and sensitivity to PARP inhibitors in high-grade serous ovarian carcinoma

Fig. 6

The CCDC6 threonine 427 and the regulatory subunit PP4R3α turned up critical for the CCDC6-dependent PARP inhibitors sensitivity. A, B In OVCAR3 cells depleted for CCDC6, upon treatment with P5091 [2.5 μM] or transfection with short hairpin RNA for CCDC6 (ShCCDC6), the Olaparib sensitivity has been evaluated following transient expression of CCDC6 wild type (Myc CCDC6), CCDC6 mutants (Myc CCDC6T427A, Myc CCDC6T427D) or empty vector (EV), at the indicated doses. Scrambled shRNA (ShCTRL) or vehicle were used as control. C OVCAR3 cells stably silenced for CCDC6 (ShCCDC6), and control cells (ShCTRL), were transfected with CCDC6 wild type, CCDC6 mutants or empty vector as in (A, B) and treated with olaparib [1 μM]. Equal amounts of cell lysates were immunoblotted with anti-γH2AX and H2AX antibodies. Anti-Myc and anti-Tubulin antibodies were employed as transfection and loading controls, respectively. D OVCAR3 cells, stably depleted for CCDC6 (ShCCDC6) or control (ShCTRL) were transfected with siRNA for PP4R3α and the olaparib sensitivity was assessed upon overexpression of CCDC6 wild type, CCDC6 mutants or empty vector, as in (A, B). E The efficacy of PP4R3α silencing or of Myc CCDC6 overexpression were assessed at Western Blot by the anti-PP4R3α or anti-Myc antibodies. Anti-Tubulin immunoblots are shown as loading control. In (A, B and D) the drugs sensitivity was determined by a modified 3-(4,5-dimethylthiazole-2- yl)-2–5-diphenyltetrazolium bromide assay, CellTiter 96 Aqueous One Solution assay (Promega), and was expressed as 50% inhibitory concentration (IC50) values. On the right side the surviving fractions of the OVCAR3 cells, as in (A, B and D), treated for 144 h with different doses of Olaparib, are shown

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