Schematic representation of the DR-GFP reporter plasmid. by the addition of cycloheximide (CHX) at 50 g/ml for the indicated times. Total protein lysates were subjected to immunoblot analysis using anti-CCDC6 or anti-PCNA antibodies. Densitometric analyses have been performed by Image J Software. The histograms represent the relative protein levels of CCDC6 against PCNA and expressed as relative intensity compared to untreated. Error bars indicate the measurement of the standard error mean. Statistical significance was verified by 2-tailed Student’s t-test (* 0.05; ** 0.01 and *** 0.001). (G, H) Immunoblot analysis of USP7, PARG, PARP1, CCDC6 and pan-ADP-Ribose in human Kuramochi, OVCAR3 and OV-90 ovarian cancer cell lines. Anti-Tubulin is shown as loading control. The different CCDC6 protein mobility on SDS-PAGE could be ascribed to cell cycle-dependent CCDC6 post-translational modifications (PTMs), as reported [30]. 13046_2022_2459_MOESM5_ESM.jpg (2.1M) GUID:?66E62D10-9CA7-4805-9340-3981D9567557 Additional file 6: Figure S3. In CCDC6-silenced Kuramochi cells (ShCCDC6), the H2AX Minnelide foci formation was rescued by CCDC6 exogenous expression upon Myc CCDC6 transient transfection (Myc CCDC6) vs empty vector (EV) as control. (A) Immunofluorescence images showing H2AX nuclear foci Minnelide formation in CCDC6-silenced Kuramochi cells, treated with Olaparib [1M] or PARGi [1M] for 48 hours and transfected with control (EV) or Myc CCDC6 expression vector. Scale bar 50m. (B) Graphs represent the percentage Minnelide of cells with more than 15 foci. Error bars indicate the standard error mean derived from three independent GPC4 experiments. Statistical significance was verified by 2-tailed Student’s t-test (* 0.05; ** 0.01 and *** 0.001). (C) The efficacy of CCDC6 silencing and the expression of Myc CCDC6 were assessed at Western Blot by the anti-CCDC6 and anti-Myc antibodies. Anti-Tubulin immunoblots are served as a loading control. 13046_2022_2459_MOESM6_ESM.jpg (584K) GUID:?236CAB6C-DB72-4BB7-A3B1-9C1BF5096F78 Additional file 7: Figure S4. In CCDC6-silenced OVCAR3 cells (ShCCDC6), the H2AX foci formation was rescued by CCDC6 exogenous expression upon Myc CCDC6 transient transfection (Myc CCDC6) vs empty vector (EV) as control. (A) Immunofluorescence images showing H2AX nuclear foci formation in CCDC6-silenced OVCAR3 cells, treated with olaparib [1M] or PARGi [1M] for 48 hours and transfected with control (EV) or Myc CCDC6 expression vector. Scale bar 50m. (B) Graphs represent the percentage of cells with more than 15 foci. Error bars indicate the standard error mean derived Minnelide from three independent experiments. Statistical significance was verified by 2-tailed Student’s t-test Minnelide (* 0.05; ** 0.01 and *** 0.001). (C) The efficacy of CCDC6 silencing and the expression of Myc CCDC6 were assessed at Western Blot by the anti-CCDC6 and anti-Myc antibodies. Anti-Tubulin immunoblots are served as a loading control. 13046_2022_2459_MOESM7_ESM.jpg (642K) GUID:?B1776EB0-A05F-4E27-B7F4-60D747045CEF Additional file 8: Figure S5. In CCDC6-silenced OV-90 cells (ShCCDC6), the H2AX foci formation was rescued by CCDC6 exogenous expression upon Myc CCDC6 transient transfection (Myc CCDC6) vs empty vector (EV) as control. (A) Immunofluorescence images showing H2AX nuclear foci formation in CCDC6-silenced OV-90 cells, treated with olaparib [1M] or PARGi [1M] for 48 hours and transfected with control (EV) or Myc CCDC6 expression vector. Scale bar 50m. (B) Graphs represent the percentage of cells with more than 15 foci. Error bars indicate the standard error mean derived from three independent experiments. Statistical significance was verified by 2-tailed Student’s t-test (* 0.05; ** 0.01 and *** 0.001). (C) The efficacy of CCDC6 silencing and the expression of myc-CCDC6 were assessed at Western Blot by the anti-CCDC6 and anti-myc antibodies. Anti-tubulin immunoblots are served as a loading control. 13046_2022_2459_MOESM8_ESM.jpg (735K) GUID:?1E385FE2-FC8A-4E85-B202-6527B67E2B86 Additional file 9: Figure S6. CCDC6 genetic depletion by short hairpin RNA (ShCCDC6) improved Olaparib sensitivity in HGSOC cells. (A, D, G) Kuramochi, OVCAR3 and OV-90 cells, transfected with ShCCDC6, or ShCTRL were treated with olaparib or PARGi at different doses for 144 hours: 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 expressed as 50% inhibitory concentration (IC50) values. (B, E, H) In P5091-treated CCDC6-depleted cells, the sensitive phenotypes were rescued by CCDC6 exogenous expression upon Myc CCDC6 transient transfection (CCDC6+) vs empty vector (EV) as control. The drugs sensitivity was determined as in A, D, G. (C,.