Supplementary Fig. 1. (A) Working model. The pluripotency transcription factor OCT4

Transcription

1 SUPPLEMENTARY FIGURE LEGENDS Supplementary Fig. 1. (A) Working model. The pluripotency transcription factor OCT4 directly up-regulates the expression of NIPP1 and CCNF that together inhibit protein phosphatase PP1 activity resulting in prb hyper-phosphorylation. (B) Left panel, real-time PCR analysis of CCNF and NIPP1 expression of OVCAR-3 cells transiently transfected with two different sirnas targeting OCT4. Control sirna values were set 100. mrna levels were normalized against ACTIN. Right panel, western blotting analysis of experimental cells using phospho prb-t356, NIPP1 and OCT4 antibodies. ACTIN was used as a loading control. (C) Top panel (left), two independent western blotting analysis of NIPP1 in OVCAR- 3 cells transfected with the indicated sirnas oligos. ACTIN was used as loading control. Numbers represent NIPP1/ACTIN ratio. Top panel (right), protein quantification of three western blotting experiments using ImageJ software. Bottom panel (left), immunofluorescence staining of NIPP1 and OCT4 protein expression in OVCAR-3 cells transfected with two different sirnas targeting OCT4 or CCNF/NIPP1. Scale bar, 10 μm. Bottom panel (right), western blotting analysis of nuclear and total lysates of OVCAR-3 cells transfected with sirnas targeting OCT4 or CCNF/NIPP1 using the indicated antibodies. HDAC3 was used as loading control. Numbers represent NIPP1/HDAC3 ratios. (D) phosphor prb-t356, NIPP1 and PP1 protein levels in SKOV-3 transiently transfected with the indicated sirnas oligos, as determined by western blotting. OCT4 is not detectable in western blotting due to reduced antibody efficiency. ACTIN was used as a loading control. (E) Immunofluorescence analysis of OCT4 expression in OVCAR-3 cell line 3 days after transfection of sirna targeting OCT4. (F) Immunofluorescence of OCT4 in OVCAR-3 subclones. (G) Comparison of OCT4 protein levels in OVCAR-3 subclones 11, 4 and 9 and mouse embryonic stem cells (mescs) using western blotting analysis. ACTIN was used as

2 loading control. (H) TGCA data set analysis. RB1 mrna expression correlates with high phosphorylation at the PP1 target sites prb (S807/T356). n, number of independent experiments carried out; error bars indicate s.d.; a one-tail Student s t test and Linear model regression test (panel H) were used to calculate significance values: *, p < 0.05; **, p < 0.01; ***, p < Supplementary Fig. 2. (A) Growth curve of OVCAR-3 cells transfected with the indicated sirnas and counted at the indicated time points (middle panel). Left panel, Representative images of experimental cells. Right panel, quantitative real-time PCR analysis of PCNA and MKI67 (Ki-67) in OVCAR-3 cells transiently transfected with sirnas targeting OCT4 or CCNF/NIPP1. Control sirna values were set 100. mrna levels were normalized against ACTIN. (B) Left panel, growth curve of SKOV-3 cells transfected with the indicated sirnas and counted at the indicate time points. Right panel, percentage of BrdU-positive cells transfected with the indicated sirnas oligos. Control sirna value was set 1. (C) Western blotting analysis of OVCAR-3 cells treated with PD for phospho prb-t356, OCT4 and NIPP1. ACTIN was used as a loading control. (D) Left panel, quantitative real-time PCR analysis of ABCB1 and ABCB10 mrnas in OVCAR-3 subclone 9 transiently transfected with the indicated sirnas oligos. Control sirna values were set 100. mrna levels were normalized against ACTIN. Right panel, chemoresistance assay of OVCAR-3 subclones 11, 4 and 9 treated with Cisplatin/Taxol, Doxorubicin, Nocodazole or Actinomycin D for 16 hours as explained in the time diagram (top panel), using the indicated drug concentrations. (E) Quantification of colony number of OVCAR-3 subclones 11, 4 and 9 transiently transfected with the indicated sirnas oligos and plated in soft-agar. (F) Soft-agar assay of OVCAR-3 cells transiently transfected with the indicated sirnas or treated with PD Left panel, representative images. Middle panel, quantification of colony number of experimental cells. Right panel, quantification of colony size of experimental cells. (G) Analysis of soft-agar

3 assay of OVCAR-3 subclone 11 transiently overexpressing HA-OCT4 or both HA-CCNF and HA-NIPP1. Left panel, anti-ha and anti-oct4 western blotting showing expression of CCNF, OCT4 and NIPP1-tagged protein in experimental cells. ACTIN was used as a loading control. Right panel, soft-agar assay quantification of colony number of experimental cells. (H) Top panel, representative images of spheroids derived from OVCAR-3 subclone 11 stably overexpressing HA-OCT4 and OVCAR-3 subclones 11, 4 and 9. Bottom panel, western blotting for OCT4 of OVCAR-3 cells stably overexpressing HA-OCT4, and subclones 11, 4 and 9. ACTIN was used as loading control. (I) Left panel, representative images of spheroids derived from OVCAR-3 cells transiently transfected with the indicated sirnas. Right panel, western blotting analysis of OCT4 and NIPP1 in OVCAR-3 cells used for spheroid formation assay after transient knock-down of OCT4 and CCNF/NIPP1. ACTIN was used as a loading control. UT, Untreated. n, number of independent experiments carried out; error bars indicate s.d.; a one-tail Student s t test was used to calculate significance values: *, p < 0.05; **, p < 0.01; ***, p < Supplementary Fig. 3. (A) Representative images of OVCAR-3 cell morphology after transfection with two different sirna oligos targeting OCT4. Black arrows indicate cells with altered morphology. (B) Left panel, protein levels of cyclin B1 (CCNB1), CDK1, p16 INK4A and OCT4 of OVCAR-3 cells in knock-down experiments using sirnas targeting different OCT4 mrna sequences (see Material and Methods), as determined by western blotting. ACTIN was used as a loading control. Middle panel, representative images of immunofluorescence staining for p16 INK4A and OCT4. Scale bar, 10 μm. Right panel, percentage of p16 INK4A under the described experimental conditions. (C) Detection of senescence markers by western blotting analysis of subclones 11, 4 and 9 using the indicated antibodies. (D) Left panel, western blotting analysis of p16 INK4A,YFP and OCT4 in OVCAR- 3 subclone 9 transiently transfected with the indicated sirnas oligos. ACTIN was used as

4 loading control. Middle panel, representative images of immunofluorescence staining for p16 INK4A and YFP-OCT4 protein levels. Scale bar, 10 μm. Right panel, immunofluorescence quantification of p16 INK4A positive cells in experimental cells. (E) Percentage of BrdUpositive SKOV-3 cells after transfection with the indicated sirna oligos. Control sirna value was set 1. (F) Left panel, western blotting analysis of SKOV-3 cells, transfected with the indicated sirnas, for the expression of cyclin B1 (CCNB1), CDK1 and NIPP1. We were not able to detect p16 INK4A protein levels due to a homozygous deletion in CDKN2A gene in SKOV-3 cells 1. ACTIN was used as a loading control. Right panel, protein expression of PARP, cleaved CASPASE 3 and NIPP1 in SKOV-3 cells transfected with the indicated sirnas. ACTIN was used as a loading control. (G) Western blotting analysis of PARP, cleaved CASPASE 3 and p16 INK4A after a 3 or 6 days knock-down of indicated genes in OVCAR-3 subclone 9. ACTIN was used as loading control. n, number of independent experiments carried out; error bars indicate s.d.; one-tail Student s t test was used to calculate significance values: *, p < 0.05; **, p < 0.01; ***, p < Scale bar, 100 µm. Supplementary Fig. 4. (A) Percentage of SKOV-3 cells with micronuclei after transient transfected with the indicated sirnas (200 cells were analysed). (B) Quantification of type I, II and III errors of OVCAR-3 subclones 11, 4 and 9 (150 mitotic cells were analysed). (C) Representative immunofluorescence images for α-tubulin of polynucleated (indicated by white arrows) OVCAR-3 subclone 9 transiently transfected with the indicated sirnas (left panel) and OVCAR-3 subclones 11, 4 and 9 (right panel). (D) Percentage of polynucleated SKOV-3 cells after knock-down of OCT4 or CCNF/NIPP1 (200 cells were analysed). (E) Quantitative real-time PCR mrna analysis of OVCAR-3 after RNAi mediated knock-down of OCT4 or CCNF/NIPP1. Left panel, real-time PCR quantification of CPC components of experimental cells. Right panel, quantitative real-time PCR validation of gene expression profiling data of OCT4 and NIPP1/CCNF co-regulated genes. Control sirna values were set

5 100. mrna levels were normalized against ACTIN. n, number of independent experiments carried out; error bars indicate s.d.; one-tail Student s t test was used to calculate significance values: *, p < 0.05; **, p < 0.01; ***, p < Scale bars, 10 µm. Supplementary Fig. 5. (A) Western blotting of SKOV-3 cells transiently transfected with the indicated sirnas. Expression of AURKB, NIPP1 and phospho H3-Ser10 was measured. ACTIN and H3 were used as loading control. (B) Protein and mrna analysis of OVCAR-3 cells transfected with two different sirnas oligos targeting OCT4. Left panel, western blotting of Aurora B, phospho H3-Ser10 and OCT4 of experimental cells. ACTIN and H3 was used as loading control. Right panel, real-time PCR for AURKB mrna levels of OVCAR-3 cells transfected with the indicated sirnas oligos. Control sirna values were set 100. mrna levels were normalized against ACTIN. (C) Western blotting analysis of untreated or Okadaic acid (OA, 15 nm) treated OVCAR-3 cells transiently transfected with sirnas targeting OCT4. Numbers represent prb T356 /ACTIN, AURKB/ACTIN, NIPP1/ACTIN, ph3 Ser10 /H3 and ph3 Ser28 /H3 ratios. (D) Representative immunofluorescence images of cells showing different number of centrosomes. (E) Western blotting of OCT4 levels in OVCAR-3 and MDA-MB-231 cells. ACTIN was used as a loading control. (F) Left panel, western blotting analysis of MDA-MB-231 cells stably expressing HA-OCT4 using the indicated antibodies. ACTIN and H3 were used as loading control. Numbers represent prb T356 /ACTIN, AURKB/ACTIN, NIPP1/ACTIN and ph3 Ser10 /H3 ratios. Right panel, quantitative real-time PCR analysis for CCNF, NIPP1 and BOREALIN mrna levels in experimental cells. Control cells values were set 100. (G) Left panel, western blotting of MDA-MB-231 cells transiently transfected with sirnas targeting OCT4 and CCNF/NIPP1 using the indicated antibodies. ACTIN and H3 were used as loading control. Numbers represent prb T356 /ACTIN, AURKB/ACTIN, NIPP1/ACTIN and ph3 Ser10 /H3 ratios. Right panel, quantitative real-time PCR analysis for OCT4, CCNF, NIPP1, AURKB, BIRC5 and

6 BOREALIN mrna levels in experimental cells. Control cells values were set 100. (H) Percentage of cells in different cell cycle phases of MDA-MB-231 cells transiently transfected with the indicated sirna oligos. (I) Top panel, representative images of micronuclei of MDA-MB-231 cells transiently transfected with the indicated vectors. Middle panel, representative images of polynucleated cells. Bottom panel, representative images of centrosomes. YFP empty vector was used as control. (J) OCT4 mrna expression levels of MDA-MB-231 cells stably transfected with the indicated vectors. Control vector values were set 100. OCT4 mrna levels were normalized against ACTIN. n, number of independent experiments carried out; error bars indicate s.d.; one-tail Student s t test was used to calculate significance values: *, p < 0.05; **, p < 0.01; ***, p < Scale bar, 10 µm. Supplementary Fig. 6. (A) Histopathologic classification, outcome and immunohistochemistry quantification (percentage of positive nuclei or cytoplasm and intensity) of 23 HG-SOC patients classified according to OCT4 levels in OCT4 high and OCT4 low as described in Supplementary Methods. (B) Western blotting analysis of OCT4 and Aurora B of the indicated HG-SOC patients. ACTIN was used as a loading control; *, non-specific band. 1 Schultz DC, Vanderveer L, Buetow KH, Boente MP, Ozols RF, Hamilton TC et al. Characterization of chromosome 9 in human ovarian neoplasia identifies frequent genetic imbalance on 9q and rare alterations involving 9p, including CDKN2. Cancer Res 1995; 55: