Fig 2: Western blot analysis showing the expression of BMI1, RING1A, RING1B, EZH2, γ-H2AX, Ub-H2A, p-CHK2, p-ATM, RNF8, RNF168, MEL18, p53BP, BRCA1 proteins in MDAMB-231cells transfected with miR-15a, miR-16 or both miR-15a/16 under etoposide-induced DNA damage conditions. Actin served as a gel loading control. Blots were cropped to enhance the representation (A,B). Immunofluorescence data showing accumulation of BMI1, ϒ-H2AX (C) RING1B (D) and Ub-H2A (E) p53BP (F) in MDAMB-231 cells transfected with miR-15a miR-16 or both miR-15a/16 under etoposide- treated conditions. Bar indicates 200 μm.

Fig 3: The clinically relevant ATR mutation is associated with a dysregulation of the ATR/M activities, endogenous replication stress and DNA damage. (A) Schematic representation of the ATR peptide with its functional domains. The “9A” frameshift mutation induces a stop codon in ATR’s ORF (Stop sign). Hence, this mutant allele is unable to code for the catalytic C-terminal domains of ATR. (B) Wild-type (WT, blue) and mutant (MUT, red) ATR clones were isolated from HCT116 by limit dilution. The poly-A region located in ATR’s exon 10 was sequenced. WT harbors the homozygous wild-type ATR sequence, whereas MUT harbors the clinically significant heterozygous frameshift mutation, leading to a downstream premature stop codon (red box). A revertant “REV” (green) clone has been obtained by genetic engineering of MUT using ZFN nucleases to restore a homozygous wild-type ATR genotype while conserving the genetic background of MUT. (C) Western Blot analysis of the ATR-CHK1 and ATM-CHK2-KAP1 pathways.GAPDH served as a loading control for each individual blot (see [Fig. S5]). A representative GAPDH panel is shown. The quantifications (relative to WT) were calculated from 3 to 5 biological replicates. Mann–Whitney tests were applied. (Full blots: [Fig. S5]) (D) DNA fiber assay performed in ATR mutant cells. IdU and CldU were sequentially added to culture media. DNA was manually spread, and fibers were observed (× 400). Representative images are shown. IdU-positive CldU tracks were measured using ImageJ (exclusion of origin firing and termination events). (n = 600, 200 fibers × 3 independent experiments). Means are displayed on the graphs. Mann–Whitney significance tests were applied. (E) γ-H2AX (red) and 53BP1 (green) immunofluorescent detection. DNA was counterstained with DAPI (blue). Cells were observed with a Zeiss Axio Imager with Apotome (× 63 objective). 53BP1 and γH2AX foci were scored in > 200 cells per condition. The experiment was repeated 3 times (total > 600 cells) and the percentage of cells harboring > 10 γH2AX or > 5 53BP1 foci were plotted on graphs. Mann–Whitney significance tests were applied.

Fig 4: Proteolysis status of CDCP1 and interactions between CDCP1 fragments in PDAC cells. A, Diagram depicting structural features of CDCP1 including three extracellular CUB-like domains (green), proteolytic processing sites at R368 and K369, amino-terminal (ATF) and carboxyl-terminal (CTF) CDCP1 fragments, and binding sites of the anti-CDCP1 antibodies 10D7 (in-house), 4115 (Cell Signaling) and 2666 (R&D systems). B, Western blot analysis under reducing conditions of nine patient-derived PDAC cells (TKCC) and two PDAC cell lines using anti-CDCP1 antibodies 4115 and 2666, and an anti-GAPDH antibody. C, Western blot analysis using anti-CDCP1 antibodies 4115 and 2666, and an anti-GAPDH antibody, of PANC-1, TKCC02, TKCC05 and TKCC10 cell lysates under reduced condition before and after enzymatic deglycosylation with N-glycosidase F (PNGase F) for 1h at 37°C. D, Flow cytometry analysis of PANC-1, TKCC02, TKCC05 and TKCC10 cells for plasma membrane CDCP1 using antibody 10D7. E, Confocal microscopy imaging of HeLa, TKCC05 and PANC-1 cells after immunofluorescent staining of CDCP1 (antibody 10D7) and co-staining of nuclei and membrane with DAPI and wheat germ agglutinin-FITC (WGA), respectively. Scale bar = 20 µm. Average signal co-localization (percent) between 10D7 and WGA are indicated. F, Anti-CDCP1 western blot analysis with the indicated antibodies of proteins immunoprecipitated with antibody 10D7 or isotype matched control antibodies. G, Analysis of fractions collected by chromatography of untreated or trypsin treated CDCP1 extracellular domain (ECD). Top, UV/Vis spectroscopy analysis of size-exclusion chromatography. Bottom, Coomassie stained gel analysis of fractions collected during chromatography.

Fig 5: Ectopic expression of miR-15a, miR-16 sensitizes breast cancer cells to DNA damage through BMI1 dependent ubiquitination pathway. Western blot analysis showing the expression of BMI1, RING1A, RING1B, EZH2, ?-H2AX, Ub-H2A, p-CHK2, p-ATM, RNF8, RNF168, MEL18, p53BP, BRCA1 proteins in MDAMB-231 cells transfected with miR-15a, miR-16 or both miR-15a/16 under etoposide-induced DNA damage conditions.. Actin served as a gel loading control. Blots were cropped to enhance the representation (A,B). Immunofluorescence data showing accumulation of BMI1, ?-H2AX (C) RING1B (D) and Ub-H2A (E) p53BP (F) in MDAMB-231 cells transfected with miR-15a miR-16 or both miR-15a/16 under etoposide- treated conditions. Bar indicates 200 µm.