Fig 2: Ectopic expression of miR-31 suppresses migration and invasion of ESCC and EAC cell lines. TE8 and FLO1 cells were transfected with pre-miR-31 containing vector (grey bars) or empty vector control (black bars). (A) Overexpression of miR-31 was verified by qRT-PCR. Pre-mature miR-31 expression was normalized to GAPDH and mature miR-31 expression was normalized to RNU6. (B) Cell migration was measured 24 hours post-transfection using Boyden chambers. (C) Cell invasion was measured 24 hours post-transfection using Matrigel-coated Boyden chambers. (D) Cell viability was measured using the WST-1 assay. (E) Cell growth was evaluated by colony formation assay and measured on the Oxford Optronix Gelcount. Results are means ± SD from at least three biological replicates.

Fig 3: Exosomes isolated from benign and malignant tumor associated ascitic supernatants increase the growth of ovarian cancer spheroids. Ovarian cancer cells SKOV3, OVCA433 HeyA8, and OVCA429 were grown in low attachment plates as spheroids as control (without exosomes), as BAE (grown with 250ug/ml exosomes from benign ascitic supernatant) or as MAE (grown with 250ug/ml exosomes from malignant ascitic supernatant). Spheroids were photographed (a) after seven days and quantitated using an Oxford-Optronix Gelcount automated cell counting instrument (Sarasota Fl.) and represented as a bar graph (b).

Fig 4: Quininib and 1,4-dihydroxy quininib reduce colony formation in Mel285 and OMM2.5 cells. Graphs show the percentage survival fraction of clones at 24 (A,C) and 96 h (B,D) post treatment with quininib (Q1), 1,4-dihydroxy quininib (Q7), montelukast (Mon), or HAMI 3379 (HAMI). Images of clones captured by GelCount™ system (Oxford Optronix) after 10 days of culture following treatment with DMSO control or 20 µM quininib (Q1) or 20 µM 1,4-dihydroxy quininib (Q7) for 24 or 96 h. Clones were stained with 0.5% crystal violet before counting. 1500 cells (Mel285) or 9000 cells (OMM2.5) were seeded and treated in duplicate in 6-well plates for each individual experiment and individual experiments were conducted three times (n = 3). Statistical analysis was performed by ANOVA with Dunnett’s post hoc multiple comparison test. Error bars are mean + S.E. *** p < 0.001; **** p < 0.0001.

Fig 5: Checkpoint proficiency and DNA repair is attenuated in the absence of PTEN. (a) FACS analysis measuring phopho-histone H3 levels and PI incorporation in replicate cultures of HCT116 WT and PTEN-/- cells with or without prior exposure to different doses of IR and incubation with nocodazole for 9 h. FACS data present the mitotic index (ratio of cells in M-phase positive for phosphorylated H3 over total number of cells × 100). The data are normalized to the nocodazole (noc) sample not exposed to IR and average±S.D. of n=3 are plotted. (b) Immunofluorescence analysis of phosphorylated H2Ax (?-H2AX) and RPA foci in HCT116 WT with or without pre-treatment with Mirin, and in PTEN-/- cells 4 h after exposure to 10 Gy IR. Quantification of the number of RPA foci per nuclei. Error bars show S.D. for three independent biological replicates. (c) Frequency of NHEJ and HR was analysed with two independent NHEJ and HR constructs in HCT116 WT and PTEN-/- cell lines. The WT cell line was either pre-transfected with control siRNA or siRNA pool against MRE11 or pre-treated with 15 µm Mirin 40 min prior to transfection with linearized constructs. The ratio of GFP+/DsRed+ cells was used to measure repair efficiency and is presented as a percentage of the untreated WT control. Error bars show s.e.m. for three independent biological replicates. ** denote P<0.01 as calculated by Student’s t-test. (d) Survival curves plotted with the number of colonies determined by the GelCount (Oxford Optronix, Abingdon, UK). % Survival was calculated relative to the cells untreated with IR. Error bars show s.d. for three independent biological replicates.