Fig 2: miR-222-3p targets PDCD10 to inhibit cell migration via EMT pathway. (A) Meta-analysis describing forest plots of PDCD10 expression as a univariate predictor of overall survival. (B) Kaplan-Meier curves for overall survival probability in 1656 OC patients with low (n=640) and high (n=1016) PDCD10 expression (analyzed with log-rank test) from KMplot (http://kmplot.com/analysis/). (C) qPCR and Western blot analyses of PDCD10 levels in FE25 and 4 different EOC cell lines. (D) Representative images of PDCD10 expression in normal and tumor ovary tissues. Bar, 100 µm (Left) and 30 µm (Right). (E) Enrichment evaluation within the PDCD10-expressing levels for predicted GSEA results of TCGA reference gene sets for high and low PDCD10 expression groups. Genes expressed in the profile datasets were ranked by fold changes (high-expression/low-expression). GSEA correlation pathways were determined by the given algorithm. Vertical bars along the x-axis of the GSEA diagram represent the positions within the ranked list of genes set in the given sets. Positive and negative GSEA curves mean positive and negative enrichments, respectively. (F and G) qPCR and Western blot analyses of PDCD10, E-cad (CDH1) and VIM expression in HO 8910 PM cells (PDCD10-overexpressing groups) and SKOV3 cells (PDCD10-silenced groups). (H) HO 8910 PM cells transduced with control vector or PDCD10 (GFP-labeled ctrl vector and PDCD10 all in green) were subjected to immunofluorescence with human-specific E-cad and VIM antibodies (in red). Bar, 10 µm. (I) Western blot analysis of PDCD10, E-cad, Vim translation levels in HO 8910 PM cells after treatment with miR-ctrl mimic or miR-222-3p mimic, in the presence or absence of PDCD10. Data are means ± SEM and are representative of three (C and F) independent experiments. *, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001, determined by unpaired two-tailed t-test.

Fig 3: p21 and ERK-independent effect of NSC59984. a, b CCM3-/- and CCM3+/+ CI-huVECs were co-cultured, treated with NSC59984 (10 µM), sorted and subjected to RNA sequencing. DMSO-treated co-cultures served as controls. Shown are the top 20 of significantly enriched biological process GO terms (n = 3 per group). c, d Co-cultures of CCM3-/- and CCM3+/+ CI-huVECs were treated with NSC59984 (10 µM) and the p21 inhibitor UC2288 (n = 4 per group; c), the ERK1/2 inhibitor SCH772984 or the MEK1/2 inhibitor U0126-EtOH (n = 3 per group; d). Mutant allele frequencies were determined after 6 days. DMSO-treated co-cultures served as controls. Fisher exact test (a, b) and one-way ANOVA with multiple comparison test (c, d) were used for statistical analyses: ***P < 0.001; ****P < 0.0001. padj = adjusted p value

Fig 4: NSC59984 efficiently blocks the abnormal proliferation of CCM3-/- CI-huVECs in co-culture. a Depicted is a scheme of the apoptosis library screening approach. Co-cultures of CCM3-/- and CCM3+/+ CI-huVECs were treated with 10 µM of each library compound on day 0 and day 3. DNA samples collected at day 6 were used to determine frequencies of CCM3 knockout alleles with amplicon deep sequencing (ADS). DMSO treatment served as control. b NSC59984 and isoalantolactone reduced the portion of CCM3 knockout alleles in co-culture. In contrast, GSK-872 further enhanced the abnormal proliferation of CCM3-/- CI-huVECs (n = 4 per group). c Bright-field microscopy demonstrated that cells treated with NSC59984 had a normal EC morphology while isoalantolactone treatment induced severe morphological changes and significant cell death (10 µM of each compound; scale bar: 200 µm). After GSK-872 treatment, cells had a compact morphology that is a known feature of CCM3-/- CI-huVECs (n = 3 per group). d No cell viability differences were observed between CCM3+/+ and CCM3-/- CI-huVECs 72 h after NSC59984 or isoalantolacone treatment, whereas CCM3+/+ CI-huVECs were more sensitive to GSK-872 treatment than mutant ECs (n = 3 per group). Comp. = library compound. padj = adjusted p value. Data are presented as mean and SD. One-way ANOVA (b) was used for statistical analyses

Fig 5: Inactivation of CCM3 gene expression in human ECs causes resistance to apoptosis and increased clonogenicity. a CCM3-/- and CCM3+/+ CI-huVECs were seeded as (near-perfect) single-cell suspensions in 6-well plates with either 250 or 100 cells per well. Colonies were stained with crystal violet after eight days. Representative images are shown for both genotypes. The plating efficiency of CCM3-/- CI-huVECs was significantly increased under both seeding conditions (n = 4 per genotype). b CCM3 inactivation did not significantly enhance proliferation of CI-huVECs under standard culture conditions (n = 9 per genotype). c CI-huVECs were treated with staurosporine (0.05 µM or 0.25 µM) to induce apoptotic cell death. After 2, 8, and 24 h, the caspase-3 activity was markedly reduced in CCM3-/- CI-huVECs (n = 3 per genotype). d A human apoptosis antibody array assay verified the reduction of active caspase-3 levels in staurosporine-treated (0.05 µM, 24 h) CCM3-/- CI-huVECs (n = 3 per genotype). Representative array membranes are shown for both genotypes. Green rectangles mark active caspase-3. No significant differences were found for other apoptosis markers. e, f The activities of caspase-8 (e) and caspase-9 (f) were also slightly reduced in staurosporine-treated (0.05 µM or 0.25 µM, 8 h) CCM3-/- CI-huVECs (n = 3 per genotype). RFU = relative fluorescence units. Data are presented as mean and SD. Student’s two-tailed t tests (a–c, e, f) were used for statistical analyses: *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001