Fig 1: Overexpression ATPIF1 restore the signaling changes caused by CCM3-KO at early stage. a change of inflammatory pathway and mitophagy in CRISPR-CCM3 EPCS. b change of Tie2, p-Tie2 and KLF4 in selected CRISPR-CCM3 EPCs and s-oeATPIF1 EPCs. c MG132 (10 μM) treatment in CRISPR-CCM3, s-oeATPIF1 and CRISPR-CCM3 + s-oeATPIF1 EPCs. d ATPIF1 mRNA expression in s-oeATPIF1 and CRISPR-CCM3 + s-oeATPIF1 EPCs, *p < 0.05, **p < 0.01, ***p < 0.001. e different changes of Tie2 and KLF4 in CIRPSR-CCM3 + s-oeATPIF1 and s-oeATPIF1 + CRISPR-CCM3 EPCs. f the change of PINK1 after s-oeATPIF1 treatment. g overexpression CCM3 in CRISPR-CCM3 EPCs for 2 days or 5 days, vector was used as negative control
Fig 2: 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
Fig 3: Overexpression of ATPIF1 restore cell proliferation, junction and migration which was impaired by CCM3-knockdown. a The changes of already proved signaling pathways in HUVECs after siCCM3 with siS100A11 or oeATPIF1 treatment. b EdU staining in HUVECs after siRNA or overexpression treatment, scale bar, 50 µm. c Immunofluorescence images about tight junction and adherent junction changes in HDMECs after siCCM3 with oeATPIF1 treatment, and the percentages of disrupted tight junctions (TJs) and adherent junctions (AJs) (3 random microscope fields were used for quantification). HUVECs monolayer migration assay (d) and Matrigel based tube formation assay (e) after siCCM3 and oeATPIF1 treatment, migration area or total branches length was measured by Image J (n = 3). *p < 0.05, **p < 0.01, ***p < 0.001. Scale bar, 50 µm
Fig 4: Chemokine signaling in wild-type ECs becomes deregulated in co-culture with CCM3-/- ECs. a A ß-integrin-mediated cell adhesion array was used to analyse the surface expression of different integrin ß subunits on CCM3-/- CI-huVECs (n = 3 per group). b, c Immunofluorescence imaging and RT-qPCR analysis verified upregulation of ITGB4 gene expression in CCM3-/- CI-huVECs (scale bar: 100 µm). Data are presented as mean and SD (n = 3 per group). d Using high integrin beta 4 expression as marker for CCM3-/- CI-huVECs, mutant ECs could be efficiently sorted from mutant/wild-type co-cultures by fluorescence activated cell sorting. Sanger sequencing of sorted ITGB4low (I) and ITGB4high (II) cell populations verified high purity of CCM3+/+ and CCM3-/- CI-huVECs, respectively. e CCM3+/+ and CCM3-/- CI-huVECs were co-cultured, sorted by FACS and analyzed by RNA sequencing. CCM3+/+ and CCM3-/- CI-huVEC mono-cultures served as controls. f, g Venn diagrams illustrate the overlap of genes significantly up- or downregulated in CCM3-/- CI-huVEC mono-cultures (group II vs group I) and genes that are significantly up- or downregulated in CCM3-/- CI-huVECs by co-culture with CCM3+/+ CI-huVECs (group IV vs group II). h Shown is a heatmap with the 20 most downregulated genes in CCM3-/- CI-huVEC mono-cultures (left column; group II vs group I). The right column illustrates how co-culture with wild-type ECs affects the expression of these genes in CCM3-/- CI-huVECs (group IV vs group II). i, j Significantly up- or downregulated genes were also subjected to gene ontology analysis. Shown are the top 20 of significantly enriched biological process GO terms (n = 3 per group for e–j). #via plasma membrane cell adhesion molecules. Multiple t tests (a), Student’s two-tailed t test (c), and Fisher exact test (i, j) were used for statistical analyses: ***P < 0.001, ****P < 0.0001. padj = adjusted p value
Fig 5: CCM3-deficient hiPSCs show no abnormal proliferation in co-culture with wild-type hiPSCs. a CCM3-/- AICS-0036 hiPSC demonstrated typical hiPSC morphology (representative images; top left, scale bar: 500 µm) and strong expression of the pluripotency markers TRA-1–60, OCT4, SOX2, and SSEA4 (representative images; middle and right panels, scale bar: 200 µm). The mEGFP-tagged cytosol of CCM3-/- AICS-0036 hiPSC is shown in the bottom left subpanel (scale bar: 50 µm). b Western blot analysis verified CCM3 inactivation in CCM3-/- AICS-0036 hiPSCs. GAPDH was used as a loading control (n = 3 per genotype). c, d CCM3-/- AICS-0036 hiPSCs were mixed with CCM3+/+ AICS-0054 hiPSCs in a 1:9 ratio and co-cultured for 8 days. CCM3+/+ AICS-0036 mixed with CCM3+/+ AICS-0054 hiPSCs served as controls (n = 3 per genotype). Representative images from day eight are shown in c (scale bar: 500 µm). Data are presented as mean and SD. Student’s two-tailed t tests (b, d) were used for statistical analyses: **P < 0.01
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