Extended Data Fig. 2: Transfer of VE-Cad and Rad51 to PMVECs damaged by MMC and transcriptional activation of ATF4 target genes in MMC-treated PMVECs.
(a) The levels of intracellular VE-Cad, Rad51, and β-actin (loading control) protein in the MMC-treated PMVECs (recipient cells; 1 × 106 cells) after the incubation with the CM from PMVECs (1 × 106 cells) treated with vehicle (CMveh) or MMC (CMMMC) for 14 h were examined by immunoblot analysis. (b) IF staining of PMVECs treated with CMveh or CMMMC (5 × 103 cells) with an anti-VE-Cad antibody (green). Cell nuclei were stained with DAPI (blue). Scale bar=10 mm. (c) CM was collected from PMVECs following two h of MMC treatment and was immediately reintroduced to the donor cells’ CM after MMC was removed from the CMMMC (-MMC, indicated in red), or it was added without removing MMC ( + MMC, indicated in blue). Duplicate samples were used. Non-specific IgG (IgG) was used as a negative control for IP. Subsequently, following a 14 h incubation period, the VRC amount in the donor cells was evaluated. (d) RNA-seq data (MMC 0 h vs 4 h) is plotted on a volcano map. X-axis and Y-axis indicate p-value (-Log10) and fold change (FC) (Log2). Annotated red dots indicate known ATF4 targets. n = 3 independent RNA samples per condition. Data are analyzed by one-way ANOVA with Tukey’s post-hoc test (c).
