Fig. 2: CRELD2 expression and secretion in ECs.
a, CRELD2 mRNA expression (RT-qPCR, normalized to GAPDH) in HCAECs exposed to simulated ischemia (20 h) or simulated ischemia (4 h) followed by reperfusion (16 h) (IR), or stimulated with Tg (2 μmol l−1), Tm (2.5 μmol l−1), IL1β (10 ng ml−1), TNF (5 ng ml−1), CCL2 (100 ng ml−1), CXCL12 (50 ng ml−1), mouse MYDGF (100 ng ml−1) or VEGFA (50 ng ml−1) for 20 h. There were 4–10 experiments conducted. Individual data points and mean values are shown. ***P < 0.001 (one-way ANOVA with Dunnett test). b,c HCAECs were cultured under control conditions (Con) or exposed to IR; exemplary immunoblots and summary data show CRELD2 expression in HCAEC lysates (normalized to alpha-tubulin (TUBA)) (b) and supernatants (Ponceau S staining used as loading control) (c). There were eight experiments conducted. ***P < 0.001 (two-sided independent-sample t-test). Mr(K) denotes molecular mass in kDa. d–f, HCAECs were infected with lentiviruses encoding a scrambled (SCR) shRNA or shRNAs targeting ATF6, IRE1α or PERK. Cells were then cultured under control conditions or exposed to IR. d, CRELD2 mRNA expression (RT-qPCR, normalized to GAPDH). There were four to six experiments conducted. Individual data points and mean values are shown. ***P < 0.001 (two-way ANOVA with Sidak test). e,f, Exemplary immunoblots and summary data showing CRELD2 expression in HCAEC lysates (e) and supernatants (f). There were five to nine experiments conducted. **P < 0.01; ***P < 0.001 (two-way ANOVA with Sidak test). g, Exemplary PCR analysis and summary data (three experiments) showing the chromatin immunoprecipitation signal of ATF6 binding to the CRELD2 promoter region in HCAECs cultured under control conditions or exposed to IR. *P < 0.05 (two-sided independent-sample t-test). h, HUVECs were transfected with expression plasmids encoding the N-terminal fragment of ATF6 (ATF6p50) or GFP (used as a control). Exemplary immunoblots and summary data showing CRELD2 expression in cell lysates and supernatants. There were five experiments conducted. *P < 0.05; **P < 0.01 (two-sided independent-sample t-test). Unless otherwise stated, individual data points, mean values and s.e.m. are shown.
