Figure 7. Vasohibin1 is a new ITAF that is active in early hypoxia.

(A, B) VASH1 knock-down was performed in HL-1 cardiomyocytes using short-interfering (siRNA) smartpools targeting VASH1 (siVASH1) or control (siControl). VASH1 mRNA level was measured by RT-qPCR (A), and VASH1 protein expression was analyzed by the capillary Simple Western method using an anti-VASH1 antibody and quantified by normalization to total proteins. The experiments were reproduced twice, giving identical results. One of the two experiments is shown (B). Knock-down of VASH-1 was performed on cardiomyocytes transduced by a set of IRES-containing lentivectors used in Figure 4. (C, D) After 8 hr of hypoxia, IRES activities (LucF/LucR ratio) were measured in cell extracts from normoxic (C) and hypoxic cardiomyocytes (D). The IRES activity values have been normalized to the control siRNA. Histograms correspond to means ± standard deviation of the mean, and a non-parametric Mann-Whitney test was used to identify significant change from control levels: *p<0.05, **p<0.01. For each IRES the mean was calculated for nine cell culture biological replicates, each of these being the mean of three technical replicates (27 technical replicates in total but the M-W test was performed with n = 9). Detailed values of biological replicates are presented in Supplementary file 5.

Figure 7—figure supplement 1. VASH1 half-life is superior to 24 hr.

(A–C) VASH1 half-life determination was performed by blocking protein synthesis with cycloheximide at 10 µg/mL, with time-course points at 0 hr, 4 hr, 6 hr, 8 hr, 16 hr and 24 hr. VASH1 (A) and P21 (B) protein stability was measured by capillary Simple Western, with normalization to the 0 hr time-course point. P21 was used as a control for its short half-life (C). Three independent experiments were performed. A representative experiment is shown.