Research Paper Volume 15, Issue 13 pp 6163—6178

Figure 4. Suppressing PRMT5 reduces the expression and stability of HIF-1α induced by hypoxia. (A) The mRNA expression of HIF-1α and PRMT5 was measured by qRT-PCR in PRMT5 depletion cells (HUVECs, n=4). *P < 0.05 vs. Scr. N.S. means not significant. (B) The HUVECs were infected with lentivirus containing scramble-shRNA or PRMT5-shRNA2, followed by CoCl₂ (200μM) treatment for 24h. The PRMT5 and HIF-α expression levels were determined by Western blotting (n=3). (C) The HUVECs were treated with vehicle or GSK591 (1μM) for five days and then were treated with CoCl₂ (200μM) for 24h. The HIF-1α expression level was determined by Western blotting (n=3). (D) The effect of different doses of GSK591 on the HIF-1α expression level induced by CoCl₂ as evaluated by Western blotting (n=3). (E) The HIF-1α stability was detected by Western blotting in PRMT5 depletion HUVECs upon cycloheximide (CHX, 20μg/mL) treatment at the indicated time points (n=3). (F) The HIF-1α stability was detected by Western blotting in GSK591-treated HUVECs upon cycloheximide treatment at the indicated time points (n=3). (G) The HUVECs were incubated with or without GSK591 (10μM) or infected with lentivirus containing PRMT5-shRNA2 and then pretreated with MG132 (10μM) and BAF-A1 (100nM) for 30 min before treating CoCl₂. The HIF-1α expression levels were evaluated by Western blotting (n=3). (H) The HUVECs were infected with lentivirus containing scramble-shRNA or PRMT5-shRNA2 and then were transfected with vector or Flag-PRMT5 before treating CoCl₂. The HIF-1α expression levels were evaluated by Western blotting (n=3).