Hypoxia-induced mRNA decay is HIF-dependent. (A) mRNA content per cell from cells grown for 16 h in normoxia, hypoxia (Hyp) or DMOG (DMOG). Each dot represents the ratio of mRNA levels in hypoxia or DMOG over normoxia in an independent experiment. The observed ratios were significantly lower than expected from a sample with a mean ratio of 1 (single sample Student's t-test, P-value = 0.033 and P-value = 0.041, respectively). (B) ARNT mRNA levels in HUVEC cells transduced with lentiviral particles expressing a shRNA to silence ARNT expression versus control (NS). The ARNT expression was on average 21% and 25% of that in control cells in normoxia and hypoxia, respectively, and significantly different to the expected value of 1 (single sample Student's t-test, P = 0.006 and P = 0.007 for cells under normoxia and hypoxia, respectively). (C) EGLN3 mRNA levels in HUVEC cells transduced with lentiviral particles expressing a shRNA to silence ARNT expression versus control (NS). The effect of hypoxia on EGLN3 induction was significantly attenuated in ARNT knockdown cells (P-value = 0.017; paired Student's t-test). (D) HUVEC cells transduced with lentiviral particles expressing a shRNA to silence ARNT (shARNT) or a control shRNA (NS) or mock infected (UT) were exposed to normoxia (Nx) or hypoxia (Hyp) for 16 h. The level of ARNT protein and tubulin were determined by immunoblot. (E) Ratio of hypoxic versus normoxic total RNA content per cell from control cells (NS) and cells whose ARNT expression was silenced (shARNT).Each dot represents a determination from a single experiment and matched observations (derived from the same experiment) are represented joined by a line. The effect of hypoxia on RNA level was significantly attenuated in ARNT knockdown cells (P-value = 0.014; paired Student's t-test). (F) EPAS1 mRNA levels in HUVEC cells transduced with lentiviral particles expressing a shRNA to silence EPAS1 expression versus control (NS). EPAS1 expression was on average 30% and 15% of that in control cells in normoxia and hypoxia, respectively, and significantly different to the expected value of 1 (single sample Student's t-test, P = 0.0021 and P = 0.0002 for cells under normoxia and hypoxia, respectively). (G) HIF1A mRNA levels in HUVEC cells transduced with lentiviral particles expressing a shRNA to silence HIF1A expression versus control (NS). HIF1A expression was on average 30% and 35% of that in control cells in normoxia and hypoxia, respectively and significantly different to the expected value of 1 (single sample Student's t-test, P = 0.0009 and P = 0.0002 for cells under normoxia and hypoxia, respectively). (H) HUVEC cells transduced with lentiviral particles expressing a shRNA to silence HIF1A (shHIF1A), EPAS1 (shEPAS1) or a control shRNA (NS) or mock infected (UT) were exposed to normoxia (Nx) or hypoxia (Hyp) for 16 h. The levels of HIF1A, EPAS1 and β-actin were determined by immunoblot. (I) Pulse-labeling of total RNA determined in control HUVEC cells (NS) and cells whose HIF1A or EPAS1 expression was silenced (shHIF1A and shEPAS1). The graph represents the logarithm of the ratio of the hypoxic (16 h) remaining radioactivity normalized by normoxic values. Each dot represents a determination from a single experiment. The effect of hypoxia on RNA level showed clear tendency to be attenuated in shHIF1A and shEPAS1 but did not reach statistical significance (shHIF1A P = 0.14; paired Student's t-test; shEPAS1 P = 0.08; paired Student's t-test). (J) EPAS1 mRNA levels of HUVEC cells transduced with a constitutively stable form of EPAS1 (EPASPP) or a stable but transcriptionally inactive form (EPASPPBH) compared with the control (PRRL). The expression of mutant forms of EPAS1 was similar (P = 0.43, paired Student's t-test). (K) ADM mRNA levels of HUVEC cells transduced with a constitutively stable form of EPAS1 (EPASPP) or a stable but transcriptionally inactive form (EPASPPBH) compared with the control (PRRL). The effect of EPASPP on ADM expression was significantly higher than that of EPASPPBH (P = 0.023, paired Student's t-test). (L) HIF1A mRNA levels of HUVEC cells transduced with a constitutively stable form of HIF1A (HIFPP) or a stable but transcriptionally inactive form (HIFPPBH) compared with the control (PRRL). The expression of HIFPPBH was higher than that of HIFPP but the difference did not reached statistical significance (P = 0.34, paired Student's t-test). (M) BNIP3 mRNA levels of HUVEC cells transduced with a constitutively stable form of HIF1A (HIFPP) or a stable but transcriptionally inactive form (HIFPPBH) compared with the control (PRRL). The effect of HIFPP on BNIP3 expression was higher than that of HIFPPBH, but the difference did not reached statistical significance (P = 0.079, paired Student's t-test). (N) Western blot to determine the overexpression of the HIFPP, HIFPPBH, EPASPP, EPASPP, or empty vector (PRRL) in samples in normoxia (Nx) or hypoxia (Hyp) for 16 h. (O,P) Ratio of hypoxic versus normoxic total RNA content per cell in HUVEC cells overexpressing a constitutively active EPAS1 (EPASPP) or HIF1A (HIFPP) and a constitutively active EPAS1 or HIF1A whose bHLH motive has been mutated (EPASPPBH, HIFPPBH). Each dot represents a determination from a single experiment and matched observations (derived from the same experiment) are represented joined by a line. The expression of EPASPP resulted in RNA level significantly reduced as compared to control cells, (P-value = 0.025, single sample Student's t-test) and the effect of each EPAS1 mutant on RNA level was significantly different (P-value = 0.025, paired Student's t-test). The expression of HIFPP resulted in RNA level reduced as compared to control cells, but did not reach statistical significance (P-value = 0.08, single sample Student's t-test) and the effect of each HIF mutant on RNA level was not significantly different (P-value = 0.23, paired Student's t-test). (Q) Ratio of remaining radioactivity present in hypoxic samples over normoxia in untreated cells (UT) and cells treated with actinomycin D (ACTD), cycloheximide (CHX) or puromycin (PURO) after pulse-labeling with tritiated uridine. Each dot represents a determination from a single experiment. A single factor ANOVA showed a significant difference among the four treatment groups UT, ACTD, CHX, and PURO (F(3,8) = 8.487, P-value = 0.007) and a posteriori Tukey test showed that all drug treatment means were significantly different than the control mean (P-value = 0.006, P-value = 0.022, and P-value = 0.047 for ACTD, CHX, and PURO, respectively).
