Figure 4: Effect of HIF-1 alpha silencing and NF-κB inhibition on HIF- 3 alpha expression. (A) The mRNA expression levels of HIF-3 alpha at different times of hypoxia were detected by RT PCR in NT shRNA and in HIF- 1α shRNA clones, in the presence or absence of parthenolide (P). Values are expressed as fold induction with respect to normoxic control (set at 1) and represent the mean ± SE of three different experiments. 18S rRNA was used as houseκeeping gene. HIF-1 alpha shRNA vs NT shRNA : *P≤0.05, **P≤0.01, ***P≤0.001. (B) Western Blot analysis of HIF-3 alpha nuclear protein was performed in NT shRNA and HIF-1α shRNA clones in the presence and absence of parthenolide (P) in normoxia (N) and at 72 hours of hypoxia (H). γ-tubuline was used as loading control. One representative experiment is shown. NT shRNA + P vs NT sh RNA: *P≤0.05, **P≤0.01, ***P≤0.001. (B bottom panel) The effect of parthenolide (P) on HIF-1 alpha nuclear protein expression was analyzed in NT shRNA clones by Western Blot in normoxia (N) and at 4 and 8 hours of hypoxia (H). γ-tubuline was used as loading control. A representative experiment is shown. (C) The effect of HIF-1 alpha silencing on NF-κB P65 (on the left) and phosphorylated NF-κB P65 (Ser276) (on the right) protein was detected by Western Blot. NT shRNA and HIF-1α shRNA clones were maintained in normoxic (N) or hypoxic conditions (H) for 30 minutes. γ-tubuline was used as loading control. A representative experiment is shown for both NF-κB P65 and pospho NF-κB P65 (Ser276).