FIGURES AND TABLES
FROM:
Antiapoptotic action of hypoxia-inducible factor-1
in human endothelial cells
Erik Z Yu, Ying-Yue Li, Xiu-Huai Liu, Elliott Kagan and Richard M McCarron
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Figure 1.
(a–d) Representative photomicrographs showing apoptotic changes in HUVECs subjected to A/R. Only occasional nontransfected cells show positive TUNEL staining (a) and immunoreactivity for activated caspase-3 (c). In contrast, the majority of cells transfected with HIF-1
siRNA duplexes demonstrate positive TUNEL staining (b) and immunoreactivity for activated caspase-3 associated with morphologic evidence of apoptotic damage (d). (e–h) Representative photomicrographs demonstrating knockdown of HIF-1 mRNA and protein expression by HIF-1 siRNA duplexes in HUVECs subjected to A/R. Many nontransfected cells demonstrate strong HIF-1 mRNA expression by in situ hybridization (e), whereas HIF-1 mRNA is barely detectable in cells transfected with siRNA duplexes (f). Strong immunoperoxidase reactivity for HIF-1 is observed in most nontransfected cells, (g) but only an occasional cell transfected with siRNA shows HIF-1 immunoreactivity (h).
Figure 2.
Group comparisons of TUNEL assays illustrating the effects of normoxia and A/R on cultured HUVECs. Transfection with HIF-1 siRNA greatly increased the number of TUNEL-positive cells subjected to anoxic stress. Results in each category represent mean
s.e.m. of three experiments. (A) Nontransfected normoxic cultures. (B) Normoxic cultures transfected with HIF-1 siRNA duplexes. (C) Cultures transfected with siRNA duplexes and subjected to A/R. (D) Cultures transfected with scrambled RNA duplexes and subjected to A/R. (E) Nontransfected cultures subjected to A/R. *Significantly different (P<0.05) compared with the other four groups.
Figure 3.
Group comparisons of immunoperoxidase staining for activated caspase-3 showing effects of normoxia, A/R, and transfection with scrambled RNA duplexes on cultured HUVEC. Transfection with HIF-1 siRNA (but not scrambled RNA) duplexes prior to A/R markedly increased the number of cells manifesting immunoreactivity for activated caspase-3. Results in each category represent means.e.m. of three experiments. (A) Nontransfected normoxic cultures. (B) Normoxic cultures transfected with HIF-1 siRNA duplexes. (C) Cultures transfected with siRNA duplexes and subjected to A/R. (D) Cultures transfected with scrambled RNA duplexes and subjected to A/R. (E) Nontransfected cultures subjected to A/R. *Significantly different (P<0.05) compared with the other four groups.
Figure 4.
Western blot analyses of HUVECs lysates for HIF-1 and activated caspase-3 showing the effects of normoxia, A/R, and transfection with scrambled RNA duplexes on cultured HUVECs. Transfection with HIF-1 siRNA duplexes prior to A/R largely attenuated HIF-1 expression, but greatly increased activated caspase-3 expression. The upper panel depicts representative immunoblots demonstrating HIF-1, activated caspase-3, and 
-tubulin protein expression. The middle panel depicts group comparisons of activated caspase-3 immunoblots. The lower panel depicts group comparisons of HIF-1 immunoblots. Results in each category represent means.e.m. of three experiments. (A) Normoxic cultures. (B) Cultures transfected with HIF-1 siRNA duplexes and subjected to A/R. (C) Cultures transfected with scrambled RNA duplexes and subjected to A/R. (D) Nontransfected cultures subjected to A/R. *Significantly different (P<0.05) compared with the other three groups. ‡Significantly different (P<0.05) compared with groups C and D.
Figure 5.
Group comparisons of Q-PCR analyses of HIF-1 mRNA expression showing the effects of normoxia, A/R, and transfection with scrambled RNA duplexes on cultured HUVEC. Transfection with HIF-1 siRNA (but not scrambled RNA) duplexes prior to A/R inhibited HIF-1 mRNA expression. Results in each group category (other than group a) represent means.e.m. of three experiments. (A) Normoxic cultures. (B) Cultures transfected with HIF-1 siRNA duplexes and subjected to A/R. (C) Cultures transfected with scrambled RNA duplexes and subjected to A/R. (D) Nontransfected cultures subjected to A/R. *Significantly different (P<0.05) compared with groups C and D.
Figure 6.
Group comparisons of HIF-1 in situ hybridization analyses illustrating the effects of normoxia and A/R on cultured HUVECs. Transfection with HIF-1 siRNA significantly decreased the amount of HIF-1 mRNA detectable in HUVECs subjected to anoxic stress. Results in each category represent means.e.m. of three experiments. (A) Normoxic cultures. (B) Cultures transfected with HIF-1 siRNA duplexes and subjected to A/R. (C) Nontransfected cultures subjected to A/R. *Significantly different (P<0.05) compared with the other two groups.
Figure 7.
Group comparisons of immunoreactivity for HIF-1 showing the effects of normoxia A/R, and transfection with scrambled RNA duplexes on cultured HUVECs. Transfection with HIF-1 siRNA significantly decreased the percentages of cells manifesting immunoreactivity for HIF-1 in response to anoxic stress. Results in each category represent means.e.m. of three experiments. (A) Nontransfected normoxic cultures. (B) Normoxic cultures transfected with HIF-1 siRNA duplexes. (C) Cultures transfected with siRNA duplexes and subjected to A/R. (D) Cultures transfected with scrambled RNA duplexes and subjected to A/R. (E) Nontransfected cultures subjected to A/R. *Significantly different (P<0.05) compared with the other four groups.
