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. 2012 Oct 22;209(11):1985–2000. doi: 10.1084/jem.20111665

Figure 3.

Figure 3.

Hypoxia controls Gal-1 expression in KS through HIF-independent, NF-κB–dependent mechanisms. (A–D) Expression of Gal-1 in KS cells transfected with or without HIF-1α siRNA or a super-repressor form of IκB-α (IκB-α-SR) and incubated under hypoxia or normoxia. (A) Promoter activity. (B) qRT-PCR of Gal-1 mRNA relative to RN18S1. AU, arbitrary units. Data are the mean ± SEM of five (A) or three (B) independent experiments. (C) Immunoblot of Gal-1, IκB-α, HIF-1α, and actin. Data are representative of four independent experiments. (D) ELISA of Gal-1 secretion. Data are the mean ± SEM of three independent experiments. (E) ELISA of Gal-1 secretion by KS cells cultured under hypoxic or normoxic conditions in the presence or absence of HIF-1α or NF-κB inhibitors. Data are the mean ± SEM of three independent experiments. (F and G) Immunoblot (F) and qRT-PCR (G) of Gal-1 expression induced by hypoxia (Hyp) in human and mouse melanoma (A375 and B16-F0), mouse breast carcinoma (4T1), and human prostate carcinoma (LNCaP) cell lines. Data are representative (F) or are the mean ± SEM (G) of three independent experiments. (H) Western blot of HIF-1α, IκB-α, Gal-1, and actin upon treatment of KS cells with CoCl2 (chemical activator of HIF-1α). Data are representative of four experiments. (I) Gal-1 promoter activity upon treatment of KS cells with CoCl2. Modulation of pGL3–Gal-1–Luciferase activity relative to Renilla expression is shown. Data are the mean ± SEM of three independent experiments. (J) ELISA of VEGF secretion by KS cells transfected with HIF-1α or scr siRNA cultured under hypoxic or normoxic conditions. Data are the mean ± SEM of three independent experiments. (K) Immunoblot of Gal-1, HIF-2α, c/EBPα, and actin in KS cells transfected with or without siRNA for HIF-2α, C/EBPα, or scrambled (scr) and incubated under hypoxia or normoxia. Data are representative of three independent experiments. (L) ELISA of Gal-1 secretion by KS cells transfected with or without siRNA for HIF-1α, HIF-2α, or C/EBPα or incubated with an NF-κB inhibitor (BAY 11–7082; 1 µM) and cultured under hypoxic or normoxic conditions. Data are the mean ± SEM of three independent experiments. (A, B, D, E, J, and L) *, P < 0.05; **, P < 0.01.