Figure 2
HIF3α activation is dependent on NF-κB activation: (a) Immunoblotting analysis of IκBα: hMSCs cultured in normoxia and treated with IL6, IFNγ, TNFα, MCP1, EGF and VEGF for 24 h. Cells were pre-treated with TCPA-1 for 1 h before cytokines supplementation. (b) Immunofluorescence analysis of HIF3α protein in hMSCs cultured in standard oxygen conditions (Normoxia) in absence and in presence of indicated cytokines and probed with antibodies against HIF3α. Cells were pre-treated with TCPA-1 for 1 h before cytokines supplementation. Scale bars: 10 μm). (c) Schematic diagram of the HIF3A promoter region, where a putative NF-KB binding site is depicted (black triangle). NFκB (RelA) binding on HIF3A promoter: ChIP analysis was performed in hMSCs cultured in standard oxygen conditions and treated with IL6, IFNγ, TNFα, MCP1, EGF and VEGF for 24 h (black bars), or pre-treated with TCPA-1 for 1 h (grey bars) before adding cytokines. As control, species matched IgG were used. Data obtained by qRT-PCR are expressed as enrichment of chromatin-associated DNA fragments immunoprecipitated by NF-κB antibody compared with input (% Input) and expressed as means ± SEM of two independent experiments performed in triplicate.
