Figure 3

GATA3 expression increases HIF-1α protein levels. (a) Western blot analysis of GATA3 and HIF-1α under normoxia (N) or hypoxia (H) for overnight with or without a proteasome inhibitor, MG132 (20 μM), as indicated. GAPDH was an internal control. OEC-M1 cells were transfected with non-targeting siRNA or GATA3 siRNA (siGATA3-1 or siGATA3-2) and A375 cells were transfected with Mock or GATA3 plasmid. (b) Real-time RT-PCR analysis of HIF-1α mRNA expression. The total RNA was extracted from cells grown under normoxia or hypoxia for overnight. The results are represented as mean±s.d. from three independent experiments. (c) Western blot analysis of GATA3 and P402A/P564A HIF-1α mutant. GAPDH was an internal control. pLKO or shGATA3/pLKO (shGATA3) expressing OEC-M1 stable transfectants as well as Mock or GATA3 overexpressing A375 stable transfectants were transfected with empty vector or HA-P402A/P564A HIF-1α/pcDNA3.1 (HA-mtHIF-1α). (d) Effects of GATA3 on HIF-1α ubiquitination. OEC-M1 cells transfected with non-targeting siRNA or GATA3 siRNA (siGATA3-2) and A375 cells transfected with Mock or GATA3 plasmid were incubated with MG132 (20 μM) under hypoxia for 6 h. Cell lysates were immunoprecipitated with anti-HIF-1α antibody and then immunoblotted with anti-ubiquitin antibody. GATA3, HIF-1α and GAPDH in whole cell lysates (input) were shown. (e) Representative images of immunohistochemical staining of GATA3 and HIF-1α in serial sections of HNSCC tissues. Scale bars, 50 μm.