Figure 7

Identification of histone-acetylation and HIF-destabilization in H23 cells upon co-treatment with panobinostat and cisplatin. (A) H23 cells were treated with increasing panobinostat concentrations under normoxic conditions and the amount of acetylated histone proteins H3 and H4 was detected by immunoblotting. (B) H23 cells were treated with different combinations of cisplatin or/and panobinostat both under normoxic (NOX) or hypoxic (HOX) conditions. The amounts of Ac-H3 and Ac-H4 were analyzed 24 hours later by immunoblotting. (C) After 24 hours treatment with increasing panobinostat concentrations immunoblotting for HIF-1α and HIF-2α was performed. (D) H23 cells were treated with cisplatin, panobinostat or with combination of both under normoxic and hypoxic conditions. After 24 hours immunoblotting for HIF-1α, class II HDACs (HDAC4, 5, 6 and 7) and HDAC1 (class I) was performed. After stripping β-actin was used as a loading control. Ac-H3 and H4, acetylated histone proteins; DMSO, vehicle control; P, panobinostat (16 nM); C, cisplatin (16 μM); P + C (16 nM panobinostat + 16 μM cisplatin); NOX, normoxia; HOX, hypoxia. (E) H23 cells were transfected with 10 nM of HIF-1α siRNA and after 24 hours knock-down effects were detected by qRT-PCR and by immunoblotting. (F) H23 cells transfected with HIF-1α siRNA were treated with different cisplatin concentrations for 24 hours under hypoxic conditions and cell viability was measured using the AlamarBlue® assay. Viability of untreated cells was set to 100%. Data were compared to cells transfected with control, non-silencing RNA (CTRL). ns, not significant; * P < 0.05; ** P < 0.01; *** P < 0.001. (G) Protein-protein interaction between HIF-1α and HDAC4 was determined by Duolink in situ proximity ligation assay and visualized by fluorescence microscopy. For negative control primary antibodies were omitted. Magnification: 200x.