Figure 4

Effect of MSA on HIF-1α protein synthesis and degradation. (A) HIF-1α synthesis was inhibited by cycloheximide but not by MSA in FaDu cells. FaDu cells were treated with 1 μM MSA and 10 μM cycloheximide alone and in combination for 1.5, 2, 3 and 24 hours at 0.5% oxygen and protein extracts were used to determine HIF-1α by western blot. β-actin expression was used as a loading control. (B) Effect of MSA on HIF-1α protein synthesis in RC2 cells. Cells were treated with cycloheximide or MSA alone and in combination for 8 h. HIF-1α was detected by western blot. β-actin was used as loading control (C) Incorporation of ³⁵ S-Methionine was not affected by MSA in RC2. Cells were treated with cycloheximide or MSA separately in duplicate samples for 5 h and ³⁵ S-Methionine (2.3 μCi/ml) was added at the last 1 h of treatment. Protein extracts (20 μg) were used to separate by SDS polyacrylamide gel electrophoresis and detected the incorporated ³⁵ S-Methionin by autoradiography. Lower panel showing the coomassie blue stained proteins as loading control. (D) Determination of ³⁵ S-Methionine radioactivity counts in cycloheximide or MSA treated RC2 cells. Protein extracts (20 μl) were used to detect ³⁵ S-methionine radioactivity in the cells by counting in Liquid Scientilator Counter. Total counts were calculated in one milligram of protein and presented the number of counts in millions as compared to untreated control cells. P < 0.05 was considered as significant. (E) HIF-1α degradation by MSA is proteasome dependent. FaDu cells which do not express constitutive HIF-1α under normoxic culture conditions were subjected to 0.5% oxygen and treated with 1 μM MSA alone and in combination with 10 μM proteasome inhibitor MG132 for 24 h. Cell extracts were prepared and the expression of HIF-l α was analyzed. Expression of β-actin was used as a loading control. (F) Proteasome independent degradation of HIF-1α in VHL mutated RC2 cells. RC2 cells were treated with MSA and MG132 alone and in combination for 8 h. In a separate experiment, 1 h pre-treatment of MG132 followed by 7 h MSA treatment was performed to see the effect of MSA on HIF-1α. Cells were processed to extract protein and HIF-1α levels were determined. Expression of β-actin was used as a loading control.