Figure 1.
SAHA represses HIF-1α induction in response to hypoxic mimics.
(A) Immunoblot analysis of HIF-1α, p53 and GAPDH protein expression from cell lysates following treatment of HuH7 cells with 5 µM SAHA, DMSO, DMSO+150 µM cobalt chloride (CoCl2) or SAHA+150 µM cobalt chloride (CoCl2) for 24 h. (B) Immunoblot analysis of HIF-1α, p53 and GAPDH protein expression from cell lysates following treatment of HuH7 cells with 5 µM SAHA, DMSO, DMSO+500 µM dimethyloxallyl glycine (DMOG) or SAHA+500 µM dimethyloxallyl glycine (DMOG) for 24 h. (C) Immunoblot analysis of HIF-1α, p53 and GAPDH protein expression from cell lysates following treatment of HuH7 cells with 5 µM SAHA or DMSO for 24 h in the presence or absence of 100 µM desferrioxamine (DFO) for 18 h. (D) Immunoblot analysis of HIF-1α, p53, HDAC7 and GAPDH protein expression from cell lysates following treatment of HuH7 cells after SAHA treatment at the indicate concentration is shown in combination with 50 µM MG132 for 4 h. (E) and Hep3B cells with 5 µM SAHA or DMSO for 24 h in the presence or absence of 50 µM MG132 for 4 h. (F) Immunoblot analysis of HIF-1α, p53, HDAC7 and GAPDH protein expression as well as the splicing of LC3 following treatment of HuH7 cells with 5 µM SAHA or DMSO for 24 h in the presence or absence of 50 µM MG132 for 4 h. (G) Immunoblot analyses of HIF-1α, p53 as well as the splicing of LC3 following treatment of HuH7 cells with 50 µM MG132+5 µM SAHA or DMSO in presence or absence of 10 mM ammonium chloride (NH4Cl) for 8 h. In all panels GAPDH is used as loading control and HDAC7 is used as control to SAHA treatment.
Figure 2.
Silencing of HDAC9 represses HIF-1α induction.
(A) Immunoblot analysis of HIF-1α, HDAC7 and GAPDH protein expression in cell lysates following siRNA-mediated silencing of HDACs 1–11 in HuH7 cells. Quantitative analysis (lower) of the level of HIF-1α in response to silencing of the indicated HDAC in HuH7 cells. Data shown denote the fold change in HIF-1α protein expression relative to scramble (Scr) control (black bar) (mean ± SD, n = 3). Asterisks indicates p<0.05 as determined by two-tailed t-test using Scr control (black bar) as the reference and # indicates p<0.05 as determined by two-tailed t-test using HDAC9 siRNA as the reference. (B) Immunoblot analysis of p53 and GAPDH protein expression in cell lysates following siRNA-mediated silencing of HDACs 1–11 in HuH7 cells. Quantitative analysis (lower) of the level of p53 in response to silencing of the indicated HDAC in HuH7 cells. Data shown denote the fold change in p53 protein expression relative to scramble (Scr) control (black bar) (mean ± SD, n = 3). Asterisks indicates p<0.05 as determined by two-tailed t-test using Scr control as the reference. (C) Immunoblot analysis of HIF-1α and GAPDH protein expression in cell lysates following siRNA-mediated silencing of HDAC9 (siHDAC9) in the presence of 5 µM SAHA+50 µM MG132 in HuH7 cells. In all panels GAPDH is used as loading control and HDAC7 is used as control to SAHA treatment.
Figure 3.
SAHA did not affect the level of HIF-1α mRNA.
(A and D) qRT-PCR analysis of p53 mRNA level in HuH7 cell line following the indicated concentration of SAHA (A) or HDAC9 and HDAC10 silencing (D). Data is shown as the fold change of the ratio of p53 to GAPDH mRNA relative to that seen for DMSO (0 µM) (A) or scrambled (Scr) siRNA control (D) (mean ± SD, n = 3). In all panels, asterisk indicates p<0.05, as determined by two-tailed t-test using scrambled siRNA (Scr) (D) or DMSO (0 mM) (A) as the reference. (B–C) qRT-PCR analysis of HIF-1α mRNA level in HuH7 cell line following the indicated concentration of SAHA (B) or HDAC9 and HDAC10 silencing (C). Data is shown as the fold change of the ratio of HIF-1α to GAPDH mRNA relative to that seen for DMSO (0 µM) (B) or scrambled (Scr) siRNA control (C) (mean ± SD, n = 3). In all panels, asterisk indicates p<0.05, as determined by two-tailed t-test using scrambled siRNA (Scr) (C) or DMSO (0 µM) (B) as the reference.
Figure 4.
eIF3G silencing reversed SAHA effect on HIF-1α repression in response to hypoxic mimic.
Immunoblot analysis of HIF-1α and GAPDH protein expression in cell lysates following siRNA-mediated silencing of eIF3 A-M, eIF4 E, G1–3 and eIF5 in HuH7 cells in presence or absence of SAHA+MG132. Quantitative analysis of the level of HIF-1α in response to silencing of the indicated eIF in HuH7 cells. Data shown denote the fold change in HIF-1α protein expression relative to MG132 treatment alone (black bar) (mean ± SD, n = 3). Asterisks indicates p<0.05 as determined by two-tailed t-test using Scr control (grey bar) as the reference and # indicates p<0.05 as determined by two-tailed t-test using MG132 (black bar) as the reference. In all panels GAPDH is used as loading control.
Figure 5.
Combined effect of eIF3H silencing and SAHA treament.
(A–B) Immunoblot analysis of HIF-1α, p53, eIF3H and GAPDH protein expression in cell lysates following siRNA-mediated silencing of eIF3H (sieIF3H) in presence or absence of SAHA+DFO (A) or SAHA+MG132 (B) in HuH7 cells. Quantitative analysis of the level of HIF-1α in response to silencing of the indicated eIF3H in HuH7 cells. Data shown denote the fold change in HIF-1α protein expression relative to DFO treatment+Scr control (A) MG132 treatment+Scr control (B) (black bar) (mean ± SD, n = 3). Asterisks indicates p<0.05 as determined by two-tailed t-test using Scr control+DFO (A) or Scr control+MG132 (B) (black bar) as the reference and # indicates p<0.05 as determined by two-tailed t-test using SAHA+MG132 (B) or SAHA+DFO (grey bar) as the reference. In all panels GAPDH is used as loading control.
Figure 6.
A model for SAHA mediated effects on HIF-1α translation.
In non-treated conditions (left panel-A), HIF-1α translation is controlled by HDAC9 and then HIF-1α mRNA is translated in protein. Following SAHA treatment (middle panel-B), HDAC9 is inhibited and we suggest that SAHA could promote mRNA and protein expression of an unknown protein (referred as X) that controls HIF-1α translation. As a result of this, the unknown protein could repress specifically HIF-1α translation. Upon the combined treatment SAHA and eIF3G silencing (right panel-C), we suggest that eIF3G may play a regulatory role in translation of the mRNA coding the unknown protein. By consequence, the silencing of eIF3G results in the inhibition of the unknown protein translation. As the expression of this unknown protein is down regulated, HIF-1α translation is not repressed anymore and could be translated de novo.