Figure 6. HIF-1α translation inhibition... | Rockefeller University Press

Figure 6.

$Figure 6. HIF-1α translation inhibition is reversed after microtubule repolymerization. (A) HeLa–GFP-Ago2 (green) cells treated with 10 µM Noc for 6 h followed by drug washout (W/O). Cells were fixed at the indicated times and immunostained for α-tubulin (white). The percentage of cells with either three or less or more than three P-bodies is plotted (mean ± SEM; n = 10 fields of view/condition). (B) HIF-1α and actin protein levels in HeLa–GFP-Ago2 cells treated with 1 µM Noc overnight and lysed after 6-h washout (6) or continued drug treatment (−) with 4-h hypoxia. (C) The percentage of HIF-1α mRNA associated with untranslating fractions (1_6) or actively translating polysomes (7_12) after 1 µM Noc treatment overnight and 6 h drug washout is plotted (mean ± SEM; n = 3; *, P < 0.05). (D) HeLa–GFP-Ago2 cells were incubated in glucose-free media for 1 h and stained for α-tubulin (white). The percentage of cells with three or less or more than three P-bodies (mean ± SEM; n = 3; *, P < 0.05) is plotted. (E) Immunoblot of HIF-1α and actin after 1-h glucose starvation and 4-h hypoxia. (F) Ago2-bound RNA extracted from control (Ctrl), glucose-starved (1 h) cells, or cells treated overnight with 1 µM Noc (Noc) and followed by 6-h washout. HIF-1α expression was quantified by qRT-PCR (mean ± SEM; n = 2). Bars, 10 µm.$

HIF-1α translation inhibition is reversed after microtubule repolymerization. (A) HeLa–GFP-Ago2 (green) cells treated with 10 µM Noc for 6 h followed by drug washout (W/O). Cells were fixed at the indicated times and immunostained for α-tubulin (white). The percentage of cells with either three or less or more than three P-bodies is plotted (mean ± SEM; n = 10 fields of view/condition). (B) HIF-1α and actin protein levels in HeLa–GFP-Ago2 cells treated with 1 µM Noc overnight and lysed after 6-h washout (6) or continued drug treatment (−) with 4-h hypoxia. (C) The percentage of HIF-1α mRNA associated with untranslating fractions (1_6) or actively translating polysomes (7_12) after 1 µM Noc treatment overnight and 6 h drug washout is plotted (mean ± SEM; n = 3; *, P < 0.05). (D) HeLa–GFP-Ago2 cells were incubated in glucose-free media for 1 h and stained for α-tubulin (white). The percentage of cells with three or less or more than three P-bodies (mean ± SEM; n = 3; *, P < 0.05) is plotted. (E) Immunoblot of HIF-1α and actin after 1-h glucose starvation and 4-h hypoxia. (F) Ago2-bound RNA extracted from control (Ctrl), glucose-starved (1 h) cells, or cells treated overnight with 1 µM Noc (Noc) and followed by 6-h washout. HIF-1α expression was quantified by qRT-PCR (mean ± SEM; n = 2). Bars, 10 µm.