American Society of Hematology

Figure 2

$Figure 2. Sal selectively increases the translation efficiency of γ-globin mRNA. (A) Representative polysome profile on day 15 after 6 hours of 12 μM Sal treatment reveals a polysome-to-monosome shift, indicative of halted translation initiation and reduced translation. (B) Western blotting shows that p-eIF2α is increased at 3 and 6 hours after 12 μM Sal treatment on day 15 relative to an untreated control. At 24 hours after treatment, p-eIF2α levels are similar between the 2 conditions. Total eIF2α and β-actin are used as loading controls. (C) After 6 hours of treatment with 12 μM Sal, γ- and β-globin translation efficiency is unchanged relative to the untreated control. Polysome profiling fractions were pooled into 7 larger fractions containing ribosome-free 40S/60S or 80S, or polysome-bound RNA ranging from lower to higher ribosome occupancy. γ- and β-globin mRNA were quantified in each pooled fraction and normalized to an exogenous luciferase mRNA control. The percent of total mRNA for each fraction was calculated. Error bars represent ± SEM of 3 independent experiments. (D) After 24 hours of 12 μM Sal treatment, the representative polysome profile on day 16 shows a slight polysome-to-monosome shift. (E) After 24 hours, 12 μM Sal significantly increases the translation efficiency of γ- and β-globin mRNA, whereas β-actin and activating transcription factor 4 translation is not significantly changed. mRNA was quantified in each pooled fraction and normalized to an exogenous luciferase control, and the percentage of total mRNA found in each fraction was calculated. Error bars represent ± SEM of 3 independent experiments. P values were determined by using an unpaired 2-tailed Student t test. *P < .05; **P < .01. (F) At 24 hours, Sal increases translation efficiency of γ-globin to a greater extent than β-globin. Percentage of mRNA in the lightest 2 polysome fractions was compared with the percentage of mRNA in the heaviest 2 polysome fractions by using a heavy-to-light polysome ratio. Error bars represent ± SEM of 3 independent experiments. P values were determined by using an unpaired 2-tailed Student t test. *P < .05. (G) The representative polysome profile at 24 hours shows ribosome dissociation and a dramatic shift to the monosome peak when 12 μM Sal and the untreated control are incubated with 1 mM puromycin (Puro). (H) Puromycin treatment after 24 hours of 12 μM Sal shifts the percentage of γ- and β-globin mRNA to lighter polysome fractions to the same extent as in the untreated control. Error bars represent ± SEM of 2 independent experiments. (I) Puromycin reduces the heavy:light polysome ratio to the same extent in 12 μM Sal-treated and untreated cells. Error bars represent ± SEM of 2 independent experiments.$

Sal selectively increases the translation efficiency of γ-globin mRNA. (A) Representative polysome profile on day 15 after 6 hours of 12 μM Sal treatment reveals a polysome-to-monosome shift, indicative of halted translation initiation and reduced translation. (B) Western blotting shows that p-eIF2α is increased at 3 and 6 hours after 12 μM Sal treatment on day 15 relative to an untreated control. At 24 hours after treatment, p-eIF2α levels are similar between the 2 conditions. Total eIF2α and β-actin are used as loading controls. (C) After 6 hours of treatment with 12 μM Sal, γ- and β-globin translation efficiency is unchanged relative to the untreated control. Polysome profiling fractions were pooled into 7 larger fractions containing ribosome-free 40S/60S or 80S, or polysome-bound RNA ranging from lower to higher ribosome occupancy. γ- and β-globin mRNA were quantified in each pooled fraction and normalized to an exogenous luciferase mRNA control. The percent of total mRNA for each fraction was calculated. Error bars represent ± SEM of 3 independent experiments. (D) After 24 hours of 12 μM Sal treatment, the representative polysome profile on day 16 shows a slight polysome-to-monosome shift. (E) After 24 hours, 12 μM Sal significantly increases the translation efficiency of γ- and β-globin mRNA, whereas β-actin and activating transcription factor 4 translation is not significantly changed. mRNA was quantified in each pooled fraction and normalized to an exogenous luciferase control, and the percentage of total mRNA found in each fraction was calculated. Error bars represent ± SEM of 3 independent experiments. P values were determined by using an unpaired 2-tailed Student t test. *P < .05; **P < .01. (F) At 24 hours, Sal increases translation efficiency of γ-globin to a greater extent than β-globin. Percentage of mRNA in the lightest 2 polysome fractions was compared with the percentage of mRNA in the heaviest 2 polysome fractions by using a heavy-to-light polysome ratio. Error bars represent ± SEM of 3 independent experiments. P values were determined by using an unpaired 2-tailed Student t test. *P < .05. (G) The representative polysome profile at 24 hours shows ribosome dissociation and a dramatic shift to the monosome peak when 12 μM Sal and the untreated control are incubated with 1 mM puromycin (Puro). (H) Puromycin treatment after 24 hours of 12 μM Sal shifts the percentage of γ- and β-globin mRNA to lighter polysome fractions to the same extent as in the untreated control. Error bars represent ± SEM of 2 independent experiments. (I) Puromycin reduces the heavy:light polysome ratio to the same extent in 12 μM Sal-treated and untreated cells. Error bars represent ± SEM of 2 independent experiments.

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