Figure 3
Figure 3. Salubrinal does not change cellular differentiation, proliferation, or globin gene expression in human primary erythroid cells. (A) Fluorescence-activated cell sorter analysis was performed with antibodies recognizing CD71 and CD235a on day 20. This representative experiment shows that salubrinal (Sal) does not change the overall differentiation profile relative to the untreated control (Untx). (B) Flow cytometry was performed in five independent experiments using two different donors to quantify CD71 and CD235a cell surface markers on day 20. Salubrinal (Sal) (3 μM and 6 μM) were compared with the untreated control. This box-and-whisker plot shows the distribution of the percent of cells that are double positive for CD71 and CD235a. The lines inside the box show the median, the boxes show the quartiles, and the whiskers show the extremes of the observed percentages. (C) Modified Wright-Giemsa staining of untreated and Sal treated cells on day 19 reveals similar stages of erythroid differentiation. Images were obtained with an Olympus BX51 microscope, 40× magnification, and Image-Pro Plus 7.0 software. (D) Sal does not alter cell growth during the treatment period. Sal was treated on days 15 and 18 and the fold increase of cell growth was compared with the untreated control on day 15. Cells were counted on days 15, 17, and 20 using trypan blue exclusion. Error bars represent ± SEM of three independent experiments (two unique donors). (E) The mRNA expression of γ- and β-globin was evaluated over the course of differentiation from day 7 through day 20. Sal treatments (3 μM and 6 μM) occurred on days 15 and 18, and the fold increase relative to the untreated control day 7 sample is shown. No significant changes in γ-globin or β-globin mRNA levels are observed. Error bars represent ± standard error of the mean of 3 independent experiments (2 different donors). (F) The AUC was calculated for γ- and β-globin mRNA expression from days 7 through 20. This representation of the data allows quantification of the total mRNA from each gene available for translation over the course of erythroid differentiation. Sal treatments (3 μM and 6 μM) on days 15 and 18 did not change the γ- or β-globin AUC relative to the untreated control. Error bars represent ± standard error of the mean of 3 independent experiments (2 different donors). (G) The AUCs for γ-globin and β-globin were compared as a γ/(γ + β) ratio. Sal treatment (3 μM and 6 μM) did not change this ratio relative to the untreated control in 3 independent experiments using 2 unique donors. Error bars denote ± standard error of the mean.

Salubrinal does not change cellular differentiation, proliferation, or globin gene expression in human primary erythroid cells. (A) Fluorescence-activated cell sorter analysis was performed with antibodies recognizing CD71 and CD235a on day 20. This representative experiment shows that salubrinal (Sal) does not change the overall differentiation profile relative to the untreated control (Untx). (B) Flow cytometry was performed in five independent experiments using two different donors to quantify CD71 and CD235a cell surface markers on day 20. Salubrinal (Sal) (3 μM and 6 μM) were compared with the untreated control. This box-and-whisker plot shows the distribution of the percent of cells that are double positive for CD71 and CD235a. The lines inside the box show the median, the boxes show the quartiles, and the whiskers show the extremes of the observed percentages. (C) Modified Wright-Giemsa staining of untreated and Sal treated cells on day 19 reveals similar stages of erythroid differentiation. Images were obtained with an Olympus BX51 microscope, 40× magnification, and Image-Pro Plus 7.0 software. (D) Sal does not alter cell growth during the treatment period. Sal was treated on days 15 and 18 and the fold increase of cell growth was compared with the untreated control on day 15. Cells were counted on days 15, 17, and 20 using trypan blue exclusion. Error bars represent ± SEM of three independent experiments (two unique donors). (E) The mRNA expression of γ- and β-globin was evaluated over the course of differentiation from day 7 through day 20. Sal treatments (3 μM and 6 μM) occurred on days 15 and 18, and the fold increase relative to the untreated control day 7 sample is shown. No significant changes in γ-globin or β-globin mRNA levels are observed. Error bars represent ± standard error of the mean of 3 independent experiments (2 different donors). (F) The AUC was calculated for γ- and β-globin mRNA expression from days 7 through 20. This representation of the data allows quantification of the total mRNA from each gene available for translation over the course of erythroid differentiation. Sal treatments (3 μM and 6 μM) on days 15 and 18 did not change the γ- or β-globin AUC relative to the untreated control. Error bars represent ± standard error of the mean of 3 independent experiments (2 different donors). (G) The AUCs for γ-globin and β-globin were compared as a γ/(γ + β) ratio. Sal treatment (3 μM and 6 μM) did not change this ratio relative to the untreated control in 3 independent experiments using 2 unique donors. Error bars denote ± standard error of the mean.

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