Figure 3.
Dominant-negative effects of SRP54 mutations are conserved in the human HL-60 cell line and lead to differentiation defects. (A) Schematic overview of the experimental setup. (B) Western blots document elevated SRP54 protein expression (top) in HL-60 cells upon transduction with mutant or wt SRP54 constructs. Nontransduced control cells (–) are shown on the left. The following expression fold changes were deduced from area counts relative to empty control as quantified by Fiji software: wt, 2.8; T115A, 2.9; T117Δ, 1.9; and G226E, 3.5. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as loading control and for normalization. (C) Representative images of cells during ATRA-driven HL-60 cell differentiation (left: blast cell, day 0; middle: lobulated neutrophil, day 3; right: multilobulated neutrophil, day 6). Photographs were taken from hematoxylin and eosin (H&E)–stained cytospots. (D) Quantification of H&E-stained cytospots after 6 days of treatment with ATRA. Criteria for classification: No lobules indicates blasts; 1 to 5 lobules indicates lobulated; >5 lobules indicates multilobed. Statistics: empty vs SRP54 (wt): blasts, lobulated, and multilobed are not significant; empty vs T115A: blasts are not significant and lobulated and multilobed have P < .05; empty vs T117Δ: blasts, lobulated, and multilobed are not significant; empty vs G226E: blasts are not significant and lobulated and multilobed have P < .005. (E) Histograms indicating CD11b surface staining on empty control, SRP54 (WT), T115A, T117Δ, and G226E transduced HL-60 cells as analyzed by flow cytometry. (F) Corresponding quantification of CD11b expression on empty control, SRP54 (WT), T115A, T117Δ, and G226E transduced cells. Plot shows the geometric (G) mean fluorescence intensity shift in the CD11b channel upon treatment with ATRA (y-axis) per indicated cell lines (x-axis). Note slightly reduced CD11b surface induction in T117Δ cells but a significant differentiation block in cells expressing T115A and G226E mutant forms of SRP54. Student t test was used for statistical analysis. diff, differentiation; FACS, fluorescence-activated cell sorting. *P < .05. Bar plots represent the mean value of the replicates. Error bars indicate the standard deviation of the mean.

Dominant-negative effects of SRP54 mutations are conserved in the human HL-60 cell line and lead to differentiation defects. (A) Schematic overview of the experimental setup. (B) Western blots document elevated SRP54 protein expression (top) in HL-60 cells upon transduction with mutant or wt SRP54 constructs. Nontransduced control cells (–) are shown on the left. The following expression fold changes were deduced from area counts relative to empty control as quantified by Fiji software: wt, 2.8; T115A, 2.9; T117Δ, 1.9; and G226E, 3.5. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as loading control and for normalization. (C) Representative images of cells during ATRA-driven HL-60 cell differentiation (left: blast cell, day 0; middle: lobulated neutrophil, day 3; right: multilobulated neutrophil, day 6). Photographs were taken from hematoxylin and eosin (H&E)–stained cytospots. (D) Quantification of H&E-stained cytospots after 6 days of treatment with ATRA. Criteria for classification: No lobules indicates blasts; 1 to 5 lobules indicates lobulated; >5 lobules indicates multilobed. Statistics: empty vs SRP54 (wt): blasts, lobulated, and multilobed are not significant; empty vs T115A: blasts are not significant and lobulated and multilobed have P < .05; empty vs T117Δ: blasts, lobulated, and multilobed are not significant; empty vs G226E: blasts are not significant and lobulated and multilobed have P < .005. (E) Histograms indicating CD11b surface staining on empty control, SRP54 (WT), T115A, T117Δ, and G226E transduced HL-60 cells as analyzed by flow cytometry. (F) Corresponding quantification of CD11b expression on empty control, SRP54 (WT), T115A, T117Δ, and G226E transduced cells. Plot shows the geometric (G) mean fluorescence intensity shift in the CD11b channel upon treatment with ATRA (y-axis) per indicated cell lines (x-axis). Note slightly reduced CD11b surface induction in T117Δ cells but a significant differentiation block in cells expressing T115A and G226E mutant forms of SRP54. Student t test was used for statistical analysis. diff, differentiation; FACS, fluorescence-activated cell sorting. *P < .05. Bar plots represent the mean value of the replicates. Error bars indicate the standard deviation of the mean.

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