American Society of Hematology

Figure 5.

$PLAG1-S dampens protein synthesis and promotes dormancy in stimulated human HSPCs. (A) OP-Puro incorporation dynamics measured as median fluorescence intensity (MFI) in cultured Lin−CD34+ cells with representative flow cytometry plots (n = 5 for 0 and 24 hours; n = 3 for 4, 48, and 72 hours). Red and blue asterisks denote statistical significance relative to previous timepoint or T0, respectively. (B) Fold difference of OP-Puro MFI relative to T0 in cultured Lin−CD34+ compared with Lin−CD34− CB fractions (n = 4 for 24 hours, n = 2 for 48 hours). Blue statistics are relative to 1× levels at T0 and red statistics are between cell types at matched time points. (C) OP-Puro incorporation by PLAG1-SOE and control Lin−CD34+ cells on day 4 of ex vivo culture (n = 8). Data from 3 experiments normalized to the average MFI in control cells per experiment. (D) Reduced size of PLAG1-SOE Lin−CD34+ cells on day 4 of ex vivo culture determined by flow cytometric MFI of FSC-H profiles (n = 7, left, each point is from a culture of an individual CB unit) and immunofluorescence microscopy (right, each point is a single cell; scale bar = 25 μm). (E) Cell-cycle analysis by Hoechst and Ki67 staining of PLAG1-SOE and control Lin−CD34+ cells on days 4 and 7 of ex vivo culture (n = 3). (F) Representative flow plots for PLAG1-SOE and control Lin−CD34+ cells stained for 7-AAD and Annexin V with apoptosis measurements of surface positivity of Annexin V on day 4 (n = 5) and day 7 (n = 4) of ex vivo culture. (G) Heatmap of log2FC of transcripts coding EIF2 subunits (bottom) and intracellular flow cytometric measures of EIF2S1 protein expression (n = 4) in PLAG1-SOE relative to control Lin−CD34+ cells on day 4 of ex vivo culture (top). (H) GSEA of the PLAG1-SOE transcriptome to curated targets of ATF4 generated by Han et al74 and used by van Galen et al73 and FPKM heatmap of ATF4 targets differentially expressed in PLAG1-SOE HSPCs (p.adj < .1). (I) Negative enrichment of gene sets related to unfolded protein response (P < .05). Data are presented as average ± SEM unless otherwise indicated. Each point represents an individual CB unit otherwise indicated. ***P < .005, **P < .01, *P < .05. See also supplemental Figure 5. FSC-H, Forward Scatter Height; n.s., not significant; p.adj, adjusted P value.$

PLAG1-S dampens protein synthesis and promotes dormancy in stimulated human HSPCs. (A) OP-Puro incorporation dynamics measured as median fluorescence intensity (MFI) in cultured Lin⁻CD34⁺ cells with representative flow cytometry plots (n = 5 for 0 and 24 hours; n = 3 for 4, 48, and 72 hours). Red and blue asterisks denote statistical significance relative to previous timepoint or T0, respectively. (B) Fold difference of OP-Puro MFI relative to T0 in cultured Lin⁻CD34⁺ compared with Lin⁻CD34⁻ CB fractions (n = 4 for 24 hours, n = 2 for 48 hours). Blue statistics are relative to 1× levels at T0 and red statistics are between cell types at matched time points. (C) OP-Puro incorporation by PLAG1-S^OE and control Lin⁻CD34⁺ cells on day 4 of ex vivo culture (n = 8). Data from 3 experiments normalized to the average MFI in control cells per experiment. (D) Reduced size of PLAG1-S^OE Lin⁻CD34⁺ cells on day 4 of ex vivo culture determined by flow cytometric MFI of FSC-H profiles (n = 7, left, each point is from a culture of an individual CB unit) and immunofluorescence microscopy (right, each point is a single cell; scale bar = 25 μm). (E) Cell-cycle analysis by Hoechst and Ki67 staining of PLAG1-S^OE and control Lin⁻CD34⁺ cells on days 4 and 7 of ex vivo culture (n = 3). (F) Representative flow plots for PLAG1-S^OE and control Lin⁻CD34⁺ cells stained for 7-AAD and Annexin V with apoptosis measurements of surface positivity of Annexin V on day 4 (n = 5) and day 7 (n = 4) of ex vivo culture. (G) Heatmap of log2FC of transcripts coding EIF2 subunits (bottom) and intracellular flow cytometric measures of EIF2S1 protein expression (n = 4) in PLAG1-S^OE relative to control Lin⁻CD34⁺ cells on day 4 of ex vivo culture (top). (H) GSEA of the PLAG1-S^OE transcriptome to curated targets of ATF4 generated by Han et al⁷⁴ and used by van Galen et al⁷³ and FPKM heatmap of ATF4 targets differentially expressed in PLAG1-S^OE HSPCs (p.adj < .1). (I) Negative enrichment of gene sets related to unfolded protein response (P < .05). Data are presented as average ± SEM unless otherwise indicated. Each point represents an individual CB unit otherwise indicated. ***P < .005, **P < .01, *P < .05. See also supplemental Figure 5. FSC-H, Forward Scatter Height; n.s., not significant; p.adj, adjusted P value.

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