Figure 2
Rescue with GATA1 WT improves erythroid differentiation and gene expression in primary R307C cells. (A) Schematic of primary cell rescue experiment. Indicated expression vectors were introduced to R307C patient bone marrow (BM) mononuclear (MNC) or healthy donor cells via lentiviral integration followed by in vitro culture. (B) Ratio of CD235+/CD235− cells as determined by flow cytometry of R307C cells transduced with indicated expression constructs after 5 days postinfection (pi). Error bars represent 1 standard error of the mean. (C) Flow cytometric plots (top row) of CD11b/CD41a and CD235a surface marker–stained populations and images of May-Grünwald-Giemsa–stained cytospin preparations (bottom row; ×63 original magnification) of R307C cells transduced with indicated expression constructs after 5 days pi. (D) Histogram plots of forward scatter of R307C cells transduced with indicated expression constructs assessed after 5 days pi. (E) Volcano plot showing differentially expressed genes comparing R307C cells transduced with mutant (R307C/H) with GATA1 WT–expressing vectors. Selected genes are indicated. Two replicates for GATA1 WT and all 4 mutant replicates were pooled for this analysis. (F) Heatmap showing results of k-means clustering of differentially expressed genes of in vitro cultured R307C patient cells transduced with indicated GATA1 constructs. Results of 2 replicates for each construct were pooled for this analysis. Color bar: z-scored expression. (G) z-score–transformed expression abundances based on RNA-seq for indicated GATA1 genotypes and selected genes with associated biological functions. Individual replicate results are depicted in the heatmap (2 per construct). (H) Bar graph showing ADA messenger RNA expression levels for indicated GATA1 genotypes. Individual replicates results are shown (dots; 2 replicates per construct). (I) Gene set enrichment analyses for indicated gene sets for differential expression analysis comparing R307C cells transduced with mutant and GATA1 WT. Two replicates for GATA1 WT and all 4 mutant replicates were pooled for this analysis. cpm, count per million; FDR, false discovery rate; Mb, metabolism; NES, normalized enrichment score; Tx, transcription.

Rescue with GATA1 WT improves erythroid differentiation and gene expression in primary R307C cells. (A) Schematic of primary cell rescue experiment. Indicated expression vectors were introduced to R307C patient bone marrow (BM) mononuclear (MNC) or healthy donor cells via lentiviral integration followed by in vitro culture. (B) Ratio of CD235+/CD235− cells as determined by flow cytometry of R307C cells transduced with indicated expression constructs after 5 days postinfection (pi). Error bars represent 1 standard error of the mean. (C) Flow cytometric plots (top row) of CD11b/CD41a and CD235a surface marker–stained populations and images of May-Grünwald-Giemsa–stained cytospin preparations (bottom row; ×63 original magnification) of R307C cells transduced with indicated expression constructs after 5 days pi. (D) Histogram plots of forward scatter of R307C cells transduced with indicated expression constructs assessed after 5 days pi. (E) Volcano plot showing differentially expressed genes comparing R307C cells transduced with mutant (R307C/H) with GATA1 WT–expressing vectors. Selected genes are indicated. Two replicates for GATA1 WT and all 4 mutant replicates were pooled for this analysis. (F) Heatmap showing results of k-means clustering of differentially expressed genes of in vitro cultured R307C patient cells transduced with indicated GATA1 constructs. Results of 2 replicates for each construct were pooled for this analysis. Color bar: z-scored expression. (G) z-score–transformed expression abundances based on RNA-seq for indicated GATA1 genotypes and selected genes with associated biological functions. Individual replicate results are depicted in the heatmap (2 per construct). (H) Bar graph showing ADA messenger RNA expression levels for indicated GATA1 genotypes. Individual replicates results are shown (dots; 2 replicates per construct). (I) Gene set enrichment analyses for indicated gene sets for differential expression analysis comparing R307C cells transduced with mutant and GATA1 WT. Two replicates for GATA1 WT and all 4 mutant replicates were pooled for this analysis. cpm, count per million; FDR, false discovery rate; Mb, metabolism; NES, normalized enrichment score; Tx, transcription.

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