Figure 4.
ARID3A promotes terminal megakaryocytic differentiation. (A) ARID3A expression (reads per kilobase of transcript per million mapped reads, RPKM) in sorted HSPCs (peripheral blood [PB] mobilized CD34+ cells; n = 4), megakaryocyte (MK; CD41+CD61+; n = 3), and erythroid cells (CD71+, n = 3) derived from PB CD34+ HSPCs after 7 days of differentiation in megakaryocyte- or erythroid-promoting culture conditions (1-way ANOVA). (B) Colony counts after replating sh-ctrl-, miR-125b-, or shArid3a-transduced murine FLCs in low Thpo methylcellulose-based CFU assays (n = 6; 2-way ANOVA). The serial replating number is shown on the x-axis. (C-D) Ratio of terminal megakaryocyte (MK, CD41+CD42d+) and erythroid cells (ERY, CD71+Ter-119+) in shArid3a- vs sh-ctrl-transduced murine FLCs (C) and in Arid3a- vs LUC-transduced FLCs (D) after 6 and 4 days of differentiation, respectively (n = 3; paired Student t test). (E) Classification of colonies after plating cDNA-transduced (Arid3a or LUC) murine FLCs in methylcellulose-based CFU assays under complete cytokine conditions (n = 3; 2-way ANOVA). CFU-G/M: granulocytic (CFU-G), monocytic (CFU-M) and granulocytic/monocytic (CFU-GM); CFU/BFU-E: erythroid. (F) Dot plot showing the percentage of mature megakaryocytes (mature MK, CD41+CD42d+), immature megakaryocytes (Immature MK, CD41+CD42−), early erythroblasts (ProE I, CD71+Ter-119+), and late erythroblasts (ProE II, CD71−Ter-119+) in the BM of mouse recipients of shRNA-transduced Ter-119− FLCs (sh-ctrl or shArid3a). shRNA+ cells shown (n = 5, unpaired Student t test). (G) Percentage of differentiated cells after transduction of human PB CD34+ HSPCs with ARID3A cDNA, normalized to LUC-transduced HSPCs (n = 6; paired Student t test). Percentage of mature megakaryocytes (CD41+/CD61+/CD42+) after 11 days in medium promoting megakaryocytic differentiation (left). Percentage of mature erythroid cells (CD71+CD235a+) after 7 days in medium promoting erythroid differentiation (right). (H) Classification of colonies after plating ARID3A- or LUC-expressing human PB CD34+ HSPCs in methylcellulose-based CFU assays under complete cytokine conditions (n = 6, 2-way ANOVA). (I) Percentage of terminally differentiated cells after transduction of human PB CD34+ HSPCs with shRNAs targeting ARID3A, normalized to sh-ctrl (n = 3; paired Student t test). Percentage of mature megakaryocytes (CD41+CD61+CD42+) after 11 days in medium promoting megakaryocytic differentiation (left). Percentage of mature erythroid cells (CD71+CD235a+) after 7 days in medium promoting erythroid differentiation (right). Data are presented as the mean ± standard deviation. n.s., not significant.

ARID3A promotes terminal megakaryocytic differentiation. (A) ARID3A expression (reads per kilobase of transcript per million mapped reads, RPKM) in sorted HSPCs (peripheral blood [PB] mobilized CD34+ cells; n = 4), megakaryocyte (MK; CD41+CD61+; n = 3), and erythroid cells (CD71+, n = 3) derived from PB CD34+ HSPCs after 7 days of differentiation in megakaryocyte- or erythroid-promoting culture conditions (1-way ANOVA). (B) Colony counts after replating sh-ctrl-, miR-125b-, or shArid3a-transduced murine FLCs in low Thpo methylcellulose-based CFU assays (n = 6; 2-way ANOVA). The serial replating number is shown on the x-axis. (C-D) Ratio of terminal megakaryocyte (MK, CD41+CD42d+) and erythroid cells (ERY, CD71+Ter-119+) in shArid3a- vs sh-ctrl-transduced murine FLCs (C) and in Arid3a- vs LUC-transduced FLCs (D) after 6 and 4 days of differentiation, respectively (n = 3; paired Student t test). (E) Classification of colonies after plating cDNA-transduced (Arid3a or LUC) murine FLCs in methylcellulose-based CFU assays under complete cytokine conditions (n = 3; 2-way ANOVA). CFU-G/M: granulocytic (CFU-G), monocytic (CFU-M) and granulocytic/monocytic (CFU-GM); CFU/BFU-E: erythroid. (F) Dot plot showing the percentage of mature megakaryocytes (mature MK, CD41+CD42d+), immature megakaryocytes (Immature MK, CD41+CD42−), early erythroblasts (ProE I, CD71+Ter-119+), and late erythroblasts (ProE II, CD71−Ter-119+) in the BM of mouse recipients of shRNA-transduced Ter-119− FLCs (sh-ctrl or shArid3a). shRNA+ cells shown (n = 5, unpaired Student t test). (G) Percentage of differentiated cells after transduction of human PB CD34+ HSPCs with ARID3A cDNA, normalized to LUC-transduced HSPCs (n = 6; paired Student t test). Percentage of mature megakaryocytes (CD41+/CD61+/CD42+) after 11 days in medium promoting megakaryocytic differentiation (left). Percentage of mature erythroid cells (CD71+CD235a+) after 7 days in medium promoting erythroid differentiation (right). (H) Classification of colonies after plating ARID3A- or LUC-expressing human PB CD34+ HSPCs in methylcellulose-based CFU assays under complete cytokine conditions (n = 6, 2-way ANOVA). (I) Percentage of terminally differentiated cells after transduction of human PB CD34+ HSPCs with shRNAs targeting ARID3A, normalized to sh-ctrl (n = 3; paired Student t test). Percentage of mature megakaryocytes (CD41+CD61+CD42+) after 11 days in medium promoting megakaryocytic differentiation (left). Percentage of mature erythroid cells (CD71+CD235a+) after 7 days in medium promoting erythroid differentiation (right). Data are presented as the mean ± standard deviation. n.s., not significant.

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