Figure 3.
Arid3a knockdown mimics the miR-125b phenotype in Gata1s FLCs. (A) Percentage of transduced (sh-ctrl or shArid3a #2 or #3) Gata1s FLCs compared with that on day 0 (n = 4, 2-way ANOVA). (B) Percentage of stem/progenitor cells (CD117+), mature megakaryocytes (MK, CD41+ CD42d+), and erythroid cells (ERY, CD71+Ter-119+) in transduced (sh-ctrl or shArid3a #2 or #3) Gata1s FLCs after 6 days of differentiation (n = 3, paired Student t test). (C-D) Serial replating of transduced (sh-ctrl or shArid3a #2 or #3) Gata1s FLCs in methylcellulose-based medium with low concentrations of Thpo (20 ng/mL). (C) The number of megakaryocyte colonies, with the serial replating number on the x-axis (n = 4, 2-way ANOVA) (C), alongside a representative image from the first plating of 4 independent experiments (D). (E) Percentage of cDNA-transduced (Arid3a or LUC) miR-125b-Gata1s FLCs normalized to that on day 0 (n = 4, unpaired Student t test). (F) Percentage of stem/progenitor cells (CD117+), mature megakaryocytes (MK, CD41+CD42d+), and erythroid cells (ERY, CD71+Ter-119+) in cDNA-transduced (Arid3a or LUC) miR-125b-Gata1s FLCs after 6 days of differentiation (n = 3, paired Student t-test). (G-H) Methylcellulose-based colony-forming assays of cDNA-transduced (Arid3a or LUC) miR-125b Gata1s FLCs in low Thpo conditions (20 ng/mL; n = 4, unpaired Student t test). (G) The number of colonies (depicted in panel H) alongside a representative image from 4 independent experiments. (I-K) Analysis of C57BL/6J recipients of shRNA-transduced (sh-ctrl or shArid3a) Gata1s FLCs, including Kaplan-Meier survival curves (log-rank test) (I), spleen weights (unpaired Student t test) (J), and representative flow cytometry plots of BM-derived leukemic cells from the diseased mice (K). shArid3a-expressing Gata1s FLC–derived blasts are highlighted in green. The average percentage of shArid3a+ blasts belonging to each immunophenotype is indicated in the corresponding gate. All data are presented as the mean ± standard deviation. *P < .05; **P < .01; ***P < .001. FP+, fluorescent protein positive; n.s., not significant.

Arid3a knockdown mimics the miR-125b phenotype in Gata1s FLCs. (A) Percentage of transduced (sh-ctrl or shArid3a #2 or #3) Gata1s FLCs compared with that on day 0 (n = 4, 2-way ANOVA). (B) Percentage of stem/progenitor cells (CD117+), mature megakaryocytes (MK, CD41+ CD42d+), and erythroid cells (ERY, CD71+Ter-119+) in transduced (sh-ctrl or shArid3a #2 or #3) Gata1s FLCs after 6 days of differentiation (n = 3, paired Student t test). (C-D) Serial replating of transduced (sh-ctrl or shArid3a #2 or #3) Gata1s FLCs in methylcellulose-based medium with low concentrations of Thpo (20 ng/mL). (C) The number of megakaryocyte colonies, with the serial replating number on the x-axis (n = 4, 2-way ANOVA) (C), alongside a representative image from the first plating of 4 independent experiments (D). (E) Percentage of cDNA-transduced (Arid3a or LUC) miR-125b-Gata1s FLCs normalized to that on day 0 (n = 4, unpaired Student t test). (F) Percentage of stem/progenitor cells (CD117+), mature megakaryocytes (MK, CD41+CD42d+), and erythroid cells (ERY, CD71+Ter-119+) in cDNA-transduced (Arid3a or LUC) miR-125b-Gata1s FLCs after 6 days of differentiation (n = 3, paired Student t-test). (G-H) Methylcellulose-based colony-forming assays of cDNA-transduced (Arid3a or LUC) miR-125b Gata1s FLCs in low Thpo conditions (20 ng/mL; n = 4, unpaired Student t test). (G) The number of colonies (depicted in panel H) alongside a representative image from 4 independent experiments. (I-K) Analysis of C57BL/6J recipients of shRNA-transduced (sh-ctrl or shArid3a) Gata1s FLCs, including Kaplan-Meier survival curves (log-rank test) (I), spleen weights (unpaired Student t test) (J), and representative flow cytometry plots of BM-derived leukemic cells from the diseased mice (K). shArid3a-expressing Gata1s FLC–derived blasts are highlighted in green. The average percentage of shArid3a+ blasts belonging to each immunophenotype is indicated in the corresponding gate. All data are presented as the mean ± standard deviation. *P < .05; **P < .01; ***P < .001. FP+, fluorescent protein positive; n.s., not significant.

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