TGFβ-mediated cell-cycle arrest is protective for sf3b1-mutant erythrocytes. (A) Schematic of experiment to determine whether inhibition of TGFβ signaling alters erythroid cell-cycle arrest or anemia in sf3b1 mutants. (B) Graph quantifying the percentage of gata1:eGFP⁺ erythrocytes in G0/G1, S, or G2/M phases of the cell cycle at 24 hpf in sf3b1 mutants treated with a DMSO control or 25 μM of the TGFβ inhibitor SB431542. Cell cycle of wild-type siblings is included for comparison. Representative flow cytometry histograms of erythrocyte DNA content as measured by DAPI fluorescence intensity on sf3b1 mutants treated with a DMSO control or 25 μM SB431542 are shown on the right. Statistical significance calculated by an ANOVA with a Bonferroni FDR multitesting correction. (C) Graph showing frequency of sf3b1 mutants with designated levels of o-dianisidine–positive oxygenated erythrocytes at 48 hpf after treatment with DMSO control or 25 μM SB431542. Total number of mutants analyzed per treatment group is listed below the graph. Images of sf3b1-mutant embryos with low-medium or low-none o-dianisidine–positive cells are shown to the right. Significance of comparison between groups determined by the Fisher's exact test. All experiments were done in biological triplicates. **P < .01, ****P < .0001. (C) O-dianisidine stain; original magnification ×4.