Ssx2ip knock down impairs the function of erythroid precursors in regenerative erythropoiesis. (A) Quantitation of Ssx2ip mRNA level in cells after retroviral infection with nontargeting shControl or 2 shRNAs targeting Ssx2ip transcript (shSsx2ip-1 shSsx2ip-2) cells. Sorted GFP⁺CD71⁺Ter119⁻ and GFP⁺CD71⁺Ter119⁺ populations are shown. (B) Flow cytometric gating using anti-CD71 and anti-Ter119 antibodies in PHZ-treated, lineage-depleted spleen cells after retroviral infection with shControl or shSsx2ip-2. R1 to R5 represent distinct stages of erythroid maturation. (C) Quantitation from R1 to R5 percentages in knockdown cells (N = 3). (D) Wright-Giemsa staining of GFP sorted spleen cells infected with shControl or shSsx2ip-2. Arrows represent prominent cell morphologies in each culture. (E) Quantitation of forward scatter (FSC) relative MFI in R1 and R2 cell populations. (F) Representative histograms (left) and quantitation (right) of Kit fluorescence intensity in knockdown or control GFP⁺ R1 and R2 cells. (G) Quantitation of BFU-E (counted on day 5) and CFU-E (counted on day 2) in GFP sorted shControl or shSsx2ip-2-expressing cells cultured in methylcellulose media containing SCF and Epo (N = 4). (H) Violin plot of BFU-E colony size (mm²) in GFP sorted shControl or shSsx2ip-2-expressing cells cultured in methylcellulose media containing SCF and Epo (N = 6). Each dot represents 1 colony. Solid line indicated median and dashed line indicated quartiles. (I) mRNA expression of erythroid-related genes Kit, Gypa, and EpoR in GFP-sorted R2 or R3 cells infected with shControl or shSsx2ip-2–expressing retrovirus (N = 3). Error bars represent SD. ∗P < .05 (two-tailed unpaired Student t test). SCF, stem cell factor.