Figure 1. A. Methylcellulose colony assay on bone marrow cells from doxycycline treated shRNA Rpl5 and control mice (n=3), (CFU-E, P=0.0003; BFU-E, P=1; GFU-GM, P=0.3); B. FACS analysis of bone marrow cells from shRNA Rpl5 and control mice. Total bone marrow cells from shRNA Rpl5 (B.1) and control (B.2) mice; viable cells were gated for analysis and sorting. We used the cell-surface markers CD71 (Y-axis) and Ter119 (X-axis) to separate mature erythroids and erythroid precursors. The most mature erythroblasts are CD71lowTer119high, while the erythroid precursors are CD71highTer119intermediate cells. In our FACS analysis the gated population comprises CD71highTer119med cells (P4) and CD71highTer119high cells (P5) from shRNA Rpl5 (B.3 and B.5) and control (B4 and B6) mice; the results here are collected from one mouse per group and are representative of 6 independent experiments; in each experiment, cells were collected from 1-3 mice for each group and sorted independently; C. Western Blot analysis of RPL5 protein expressions in CD71highTer119high cells. There was a significant decrease in the expression level of RPL5 in the treated shRNA Rpl5 compared to the control mice; D. Western Blot analysis of GATA1 protein expressions in CD71highTer119high cells. There was a significant decrease in the expression of GATA1 in the shRNA Rpl5 group compared to the control mice. In these experiments, GAPDH was used as a loading control, and they are representative of 3 independent experiments.