Validation assays for identified hits. (A) Schema of experimental design. (B) Long-term contribution of GFP⁺ (shLuc⁺, shJarid1b⁺, or NA10hd⁺) cells to peripheral blood reconstitution of recipients; GT >80% for all conditions. Left panel: recipients of day 0 cells; central and right panels, recipients of day 7 cells. NA10hd, cells engineered to overexpress NUP98Hoxa10-homeodomain fusion protein and GFP. (C) Evaluation of Jarid1b knockdown in GFP⁺shJarid1b-transduced cells compared with shLuc controls. Results represent average ± SEM; n = 4). Relative transcript quantities (RQ) values determined for 6 hairpins. Bars with dashed lines correspond to shRNA constructs (sh5,6 and 1) that did not achieve significant (ie, >30%) knockdown of Jarid1b in BM HSC. (D) Long-term contribution of GFP⁺ (shLuc⁺, or shMoz⁺, or shJhdm1f⁺) cells to peripheral blood reconstitution in recipients of day 0 cells. Each recipient received a one-quarter of the transduced cell population, or twice the number of input cells transplanted for validation experiment shown in Figure 3A. (E) Short-term (3 weeks) contribution of GFP⁺ (shLuc⁺- or shJhdm1f⁺-transduced) cells to peripheral blood reconstitution in recipients of day 0 cells. GT = 99% for all conditions. Experiment (as in Figure 4D) was repeated to include all hairpins against Jhdm1f. (F) Evaluation of Jhmdm1f knockdown in GFP⁺shJhdm1f-transduced cells compared with shLuc controls. Results represent average ± SEM (n = 3). RQ values determined for 5 hairpins. (G) Summary of screen results.