Figure 3.

BCL11A-dependent and independent cooperativities after combinatorial HRI depletion and HbF pharmacologic induction. (A) BCL11A transcript levels (normalized to DMSO control) in healthy CD34⁺ donors, as measured by qRT-PCR after shRNA scrambled control or HRI depletion with 2 independent shRNAs combined with either vehicle control or HbF pharmacologic induction (50 μM hydroxyurea [HU], 1 μM pomalidomide [Pom], or 0.125 μM UNC0638). (B) LRF/ZBTB7A transcript levels (normalized to DMSO control) as measured by qRT-PCR. Each symbol (circle, square, triangle) represents a biologically independent sample. (C) Representative western blot after shRNA scrambled control or HRI depletion with HRI shRNA #2 combined with HbF pharmacologic inducers. (D) Western blot quantification of BCL11A protein levels and LRF/ZBTB7A protein levels from 3 independent biological samples, normalized to total protein levels. Statistical analyses by 2-way analysis of variance. Significance vs DMSO control. Three independent biological replicates for all experiments. Error bars represent standard deviation. ***P < .001. (E) Hierarchical clustering using all genes (unsupervised) of RNA-Seq data after shRNA-mediated HRI depletion combined with vehicle control, 1 μM pomalidomide, or 0.125 μM UNC0638. (F) Principal component analysis of RNA-Seq data (red, donor replicate #1; blue, donor replicate #2). Percentage variance represented by each principal component axis. (G) RNA-Seq data visualized as log₂ fold change in expression of known HbF regulators. DMSO served as the pharmacologic vehicle control. Two independent biological replicates for RNA-Seq analysis. **Absolute change >1.5-fold and adjusted P < .05.