NELF depletion leads to a genome-wide loss of transcription in progenitor cells. (A) Western blot of NELF-B and -E in human CD34⁺ HSPCs transfected with a scramble siRNA (ctrl), or siRNA targeting NELF-B or -E. Protein extraction was done on day 3 posttransfection. GAPDH is used as a loading control. (B) Quantification of western blot in A by imageJ. Protein level is normalized to GAPDH and presented as fold change relative to control samples (n = 3; mean ± SEM; **P < .01). (C) Metagene analysis showing Pol II occupancy measured by GRO-seq on both sense and antisense strands in human CD34⁺ HSPCs transfected with control-siRNA (ctrl) or siRNA targeting NELF-E (NE-KD). Cells were collected on day 3 posttransfection. (D) Boxplot analysis to compare PI between cells transfected with a control-siRNA (ctrl), or siRNA targeting NELF-B (NB-KD) or -E (NE-KD). (E) Cumulative distribution function analysis to compare PI distribution in cells transfected with a control-siRNA (ctrl), or siRNA targeting NELF-E (NE-KD). (F) Venn diagram showing the overlap of upregulated (“up”) and downregulated genes (“down”) between NELF-B and NELF-E knockdown cells.