Mcl-1 is required for maintaining the primitive lin⁻ CD34⁺CD38⁻ SRC-enriched phenotype in vitro. (A) Experimental strategy used to examine the role of Mcl-1 in the viability and function of primitive human HPCs in vitro. (B) Knockdown of the human Mcl-1 transcript in lin⁻ CB hematopoietic cells. Error bars represent the mean ± SEM of 3 independent experiments. (C) Knockdown of human Mcl-1 protein expression in lin⁻ CB hematopoietic cells. Protein expression is the mean fluorescence intensity (MFI) relative to the isotype control. Error bars represent the mean ± SEM of 3 independent experiments. (D) Analysis of the effect of Mcl-1 knockdown on the viability of human lin⁻CD34⁺CD38⁻ and CD34⁺CD38⁺ CB hematopoietic cells at 24 hours after transduction and after 1 week of ex vivo culture after transduction. Error bars represent the mean ± SEM of 3 independent experiments. (E) Average frequency of viable (7AAD⁻) annexin V⁺ human lin⁻ hematopoietic cells 24 hours after transduction with the empty vector or Mcl-1 shRNA-expressing vector. Error bars represent SEM (n = 3). (F) Analysis of the effect of Mcl-1 knockdown on primitive cell phenotypes within the human HSC hierarchy. Analysis was performed on transduced lin⁻ cells isolated from 7 independent CB donors. (G) Representative examples of flow cytometric analyses used to assess the effects of Mcl-1 knockdown on the primitive human hematopoietic phenotypes. Percentages represent frequency of total human cells with indicated cell surface phenotype. (H) Analysis of the effect of Mcl-1 knockdown on the frequency of human hematopoietic progenitors. Progenitor frequency is expressed as the number of hematopoietic colonies scored after 12 to 14 days per 1000 cells plated. Error bars represent the mean ± SEM of 4 independent experiments.