![Figure 6. BLVRBS111L hematopoietic effects on ROS accumulation and lineage fate. (A-B) CD34+ HSCs transduced with Lv/BLVRBWT, Lv/BLVRBS111L, or Lv/Control were puromycin-selected and expanded for 48 hours prior to terminal differentiation (day 0). Genetically modified cells were assessed for viability using (A) trypan blue exclusion (N = 4) or (B) cell quantification and flow cytometry. Cell quantification (N = 6) is expressed as relative fold-increase to control, and flow cytometric quantification was determined using gated, live (7-AAD negative) CD34+ cells (N = 4); flow cytometry gate (forward scatter [FSC], size vs side scatter [SSC], side scatter as a measure of complexity) used for quantification of 7-AAD negative CD34+ cells and for ROS tracking is shown. (C-D) Genetically modified CD34+ HSCs were puromycin selected and plated for determination of lineage fate using (C) multipotential progenitor assays (N = 4 experiments) or (D) CFU-MK determinations (N = 3 experiments). (E) Representative flow cytometric analysis of developing MKs dually labeled for ROS accumulation and CD41 at discrete time points (LV/control-infected CD34+ HSCs are shown). (F) Cumulative distribution plots across ROShigh subsets generated by flow cytometric quantification of MK phenotypic parameters (ROS and CD41 [Tpo cultures], or CD41 [CD41+/GlyA− [bilineage Tpo/Epo cultures]) are displayed by culture conditions and time points for genetically-modified CD34+/BLVRBWT, CD34+/BLVRBS111L, and CD34+/Control; results are from a single representative experiment repeated on 3 occasions; P values for pairwise comparisons were calculated over 200 bootstrapped samples. (G) Percentage of CD41−/GlyA+ erythroid cells in day 10 bilineage cultures (N = 3). (H) UV-visible spectroscopy of day 10 bilineage cultures (normalized to the peak Soret absorbance of oxyhemoglobin [λ414]) demonstrates no differences in methemoglobin (λ630) accumulation across the genotypes. For all panels except F, results are expressed as mean ± SEM; *P < .05, **P < .01, ***P < .001, N.S., not significant.](https://ash.silverchair-cdn.com/ash/content_public/journal/blood/128/5/10.1182_blood-2016-02-696997/4/699f6.jpeg?Expires=1723666029&Signature=C1F40bM24VIlquvsY~Qh~Ex2Qt2IFTbNx6jIe4b5oCbuMmye0y-UDqV46xWTtkmp--UszdttZrASwhE1kEUKjIeJ7nknui~3KJNU52bFFxMcZu2EXLXEhk3n7V7GB1vTxLVRwXWg0Nxw0UV~iToqBHRT0X3nfTDbb7OdMtOPH3S6Me6BctFe-uW2T8Li28onaRO1PL4E8Wkxd8rLxDlS0vg6mmRkn3mZpha8QoiMnB-s53TP3GVUZPIZOnXCMY5FseyCLRLMhPu9pf1pVVljL0RpKKU23sQgUp3b8ci8Hc7VXVCsuwzJ8TdRuKf2QGFhW7O9ptQsFG5FwrXvV876wA__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
BLVRBS111L hematopoietic effects on ROS accumulation and lineage fate. (A-B) CD34+ HSCs transduced with Lv/BLVRBWT, Lv/BLVRBS111L, or Lv/Control were puromycin-selected and expanded for 48 hours prior to terminal differentiation (day 0). Genetically modified cells were assessed for viability using (A) trypan blue exclusion (N = 4) or (B) cell quantification and flow cytometry. Cell quantification (N = 6) is expressed as relative fold-increase to control, and flow cytometric quantification was determined using gated, live (7-AAD negative) CD34+ cells (N = 4); flow cytometry gate (forward scatter [FSC], size vs side scatter [SSC], side scatter as a measure of complexity) used for quantification of 7-AAD negative CD34+ cells and for ROS tracking is shown. (C-D) Genetically modified CD34+ HSCs were puromycin selected and plated for determination of lineage fate using (C) multipotential progenitor assays (N = 4 experiments) or (D) CFU-MK determinations (N = 3 experiments). (E) Representative flow cytometric analysis of developing MKs dually labeled for ROS accumulation and CD41 at discrete time points (LV/control-infected CD34+ HSCs are shown). (F) Cumulative distribution plots across ROShigh subsets generated by flow cytometric quantification of MK phenotypic parameters (ROS and CD41 [Tpo cultures], or CD41 [CD41+/GlyA− [bilineage Tpo/Epo cultures]) are displayed by culture conditions and time points for genetically-modified CD34+/BLVRBWT, CD34+/BLVRBS111L, and CD34+/Control; results are from a single representative experiment repeated on 3 occasions; P values for pairwise comparisons were calculated over 200 bootstrapped samples. (G) Percentage of CD41−/GlyA+ erythroid cells in day 10 bilineage cultures (N = 3). (H) UV-visible spectroscopy of day 10 bilineage cultures (normalized to the peak Soret absorbance of oxyhemoglobin [λ414]) demonstrates no differences in methemoglobin (λ630) accumulation across the genotypes. For all panels except F, results are expressed as mean ± SEM; *P < .05, **P < .01, ***P < .001, N.S., not significant.
