Figure 6
Figure 6. Inhibition of erythroid colony formation from PV patients by the Jak2V617F-specific shRNA and rescue by EPO. (A) Percentage of Jak2V617F-positive BFU-E and EEC colonies isolated from 4 patient CD34+ cell cultures. Genomic DNA from individual colonies was analyzed for Jak2V617F or Jak2WT content by allele-specific PCR. Main zygosity of progenitor cells for the mutation is indicated (Hom., homozygous; Het., heterozygous; NS, no added EPO). (B) Western blot analysis of JAK2 and β-actin (loading control) protein expression levels from infected (GFP+) cells cultured 24 hours or 48 hours after infection in the presence of 1 U/mL EPO using 2 control, PV1, and PV3 patient CD34+ blood cells. Results show efficient Jak2V617F gene silencing in PV patient cells. (C) Western blot analysis of JAK2, phospho-STAT5, STAT5, and β-actin protein expression levels from infected cells (prepared as in B) using PV2 and PV4 patient cells. Results show efficient Jak2V617F gene silencing and a decrease in STAT5 phosphorylation in PV patient cells. (D) CD34+ cells from PV patients and 2 controls were infected with the pRRL-mt4 (■) or control pRRL-Scr (□) lentivirus, and GFP-positive cells were plated at 2 × 103 cells in methylcellulose semisolid culture medium in the presence of SCF, IL-3, and with (+) or without (−) EPO. The number of BFU-E–derived colonies from pRRL-mt4 or control pRRL-Scr lentivirus-infected cells was analyzed after 14 days of culture. (E) Number of BFU-E–derived colonies from pRRL-mt4 or control pRRL-Scr lentivirus-infected cells analyzed after 14 days of culture in the absence or the presence of 10 mU/mL, 100 mU/mL, or 1000 mU/mL EPO. (D-E) Results are expressed in mean numbers of colonies (± SD) per 2 × 103 plated cells from triplicate cultures of single experiments. **P ≤ .05 and ***P ≤ .005. They show efficient and selective inhibition of colony formation from PV patient cells by the Jak2V617F-specific shRNA. (E) The presence of high concentrations of EPO (100 and 1000 mU/mL) blocked the inhibition induced by the shRNA.

Inhibition of erythroid colony formation from PV patients by the Jak2V617F-specific shRNA and rescue by EPO. (A) Percentage of Jak2V617F-positive BFU-E and EEC colonies isolated from 4 patient CD34+ cell cultures. Genomic DNA from individual colonies was analyzed for Jak2V617F or Jak2WT content by allele-specific PCR. Main zygosity of progenitor cells for the mutation is indicated (Hom., homozygous; Het., heterozygous; NS, no added EPO). (B) Western blot analysis of JAK2 and β-actin (loading control) protein expression levels from infected (GFP+) cells cultured 24 hours or 48 hours after infection in the presence of 1 U/mL EPO using 2 control, PV1, and PV3 patient CD34+ blood cells. Results show efficient Jak2V617F gene silencing in PV patient cells. (C) Western blot analysis of JAK2, phospho-STAT5, STAT5, and β-actin protein expression levels from infected cells (prepared as in B) using PV2 and PV4 patient cells. Results show efficient Jak2V617F gene silencing and a decrease in STAT5 phosphorylation in PV patient cells. (D) CD34+ cells from PV patients and 2 controls were infected with the pRRL-mt4 (■) or control pRRL-Scr (□) lentivirus, and GFP-positive cells were plated at 2 × 103 cells in methylcellulose semisolid culture medium in the presence of SCF, IL-3, and with (+) or without (−) EPO. The number of BFU-E–derived colonies from pRRL-mt4 or control pRRL-Scr lentivirus-infected cells was analyzed after 14 days of culture. (E) Number of BFU-E–derived colonies from pRRL-mt4 or control pRRL-Scr lentivirus-infected cells analyzed after 14 days of culture in the absence or the presence of 10 mU/mL, 100 mU/mL, or 1000 mU/mL EPO. (D-E) Results are expressed in mean numbers of colonies (± SD) per 2 × 103 plated cells from triplicate cultures of single experiments. **P ≤ .05 and ***P ≤ .005. They show efficient and selective inhibition of colony formation from PV patient cells by the Jak2V617F-specific shRNA. (E) The presence of high concentrations of EPO (100 and 1000 mU/mL) blocked the inhibition induced by the shRNA.

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