Page 159 - 2021_06-Haematologica-web

CAR promotes hematopoietic regeneration
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Figure 6. CAR enhances regeneration by stimulating Notch signaling. (A) Left panel: representative flow cytometry analyses of Numb negative and positive staining of LSKFC populations of 5-fluorouracil (5-FU) treated mice. Right top panel: Quantification of Numb negative staining in LSKFC. Right bottom panel: Quantification of Numb mRNA levels in LSKFC populations of 5-FU treated mice (the mRNA levels were calculated based on the 20-hour group, and experiments were repeated three times). (B) Top panel: representative flow cytometry analyses of Numb negative and positive staining in LSKFC populations at 36 hours after 5-FU treatment. Bottom panel: quantification of percentage of Numb negative LSKF cells in wild-type (WT) and CAR conditional knockout (cKO) cells, n=5 mice per group. (C) Numb and mouse CAR co-staining in LSKFC population of mice at 36 hours after 5-FU treatment. (D) Survival curves of WT and CAR cKO mice after injection with 300 mg/kg 5-FU and Notch signal inhibit DAPT, n=9-12 mice per group. (E) Right panel: representative flow cytometry analyses of membrane Notch1/2 staining in LSKFC populations at 36 hours after 5-FU treatment. Left top panel: quantification of percentage of Notch1-stained cells in LSKFC populations. Left bottom panel: quan- tification of percentage of Notoch2-stained cells in LSKFC populations, n=9 mice per group. (F) mRNA levels of CAR Notch target genes hes1, hey1, and myc in LSKFC populations before (right panel) and 36 hours after 5-FU treatment (left panel). (G) Notch1 and mCAR co-staining in LSKFC population of mice at 36 hours after 5- FU treatment. (H) Flow chart of experiment used to test whether Notch, LNX1 or LNX2 is involved in CAR function. DN-MAML, DN-LNX1 or DN-LNX2 was expressed in Lin- BM cells from CAR cKO mice without treatment of tamoxifen (UBC-Cre-ERT2/CARloxp/loxp), and these cells were used in repopulation assays. The recipient mice were treated with tamoxifen as CAR cKO group and treated with vehicle as WT group. (I and J) Percent repopulation of peripheral blood (PB) (left panels), the Mac1+ population (middle panels), and the B220+ population (right panels) by cells, n=8-10 mice per group. *P<0.05.
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