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. 2021 Nov 8;36(3):701–711. doi: 10.1038/s41375-021-01462-4

Fig. 3. Loss of CSF2RB attenuates FLT3-ITD-dependent transformation and proliferation and prolongs survival in xenograft and bone marrow transplantation models.

Fig. 3

A–D Immunodeficient Rag2/Il2rg-mutant mice were sublethally irradiated and injected intravenously with FLT3-ITD-positive MOLM-13 cells stably expressing CSF2RB shRNA (n = 12) or control shRNA (n = 10) together with luciferase. One animal was excluded as no signs of engraftment could be detected at any time point. A Mice were injected intraperitoneally with luciferin and subjected to bioluminescence imaging, as indicated. B Quantification of bioluminescence is shown. P value was calculated using a two-sided Mann–Whitney test. C Survival curve of mice transplanted with MOLM-13 cells expressing CSF2RB shRNA or control shRNA. P values were calculated by Mantel–Cox test, n represents biologically independent mice. D The same MOLM-13 cells were subjected to lysis, SDS–PAGE, and western blot using the indicated antibodies. E–G FLT3-ITD positive MOLM-13 cells expressing CSF2RB shRNA or control shRNA were injected subcutaneously into the flank (n = 3 for both groups) of immunodeficient Rag2/Il2rg-mutant mice. E Tumor size was measured at given days by caliper. F Tumor weight was measured after sacrificing the animals on day 17 (p value of 0.0014 calculated by F test). G Sectioned tumors were fixed in 4% paraformaldehyde and then subject to immunohistochemical staining (by labeled Streptavidin–Biotin method) with the indicated antibodies, using hematoxylin for counterstaining. Representative images are shown (Axio Imager.M2; EC Plan-Neofluar ×10/0.3), scale bar = 100 µm. H, I Bone marrow of Csf2rb/Csf2rb2 double-knockout mice and wildtype littermates was transduced with FLT3-ITD or empty vector with GFP as transduction control. In all, 3000 GFP-positive sorted cells were cultured in methylcellulose ± cytokines (SCF, IL-3, IL-6, EPO, insulin, transferrin, iron-saturated). Representative images of one experiment (H) and analysis of three independent experiments (I) are shown. J Lethally radiated C57/BL6 littermates were transplanted with bone marrow from Csf2rb/Csf2rb2 double knockout or wildtype littermates transduced with FLT3-ITD 598/599[12] construct with GFP as transduction control; non-transduced bone marrow was supplemented to equalize the number of GFP-positive cells (n = 6 for both groups). The recipients’ survival is shown, with day 246 after transplantation as an endpoint. P value was calculated by Mantel–Cox test; n represents biologically independent mice.