Journal: Science Advances

Article Title: PI3-kinase deletion promotes myelodysplasia by dysregulating autophagy in hematopoietic stem cells

doi: 10.1126/sciadv.ade8222

Figure Lengend Snippet: ( A ) Representative LC3II flow cytometry histograms and LC3II MFI quantification of WT and TKO HSCs on the right. After starvation, cells were either not treated (NT, PBS only) or treated with rapamycin (RAPA). ( B ) Quantification of GEMM colonies formed by WT and TKO BM cells in methylcellulose upon rapamycin (RAPA; 20 ng/ml) or metformin (MET; 50 mM) treatment ( N = 4 per group). NT, no drug treatment (PBS only). ( C ) Experimental design of noncompetitive BMT with in vivo treatment with RAPA or MET (created with Biorender.com ). At each time point, at least five animals per treatment group were analyzed. ( D ) Absolute numbers of donor-derived WT ( N = 8) or TKO ( N = 5 per group) LT-HSC and ST-HSC after 8 weeks of treatment. ( E ) Representative flow cytometry plots and ( F ) quantification of the number of donor-derived FLK2 + LSK cells in the BM after 8 weeks of in vivo treatment with rapamycin (RAPA; 15 mg/ml) or metformin (MET; 5 mg/ml). (A and D to F) Immunophenotypic populations were defined as follows: Flk2 + LSK, Lin − Sca1 + cKit + Flk2 + ; HSCs, Lin − Sca1 + cKit + Flk2 − CD48 − ; ST-HSCs, Lin − Sca1 + cKit + Flk2 − CD48 − CD150 − ; and LT-HSCs, Lin − Sca1 + cKit + Flk2 − CD48 − CD150 + . Significance was determined using t test (D and F) or one-way ANOVA with Tukey’s multiple comparisons test (B). * P ≤ 0.05, ** P ≤ 0.01, and *** P ≤ 0.001.

Article Snippet: Rapamycin (Santa Cruz Biotechnology, catalog number SC-3504) was diluted in ethanol at a concentration of 15 mg/ml.

Techniques: Flow Cytometry, In Vivo, Derivative Assay