Fig 1: Becn1 deficiency does not reduce the autophagy ability of HSCs. (A) Representative western blot showing LC3-II transition in c-Kit+ cells from Becn1vKO and WT control. 100,000 c-Kit+ cells were treated with 30 µM chloroquine (CQ) or mock treatment for 4 h. Cell lysates were subjected to immunoblot using indicated antibodies. The intensity of the protein signal was measured by Image J. (B and C) The histograms show the transient autophagic level (MFI) (B) and autophagic flux (?MFI) (C) of LSKs from WT and Becn1vKO mice by using Cyto-ID dye. 7500 freshly isolated LSKs were cultured for 24 h (B), and 9000 freshly isolated LSKs were cultured for 3 h before 15 µM chloroquine or mock treatment for another 21 h (C). Samples were stained with Cyto-ID dye and analyzed the fluorescence intensity of GFP by flow cytometry. ?MFI was calculated as what was mentioned in the methods. n = 3 repeats per group, data are shown as mean ± SD. (D and E) The histograms display autophagic flux (D) and relative fluorescence intensity of RFP to GFP (E) of HSCs (CD48-SK) from WT and Becn1vKO mice by GFP-LC3-RFP-LC3?G plasmid. 10,000 fresh isolated LSKs form WT and Becn1vKO mice were infected with GFP-LC3-RFP-LC3?G plasmid for 3 days, and then stained with the surface marker of HSC (CD48, Sca-1, c-Kit) before analyzing the GFP and RFP fluorescence intensity by flow cytometry. The autophagic flux was indicated by the relative fluorescence intensity of RFP to GFP and normalized to the WT group. n = 3 repeats per group, data are shown as mean ± SD for (D). The strategy to calculate the autophagic flux by using GFP-LC3-RFP-LC3?G plasmid is presented in sup. Fig. 4C.