Extended Data Fig. 10: eNOS regulates regenerative functions of NO^Hi HSCs, at least in part, via modulation of autophagic activities.

a, Flow cytometry analysis of the levels of LC3B, CYTO-ID, LAMP1, CTSA, LAMTOR1, and LAMTOR2 in NO^Hi and NO^Low HSCs. LAMP1 and CYTO-ID: MFI ratios to the average of MFIs of NO^Low HSCs. LC3B, CTSA, LAMTOR1 and LAMTOR2: Frequencies measured in comparison with isotype control antibody staining. Right: representative histograms. b, Representative immunostaining images demonstrating the distribution of NO, LC3B, and CD200R signals within NO^Hi and NO^Low HSCs. Sorted NO^Hi and NO^Low HSCs were fixed and stained with Alexa 568-conjugated LC3B antibodies. Basal autophagic activities were evaluated immediately after HSC sorting, while induced autophagy was evaluated following ex vivo incubation for three hours after sorting. CD200R antibody staining was performed before the sorting. Green: DAF-FM. Red: Alexa 568-LC3B. Cyan: Alexa 647-CD200R. Both under basal and induced conditions, NO^Hi HSCs exhibit higher intensities of LC3B+ signals when compared to NO^Low HSCs, and NO signals within NO^Hi HSCs frequently localize at LC3B+ autophagosomes. Following ex vivo culture, CD200R signals aggregate near NO + LC3+ autophagosomes. c, Flow cytometry analysis of the levels of LC3B, CTSA and CYTO-ID in NO^Hi HSCs isolated from tamoxifen-treated Pdzk1ip1^creER × eNOS^flox/flox mice. Right: Representative histograms. Left: ratio of MFI to the average of MFIs of HSCs from tamoxifen-treated Pdzk1ip1^creER × eNOS^wt/wt mice. Long bones were harvested four days after tamoxifen treatment (oral gavage, 1 mg). NO^Hi HSCs are defined as HSCs that exhibit DAF-FM signals at levels in the top 40% of whole HSCs in tamoxifen-treated Pdzk1ip1^creER × eNOS^wt/wt mice. d, CYTO-ID staining levels in HSCs in tamoxifen-treated VECad^creERT2 × CD200^flox/flox mice. N = 6-8/group, pooled from two independent experiments. The results were analysed by Student’s t-test. e, Donor chimerism in SJL recipients 16 weeks post-transplantation of NO^Hi HSCs from Pdzk1ip1^creER × Atg7^flox/flox mice (Atg7KO). f, NO^Hi HSC frequencies and numbers in tamoxifen-treated Pdzk1ip1^creER × Atg7^flox/flox mice. N = 4–6/group. The results were pooled from five independent experiments. Tamoxifen treatment was performed four days before the analysis. Student’s t-test. g, Flow cytometry analysis of the expression levels of DLL4 in capillaries isolated from tamoxifen-treated Pdzk1ip1^creER × eNOS^flox/flox mice. Right: representative histograms. Left: ratio of MFI to the average of MFIs in capillaries from control tamoxifen-treated Pdzk1ip1^creER × eNOS^wt/wt mice. Long bones were harvested four days after tamoxifen treatment. Capillaries were defined as cells that exhibited the top 20% levels of CD200 in CD31+Sca1 + CD45- cells in each analysis. N = 4–6. Student’s t-test. All data are presented as mean ± SD. Asterisks indicate significant differences (p-value < 0.05, *; <0.01, **; <0.001, ***; <0.0001, ****; N.S., >0.05). See Supplementary Notes for sample sizes, experimental replicates, and statistical analyses. See the Source Data file for individual p-values.

Source Data