Fig. 6: TGFβ inhibition induces a reduction of autophagic activity.

A Normalized UMI counts of selected genes, differentially expressed between th3/+ HSCs (red) and wt HSCs (blue) purified by FACS²³. Results are represented as Fold respect to wt (th3/+ n = 3; wt n = 4). *p < 0.05, by two-tailed unpaired t-test; mean ± SEM is shown. B Normalized UMI counts of selected genes, involved in TGFβ signaling, differentially expressed between th3/+ HSCs (red) and wt HSCs (blue)²³. Results are represented as Fold respect to wt (th3/+ n = 3; wt n = 4). *p < 0.05, **p < 0.01, by two-tailed unpaired t-test; mean ± SEM is shown. C Normalized UMI counts of selected genes, involved in autophagy pathway, differentially expressed between th3/+ HSCs (red) and wt HSCs (blue)²³. Results are represented as Fold respect to wt. (th3/+ n = 3; wt n = 4). *p < 0.05, **p < 0.01, by two-tailed unpaired t-test; mean ± SEM is shown. D Percentage of Cyto-ID⁺ in LT-HSCs in wt (n = 10) and treated th3/+ mice (n = 11) from n = 2 independent experiments. p-value by two-tailed unpaired t-test; mean ± SEM is shown. E Evaluation of TGFβ1 in th3/+ and wt BM fluid by ELISA (wt n = 8; th3/+ n = 8). p-value by two-tailed unpaired t-test; mean ± SEM is shown. F Experimental plan for in vivo treatment of wt mice with TGFβi inhibitor. Image created with BioRender.com. (https://BioRender.com/ho2ypan). G Frequencies of LT-HSCs in untreated animals (ctrl n = 9) and after in vivo treatment with TGFβi inhibitor (n = 12), from n = 2 independent experiments; p-value by one-tailed unpaired t-test; mean ± SEM is shown. H Frequencies of MPP^Mk/E (in untreated animals (ctrl n = 9) and after in vivo treatment with TGFβi inhibitor (n = 12), from n = 2 independent experiments; p-value by two-tailed unpaired t-test; mean ± SEM is shown. I Klf1 expression measured on sorted LT-HSCs from ctrl (n = 5) and treated wt mice (n = 9) from n = 3 independent experiment. p-value by Mann-Whitney test; mean ± SEM is shown. J Percentage of Cyto-ID⁺ in LT-HSCs in ctrl wt (n = 5) and treated wt mice (n = 5). p-value by Mann-Whitney test. mean ± SEM is shown. K Map1lc3b gene expression measured on sorted LT-HSCs from ctrl (n = 9) and treated wt mice (n = 10). p < 0.0001 by two-tailed unpaired t-test; mean ± SEM is shown. The percentage of Cyto-ID⁺ in LT-HSCs (L) in untreated (n = 6) and after in vitro treatment of th3/+ LT-HSCs with TGFβ1 molecule (n = 6 samples) or Bafilomycin A (n = 6 samples), as control. Each sample is a pool of 4-6 mice. (n = 3 independent experiments). p-values by one-way ANOVA, with Dunnett’s multiple comparison test. Mean ± SEM is shown. M Quantification of Cyto-ID level dye levels (MFI) in untreated (n = 6 samples) and after in vitro treatment of th3/+ LT-HSCs with TGFβ1 molecule (n = 6 samples) or Bafilomycin A (n = 5 samples), as control. Values are shown as fold versus the untreated one. p-value by Mann-Whitney test. mean ± SEM is shown. N Frequencies of LT-HSCs, MPP^Mk/E in HSPC at 12–20 weeks after transplantation from th3/+ (n = 5) and Ulk1-/- th3/+ (n = 6) mice from n = 2 independent experiments P-values by two-tailed unpaired t-test; mean ± SEM is shown. O Gene expression of Klf1 was measured on sorted LT-HSCs from th3/+ (n = 5) and Ulk1-/- th3/+ (n = 6) mice from n = 2 independent experiments. Relative expression on HPRT. th3/+, red; Ulk1-/- th3/+ green. Two-tailed unpaired t-test; mean ± SEM is shown. P Gene expression of Klf1 was measured on sorted MPP^Mk/E (P from th3/+ (n = 5) and Ulk1-/- th3/+ (n = 6) mice from n = 2 independent experiments. Relative expression on HPRT. th3/+, red; Ulk1-/- th3/+ green. Two-tailed unpaired t-test; mean ± SEM is shown. Source data are provided as Source Data file.