Fig 2: In vivo antifibrotic effects of various liposome preparations in the CDAHFD-induced fibrosis model.a Schematic of the CDAHFD-induced fibrosis model and treatment timelines for the various liposomes. b Representative images of liver sections stained for FAP (green), αSMA (red), and DAPI (blue) (n = 5 biologically independent mice per group). Scale bar: 50 μm. c, d FAP expression levels on HSCs from normal or CDAHFD-induced fibrotic liver were evaluated by flow cytometry (n = 5 biologically independent mice per group). Significant differences were assessed using a one-way ANOVA with Tukey test (Tukey’s test PCDAHFD(+) - Normal < 0.0001). Results are presented as mean ± S.D.. e, f Representative Sirius red-stained liver images for the evaluation of liver fibrosis and quantification of the Sirius red-positive area (n = 5 biologically independent mice per group). Significant differences were assessed using a one-way ANOVA with Tukey test (Tukey’s test PPRL–normal < 0.0001, PPRL–untreated < 0.0001, PPRL–PL < 0.0001, PPRL–ML < 0.0001, PPRL–SCL < 0.0001). In box plots, boxes show the 25th–75th percentile with the median, and whiskers show the minimum–maximum. g Top panel shows representative Masson’s trichrome-staining of liver. Bottom panel displays pseudocolored images by InForm 2.2.1 analysis, distinguishing fibrotic regions (blue) from normal tissue (red) (n = 5 biologically independent mice per group). Scale bar: 50 μm. h Quantification of Masson’s trichrome-stained connective tissue area calculated from five randomly selected fields per sample (n = 5 biologically independent mice per group). Significant differences were assessed using a one-way ANOVA with Tukey test (Tukey’s test PPRL–normal < 0.0001, PPRL–untreated < 0.0001, PPRL–PL < 0.0001, PPRL–ML < 0.0001, PPRL–SCL < 0.0001). In box plots, boxes show the 25th–75th percentile with the median, and whiskers show the minimum–maximum. i Collagen content of fibrotic liver samples, as measured using hydroxyproline assays. Significant differences were assessed using a one-way ANOVA with Tukey test (Tukey’s test PPRL–normal = 0.1877, PPRL–untreated < 0.0001, PPRL–PL < 0.0001, PPRL–ML < 0.0001, PPRL–SCL = 0.0001). Results are presented as mean ± S.D. Blood was collected for serum analysis of AST (j) and ALT (k) (n = 5 biologically independent mice per group). Significant differences were assessed using a one-way ANOVA with Tukey test (Tukey’s test AST: PPRL–normal = 0.7672, PPRL–untreated = 0.0002, PPRL–PL = 0.0002, PPRL–ML = 0.0005, PPRL–SCL = 0.0003, ALT: PPRL–normal = 0.9835, PPRL–untreated < 0.0001, PPRL–PL < 0.0001, PPRL–ML < 0.0001, PPRL–SCL < 0.0001). Results are presented as mean ± S.D. CDAHFD, a choline-deficient, L-amino acid-defined high-fat diet; HSC hepatic stellate cell; UT untreated group. PL plain PEGylated liposomes; ML maleimide-activated PEGylated liposomes; SCL scrambled cys-promelittin peptide-tagged liposomes; PRL promelittin-modified liposomes; CD26 cluster of differentiation 26; CD31 cluster of differentiation 31; Ck7 cytokeratin7; αSMA alpha-smooth muscle actin; ALT alanine transaminase; AST aspartate transaminase. Source data are provided as a Source Data file.

Fig 3: In vivo antifibrotic effects of various liposome preparations in the CCl4-induced liver fibrosis model.a Schematic of the CCl4-induced liver fibrosis model and treatment timelines for the various liposomes. b Immunohistochemical staining for FAP in liver tissues from normal and CCl4-induced liver fibrosis mice (n = 5 biologically independent samples per group). Scale bar: 50 μm. c, d FAP expression levels on HSCs from normal or CCl4-induced fibrotic liver were analyzed by flow cytometry (n = 5 biologically independent mice per group). Significant differences were assessed using a one-way ANOVA with Tukey test (Tukey’s test PCCl4(+) - Normal < 0.0001). Results are presented as mean ± S.D. e, f Representative Sirius red-stained liver images for the evaluation of liver fibrosis, and quantification of the Sirius red-positive area (n = 5 biologically independent mice per group). Significant differences were assessed using a one-way ANOVA with Tukey test (Tukey’s test PPRL–normal < 0.0001, PPRL–untreated < 0.0001, PPRL–PL < 0.0001, PPRL–ML < 0.0001, PPRL–SCL < 0.0001). In box plots, boxes show the 25th–75th percentile with the median, and whiskers show the minimum–maximum. g Upper panel shows representative Masson’s trichrome-stained images of CCl4-induced fibrotic liver. The lower panel displays pseudocolored images by InForm 2.2.1 analysis, distinguishing fibrotic regions (blue) from normal tissue (red) (n = 5 biologically independent mice per group). Scale bar: 50 μm. h Quantification of the connective tissue area under Masson’s trichrome staining, as calculated from five randomly selected fields per sample (n = 5 biologically independent mice per group). Significant differences were assessed using a one-way ANOVA with Tukey test (Tukey’s test PPRL–normal = 0.3374, PPRL–untreated < 0.0001, PPRL–PL < 0.0001, PPRL–ML < 0.0001, PPRL–SCL < 0.0001). In box plots, boxes show the 25th–75th percentile with the median, and whiskers show the minimum–maximum. i The severity of liver fibrosis for each group, assessed using the METAVIR scoring system (n = 5 biologically independent mice per group). Significant differences were assessed using a one-way ANOVA with Tukey test (Tukey’s test PPRL–normal = 0.2537, PPRL–untreated = 0.0073, PPRL–PL = 0.0270, PPRL–ML = 0.0270, PPRL–SCL = 0.0270). Results are presented as mean ± S.D. j Collagen content of fibrotic liver samples, as measured using hydroxyproline assays. Significant differences were assessed using a one-way ANOVA with Tukey test (Tukey’s test PPRL–normal = 0.1882, PPRL–untreated = 0.0002, PPRL–PL = 0.0005, PPRL–ML = 0.0012, PPRL–SCL = 0.0014). Results are presented as mean ± S.D. Blood was collected for serum analysis of AST (k) and ALT (l) (n = 5 biologically independent mice per group), UT: untreated group. Significant differences were assessed using a one-way ANOVA with Tukey test (Tukey’s test AST: PPRL–normal = 0.3160, PPRL–untreated < 0.0001, PPRL–PL < 0.0001, PPRL–ML < 0.0001, PPRL–SCL < 0.0001, ALT: PPRL–normal = 0.9998, PPRL–untreated < 0.0001, PPRL–PL < 0.0001, PPRL–ML < 0.0001, PPRL–SCL < 0.0001). Results are presented as mean ± S.D. m Representative immunofluorescence images of liver tissues stained with various cell markers including F4/80 (macrophage), CD26 (hepatocyte), CD31 (endothelial cell), Ck7 (cholangiocyte), and αSMA (aHSC). n Populations of cells were analyzed using a VECTRA tissue analyzer and the InForm 2.2.1 image analysis software (n = 5 biologically independent mice per group). Significant differences were assessed using a one-way ANOVA with Tukey test (Tukey’s test n.s.: P > 0.05, αSMA: PPRL–untreated < 0.0001, PPRL–PL < 0.0001, PPRL–ML < 0.0001, PPRL–SCL < 0.0001). In box plots, boxes show the 25th–75th percentile with the median, and whiskers show the minimum–maximum. Scale bar: 50 µm. HSC hepatic stellate cell; UT untreated group; PL plain PEGylated liposomes; ML maleimide-activated PEGylated liposomes; SCL scrambled cys-promelittin peptide-tagged liposomes; PRL promelittin-modified liposomes; CD26 cluster of differentiation 26; CD31 cluster of differentiation 31; Ck7 cytokeratin7; αSMA alpha-smooth muscle actin; ALT alanine transaminase; AST aspartate transaminase. Source data are provided as a Source Data file.

Fig 4: GP38 and SCA1 expression on TMC subsets. (A) Total thymi cells from 1-week-old mice were isolated, and total TMCs (CD45−EpCAM−) were analysed by flow cytometry. t-SNE representation of the expression of CD31, CD140α, CD140β, GP38, SCA-1, Ly51 and αSMA. (B) Three main clusters were identified: cluster 1 (CD31+), cluster 2 (CD140α+β+) and cluster 3 (CD140α−β+). Clusters 2 and 3 were respectively subdivided into cluster 2.1 (CD140α+β+GP38+SCA1−) and 2.2 (CD140α+β+GP38+SCA1+); and 3.1 (CD140α−β+Ly51+αSMA−) and 3.2 (CD140α−β+Ly51+αSMA+). (C) TMCs (CD45−EpCAM−CD31−) were analysed for the indicated markers, and sub-cluster 2.1 (red gate), 2.2 (green gate), 3.1 and 3.2 (light- and dark-blue gates) were identified. (D) Analysis of GP38, SCA-1 and DPP4 expression in TFA (red gate) and TFB (green gate) populations at E14, E17, 1 week old (W) and 4W. Numbers in plots indicate the frequency of cells found within each gate. Plots are of a representative analysis per time point. (E) Bar graphs showing mean+s.d. of the frequency and cellularity of TFA and TFB subsets, of three independent analyses per time point. Differences in TF subsets, CD140α+β+GP38+SCA1− (red) and CD140α+β+GP38+SCA1+ (green), were statistically analysed at different ages: ***P<0.001, **P<0.01, *P<0.05.

Fig 5: Lats1/2 deficiency promotes cell fate conversion from adipocytes to myofibroblasts.a Immunoblot analysis of protein expression of GFP in SVF and adipocyte from 8-week-old AdipoqCreRosa26mTmG scWAT. Independent experiments were performed twice with similar results. b Flow cytometry analysis of percentages of live CD45−CD31−GFP+DPP4+ cells in scWAT SVF of P21 L1L2-FFLSL-CAS9-EGFP or L1L2-AKOLSL-CAS9-EGFP reporter mice (n = 5). c Representative sections of scWAT of P14 or P28 L1L2-AKOLSL-CAS9-EGFP mice stained for αSMA (red), DPP4 (purple), nucleus (DAPI, blue) and GFP (green). Arrowheads indicate examples of GFP+DPP4+ cells. Arrows point to GFP+αSMA+ cells. Independent experiments were performed three times with similar results. d Sorted cell subsets from P21 L1L2-AKOmTmG scWAT SVF were plated and stained for αSMA (AF647, red) and nucleus (DAPI, blue). e Quantification of percentages of GFP+DPP4+ (n = 6), GFP+DPP4- (n = 7), GFP-DPP4+ (n = 7), GFP-DPP4- (n = 7) cells. Dots represent cell percentages of random visual fields from P21 L1L2-AKOmTmG mice. Independent experiments were performed twice with similar results. f Sorted GFP+DPP4+ cells from P21 L1L2-AKOLSL-CAS9-EGFP scWAT SVF were treated with TGF-β1 (10 ng/ml) for 0 h or 24 h. Cells were stained with αSMA (red), DPP4 (purple), nucleus (DAPI, blue) and GFP (green). An arrowhead points to the GFP+αSMA−DPP4+ cell. Independent experiments were performed twice with similar results. g Sorted GFP+DPP4+ cells from L1L2-AKOLSL-CAS9-EGFP reporter scWAT SVF were analyzed for DPP4 expression in GFP+ cells before or after transplantation into scWAT of 8-day-old L1L2-AKO recipient mice. One representative transplant from n = 3 biological replicates. Data are means ± SEM. One-way ANOVA with Bonferroni’s multiple-comparisons test in e; two-tailed unpaired Student’s t test in b. *P < 0.05, **P < 0.01, ***P < 0.001. Exact P values are provided in a Source data file.