Fig 2: Obesity decreases downstream targets of the TGFß1 pathway in mammary epithelial cells. A, Weight of 3-week-old FVB/N or 8-week-old C57BL/6 female mice fed a control diet (CD, n = 6) or high-fat diet (HFD, n = 6; *P < .05; repeated measures ANOVA with Sidak's multiple comparison test). B, Percent pSMAD2+ cells in mouse mammary ducts (n = 5 mice/group). C, Percent SMAD4+ cells in mouse mammary ducts (n = 5 mice/group). D, Weight of 8-week-old (adult) FVB/N female mice fed CD or HFD for 16 weeks (n = 5 mice/group). E, Percent pSMAD2+ cells in mammary ducts of adult FVB/N mice (n = 5 mice/group). F, TGFß1 expression in mouse mammary ducts (n = 5 mice/group). G, Fold change of expression of TGFß1 and TGFßRII relative to HPGRT in epithelial cells isolated from mouse mammary glands (n = 3 mice/group). H, Percent pSMAD2+ cells in breast ducts of women (n = 6 tissue samples/group). I, TGFß1 expression in human breast ducts (n = 5 tissue samples/group). Data analyzed with Student's t test, mean ± SEM. Magnification bar = 50 µm

Fig 3: Percentages of cells (mean ± SD) in the different phases of cell cycle in untreated control cells, and following treatments with Act-A, Act-AB, Act-B or FS for 24 h in the (A) Smad4-intact SW480 and (B) Smad4-negative HT29 cell lines (a = p < 0.05 compared with control untreated cells; b = p < 0.05 compared with Act-A; c = p < 0.05 compared Act-AB and d = p < 0.05 compared with Act-B).

Fig 4: Expression of TGF-ß1, Smad2, Smad3, Smad4, Smad6 and a-SMA in the NB and control groups: (a) Western blot analysis of TGF-ß1 and a-SMA. The gray value ratios of TGF-ß1 and a-SMA to ß-actin between the two groups were compared. Representative western blots were cropped from the same blots. Original blots are presented in Supplementary material. (b) Immunohistochemical staining of Smad2, Smad3, Smad4, and Smad6. The brownish-yellow area in the figure indicates that positive staining for the target protein. The AOD values of each Smad protein were compared between the two groups. The bars indicate 20 µm. The data are shown as the means ± SD. **p < 0.01; ***p < 0.001; ****p < 0.0001 versus the control. Student’s t test. AOD, average optical density.

Fig 5: Smad4 regulates diencephalic astrocyte proliferation and neurosphere formation AReactome pathway analysis for all 174 transcription factor genes with higher expression in DIE (upper) and all 205 genes higher in CTX GM (lower) astrocytes.Bt-SNE visualization of DIE astrocytes isolated by ACSA-2 MACS (Fig 1A) showing Smad4 expression (scaled log-normalized read counts).CExample of a Smad4/GFP double-positive astrocyte (indicated by arrowhead) in a single optical section of DIE from 3-mo-old GLASTCreERT2/eGFP mice 21 dpt immunostained as indicated on top of the panel. Orthogonal projections of the area highlighted in left panel of C shown at higher magnification in the right panel. Scale bars: 15 µm (left panel) and 10 µm (right panel).DExample of a proliferating (PCNA+, arrowhead) astrocyte in a single optical section of DIE from 3-mo-old GLASTCreERT2/eGFP mice 21 dpt immunostained as indicated on top with single channels shown in middle and right panels. Scale bars: 15 µm.EHistogram depicting percentage of PCNA-positive cells of all GFP-positive cells in the DIE of the genotype indicated on the x-axis (n = 6 animals per genotype; 5 ROI covering the diencephalon on a total of 3 slides were analysed per animal; PCNA-negative and PCNA-positive cells comparing wt/wt vs. fl/fl animals, adjusted P-value = 0.002).F, GHistograms depicting percentage of the neurosphere-generating cells obtained from the DIE (F) or SEZ (G) of GLASTCreERT2/Smad4fl/fl/eGFP or GLASTCreERT2/Smad4WT/WT/eGFP, as indicated on the x-axis (n = 3–4 animals for SEZ, 4 to 8 technical replicates analysed per animal; 4 animals for DIE, 8 technical replicates analysed per animal).HHistogram depicting the number of secondary neurospheres generated from 10,000 Smad4f/f neurosphere cells at 7 days after infection with MLV-based retrovirus containing either CAG-NLS-Cre or only CAG-GFP (n = 11 cultures/animals, 3 technical replicates per condition analysed; control vs. Cre, P-value = 0.0149). Note the decrease in DIE astrocyte proliferation in vivo (E) and in neurosphere formation upon Smad4 deletion (F, H). Data information: In E, F, G and H data are presented as mean ± SEM. F: GFP-negative and GFP-positive cells comparing wt/wt vs fl/fl animals, adjusted P-value = 0.0408; G: GFP-negative and GFP-positive cells comparing wt/wt vs fl/fl animals, ns). Each dot represents one n. *P < 0.05, **P < 0.01 (E, F, G: nonparametric 2way ANOVA followed by Sidak's multiple comparisons test, H: nonparametric Mann–Whitney test).