Fig 1: Additional experiments and computational analysis of fibronectin and TGF-B signaling in the APOE4 pericyte-to-myofibroblast transition.A. Fibronectin deposition near αSMA staining in APOE4/4-TR mice. The percentage of fibronectin area within 2.5 µm of αSMA-positive area relative to the total fibronectin-positive area was quantified. Data points represent mean values from individual mice (n = 6 images per mouse, n = 4 mice). Bars are mean group values ± SEM. P-values were calculated using a one-sample t-test comparing the APOE4/4-TR mean to 50%. B. Comparison of FN1 gene expression in fibroblasts versus myofibroblasts. Gene expression was aggregated per individual. Data points represent individuals. Bars are group means ± SEM. P-values were calculated using a paired two-sample Student’s t-test. C. Association of FN1 expression in mural cells with AD phenotypes. Mural cell FN1 expression was aggregated per individual from the Haney dataset. A global pathology score was calculated for each individual by averaging their amyloid plaque, neurofibrillary tangle, infarct, and Lewy body scores reported in the source metadata. These pathology scores were classified as low (bottom 75%) or high (top 25%). Mural cell FN1 expression was then compared between APOE genotypes (n = 8 per genotype) and between the low (n = 11, one outlier removed via Grubbs’ test) and high (n = 4) pathology groups. For each comparison, FN1 expression was normalized to APOE3/3 or the low pathology group, respectively. Data points represent individuals. Bars are group means ± SEM. P-values were calculated using unpaired two-sample Student’s t-tests. The association between individual mural cell FN1 expression and age at AD diagnosis (n = 16) was assessed via Pearson correlation analysis. D. miBrain experiments performed with additional isogenic pairs of iPSC-derived mural cells (iMCs) as previously described in Extended Figure 3B. ADRC scale bar, 25 µm. SAD scale bar, 50 µm. The area positive for fibronectin was normalized to nuclei and expressed relative to APOE3/3. Data points represent mean values from individual miBrains (n = 6 images per miBrain, n = 4–6 miBrains per genotype). E. Validation of FN1 shRNA knockdown. qRT-PCR was performed on APOE4/4 iMCs in monoculture. Data points represent mean values from individual wells of iMCs normalized to scramble (3 qRT-PCR replicates per well, n = 3 wells per condition). Bars are mean group values ± SEM. P-values were calculated using unpaired two-sample Student’s t-tests. F-I. Additional outputs of NicheNet analysis from Figure 4A, B. J. Representative images of iMCs with and without TGFβ1 stimulation. Scale bar, 25 µm. For the control group, APOE3/3 and APOE4/4 iMCs were left untreated. For the experimental group, APOE3/3 iMCs were treated with 50 ng/mL TGFβ1 for 96 hours. iMCs were then fixed and stained for PDGFRβ and SMAD2. SMAD2 intensity inside the nuclear mask was quantified and expressed relative to APOE3/3 control. Data points represent mean values from individual wells of iMCs (6 images per well, 4 wells per condition). Bars are mean group values ± SEM. P-values were calculated using a one-way ANOVA with Dunnett’s test for multiple comparisons. K. Representative images of myofibroblast phenotypes in iMC monocultures after TGF-β inhibition. Scale bar, 50 µm. For the control group, APOE4/4 iMCs were left untreated. For the experimental group, APOE4/4 iMCs were treated with 10 µM SB431542 for 96 hours. iMCs were then fixed and stained for NG2, αSMA, and fibronectin. Intracellular αSMA and fibronectin areas were quantified as the positively stained regions within the NG2 mask, normalized to the total NG2 area, and expressed relative to APOE4/4 levels. Data points represent mean values from individual wells (n = 6 images per well, n = 4 wells per condition). Bars are mean group values ± SEM. P-values were calculated using unpaired two-sample Student’s t-tests.
Fig 2: Myofibroblast-derived fibronectin drives amyloid accumulation in APOE4 models.A. Representative images of small vessels in the corpus callosum of APOE3/3-TR and APOE4/4-TR mice stained for lectin, αSma, and fibronectin. Scale bar, 10 µm. B. Representative images of APOE3/3 and APOE4/4 miBrains stained for PECAM and fibronectin. Scale bar, 50 µm. C. Quantification of FN1 expression across cell types in the cerebrovascular atlas. FN1 expression was aggregated per cell type for each individual. Individuals missing more than one cell type were excluded from the analysis. Data points represent individuals (n = 57). Bars are group means ± SEM. P-values were calculated using a repeated measures mixed-effects analysis with the Geisser-Greenhouse correction and Dunnett’s multiple comparisons test. D. Representative images of isogenic pairs of APOE3/3 and APOE4/4 miBrains stained for three amyloid antibodies targeting different epitopes of the amyloid-β peptide. Scale bar, 25 µm. E. Representative images of isogenic APOE3/3, APOE4/4, and APOE4/4 miBrains integrated with APOE3/3 iMCs stained for PECAM, fibronectin, and amyloid (D54D2). Scale bar, 25 µm. Data represent two independent experiments. In the first experiment, APOE3/3 and APOE4/4 were compared using an unpaired two-sample Student’s t-test. In the second experiment, APOE4/4 was compared to APOE4/4 with APOE3/3 iMCs using an unpaired two-sample Student’s t-test. P-values shown reflect these independent comparisons. For clarity, only the APOE4/4 group from the second experiment is shown. Representative images are from the corresponding experiment in each condition. F. Schematic of FN1 knockdown experiment in APOE4/4 miBrains. G. Representative images of APOE4/4 miBrains integrated with either iMCs transduced with scramble shRNA or shRNA targeting FN1 stained for fibronectin and amyloid (12F4). Scale bar, 25 µm. H. Treatment of encapsulated APOE3/3 iPSC-derived endothelial cells (iECs) with fibronectin and amyloid mixtures. Scale bar, 50 µm. For the control group, iECs were left untreated. For the experimental groups, iECs were treated with either 20 nM amyloid-β 1–40 and 1–42 or 20 nM amyloid-β 1–40 and 1–42 mixed with 50 ng/mL fibronectin for 96 hrs. iECs were then fixed and stained for amyloid (D54D2). For all panels, the area positive for αSMA, fibronectin, or amyloid immunoreactivity was quantified, normalized to nuclei, and expressed relative to APOE3/3 (panels B-D, H) or APOE4/4 (panels E, G). Data points represent mean values from individual miBrains (n = 6 images per miBrain, n = 6 miBrains per condition) or mice (n = 6 images per mouse, n = 4 mice per genotype). Bars are mean group values ± SEM. Unless stated otherwise, p-values were calculated using unpaired two-sample Student’s t-tests (panels B-D, G) or a one-way ANOVA with Dunnett’s multiple comparisons test (panel H).
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