Fig 1: Labeling of ß3 integrin mouse anterior segments. Sections of mouse anterior segments labeled with rabbit antibody against ß3 integrin (Abcam ab197662) (A) or a control antibody (IgG) (B). The TM-Schlemm's canal (SC) outflow pathway is outlined by the yellow dotted rectangle. The asterisk indicates SC. Micrographs are representative of several anterior segments. Micrograph shown here is from a Cre+/- ß3flox/+ littermate not treated with tamoxifen. Scale bar: 20 µm.
Fig 2: Integrin ß3 modulates SMC transdifferentiation. a–c Mice were fed a HFD for 6 or 16 weeks as indicated, and then transverse aortic root sections were stained. In a, b sections from ApoE(-/-), SMMHC-CreERT2, ROSA26R(mTmG/+) mice were stained for SMA, GFP (fate marker), nuclei (DAPI), and either integrin ß3 (a) or CD36 (b). Dashed yellow lines separate cap from core (a, b) and core from media (b). n = 5. In c sections from ApoE(-/-) mice that were also wild type or null for Itgb3 were stained for SMMHC, CD68, and nuclei (DAPI). n = 3. Boxed regions (a, c) are shown as close-ups on right; in c CD68+SMMHC+ cells in the media (arrowheads) and plaque (arrows) of the Itgb3 null atherosclerotic aorta are indicated. Med, tunica media; Lu, lumen; Pl, plaque. Scale bars, 25 µm. d–h Aortic SMCs were isolated from ApoE(-/-) mice and then subjected to siRNA-mediated knockdown with si-Itgb3 vs. scrambled (Scr; d–f) or with si-Itgb3, si-Tlr4 vs. si-Itgb3 (g, h). Levels of indicated transcripts from qRT-PCR are relative to Gapdh and normalized to either Scr in d, f or to si-Itgb3 in g, h. For d, g, n = 4–5 in duplicate. In e silenced SMCs were cultured with DiI-conjugated ox-LDL for 10 h and stained with DAPI; n = 5. In f, h silenced SMCs were exposed to soluble cholesterol:methyl-ß-cyclodextrin complexes for 3 days, and then mRNA levels were assessed; n = 4–7 in duplicate. *, **, ***, ^, **** vs. control (Scr in d, f and si-Itgb3 in g, h), p < 0.05, p < 0.01, p < 0.005, p < 0.001, and p < 0.0005, respectively. NS, not significant. Student’s t-test was used, and error bars represent standard deviations. i Schematic of the effect of ß3 reduction via TLR4 and CD36 in SMCs on transdifferentiation
Fig 3: Integrin ß3 in bone marrow-derived cells regulates SMC clonality. a, b Itgb3(-/-), ApoE(-/-), SMMHC-CreERT2, ROSA26R(Rb/+) mice were fed a HFD for 6 weeks. In a aortic root sections were stained with DAPI and directly imaged for Rb colors. Note, multiple patches of smooth muscle-derived plaque cells of different colors with limited mixing contrasts with monoclonal plaques in Itgb3(+/+) mice (Fig. 2a, b). In b percentage of labeled cells of each color in media and plaque is shown; n = 3 mice, 6 plaques. Chi-square test was used; NS, not significant. c Percent of plaque cells at 6 weeks of HFD labeled by any color were compared in ApoE(-/-), SMMHC-CreERT2, ROSA26R(Rb/+) mice that were also Itgb3 wild type or null. Itgb3(+/+) data is also shown in Fig. 2d. d–f ApoE(-/-), SMMHC-CreERT2, ROSA26R(Rb/+) mice were induced with tamoxifen, lethally irradiated, and transplanted with Itgb3(+/+), ApoE(-/-) (d) or Itgb3(-/-), ApoE(-/-) (e) bone marrow prior to HFD. In d, e after 12 weeks of HFD, aortic root sections were stained with DAPI and imaged for Rb colors. In f mice were fed a HFD for 6–12 weeks, and percentage of labeled cells of each Rb color in media and plaque were quantified; n = 5 recipient mice, 6 plaques for each donor genotype. g–j ApoE(-/-) SMC were cultured in medium conditioned by bone marrow-derived macrophages from Itgb3(+/+), ApoE(-/-) or Itgb3(-/-), ApoE(-/-) mice for 0 and 8 h or 48 h. In g, h bright-field images and percent of SMC coverage at 8 h of uncovered area at 0 h are shown, respectively; n = 4. In i, j EdU was added to culture medium for the last 8 h of the 48 h incubation, and then SMCs were stained for EdU and DAPI and percent of cells expressing EdU was quantified; n = 4. Student’s t-test was used. k Schematics of plaques in ApoE(-/-), SMMHC-CreERT2, ROSA26R(Rb/+) mice that are Itgb3 wild type or null were induced with tamoxifen, transplanted with ApoE(-/-) bone marrow fed a HFD for 12 weeks. Error bars represent standard deviations. Med, tunica media; Pl, plaque. Scale bars, 25 µm (a, d, e, i), 100 µm (g)
Fig 4: FUT5 can regulate protein glycosylation to promote ICC progression. (A) and (B) Glycosylation sites found by the MS. (C) and (D) Grouped Gene Ontology of the differential glycosylation sites. (E) Several glycosylation sites which downregulate most when FUT5 was knockdown. (F) Volcano plot of protein expression measured by the MS. (G) Protein expression of those in (E) (Several proteins are not identified by MS may because of its low abundance). (H) Lectin enrichment assay shows change of the fucosylation level of ITGB3.
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