Fig 1: Internalization of L. interrogans into cells by CAV1/ITGB1-PI3K/FAK-mediated MF-dependent endocytosis.(A) Leptospires in the cells infected with L. interrogans strain Lai for the indicated times, examined by confocal microscopy (scale bars = 5 µm). The blue plaques indicate the nucleus. The red spots around the nucleus indicate the intracellular leptospires. (B) Statistical summary of red fluorescence intensity reflecting the leptospires in the infected cells for the indicated times. Statistical data from experiments such as shown in (A). Bars show the means ± SD of three independent experiments. The red fluorescence intensity values from the cells without infection (before infection) were set as 1.0. (C) Lep-vesicles in the cells infected with L. interrogans strain Lai for 4 hr, examined by transmission electron microscopy (scale bars = 0.5 µm). The arrows indicate the intracellular leptospires in the membrane-bound vesicles. (D) Decrease of leptospires in the different inhibitor- or siRNA-treated cells infected with L. interrogans strain Lai for 4 hr, examined by confocal microscopy (scale bars = 5 µm). The legends are the same as shown in (A). (E) Statistical summary of red fluorescence intensity reflecting the leptospires in the different inhibitor- or siRNA-treated cells infected with L. interrogans strain Lai for 4 hr. Statistical data from experiments such as shown in (D). The other legends are the same as shown in (B). 10.7554/eLife.44594.005Figure 1—source data 1.Representative source data for Figure 1B.
Fig 2: LRP6 regulates the muscle-specific splicing of integrin-ß1D during the development of striated muscle. (A) Time-lapse microscopy of C2C12 myoblasts throughout the proliferation and myodifferentiation phases. GM: growth medium; DM: differentiation medium. Scale bar: 10 µm. Quantitative PCR (B) and Western blotting (C) analysis of LRP6 and myodifferentiation markers at indicated time points. C-left: typical blots; C-right: pooled data. *P < .05 compared with Ctrl, # P < .05 compared with groups other than Ctrl. (E) Measurement of integrin-ß1 transcripts and proteins in C2C12 cells. Upper: examination of integrin-ß1 transcripts during myodifferentiation using gel electrophoresis; Middle and Lower: Western blotting examination of integrin-ß1 (ITGB1) and ITGB1D during myodifferentiation (middle) and under Lrp6-deficient conditions (lower). E-middle and -lower left: typical blots; E-middle and -lower right: pooled data. *P < .05 compared with Ctrl. Representative images from five independent experiments with similar results are shown
Fig 3: (a) TEM of exosomes showing spheroid double-membrane-bound morphology (arrows) with a diameter of 40–100 nm; (b) Exosomes were also detected in cardiac tissues by PKH26; (c) Western blot for exosome characterization; (d) Histogram of exosome characterization for CD81, CD63, CD83 indicated significant differences (p ˂ 0.05) *, significant compared to the first passage at p ˂ 0.05; #, significant compared to the second passage at p ˂ 0.05. Data are shown as mean ± SEM; (e) Western blot for exosome localization in cardiac tissues; (f) Histogram of exosome localization in groups III and IV for CD29, CD44, and CD73. Data are shown as mean ± SEM. Prussian blue staining of cardiac tissues proved the absence of blue staining in un-injected animals (g) and iron oxide particles stained blue in the myocardium of exosome-injected animals in the protective (h) and curative (i) groups.
Fig 4: rBMSC characterization.An example of rBMSC phenotype and functional analysis. (A) Flow cytometry analysis of MSCs was performed with FITC-conjugated antibodies (red histograms) and PE-conjugated antibodies (green histogram) to detect cell surface markers. The expression of isotype controls is shown in the black histograms. Flow cytometry analysis revealed typical CD44, CD29 and CD90 mesenchymal surface-marker expression, without CD45 and CD34 hematopoietic surface markers, and confirmed that only mesenchymal cells had been isolated (CD45-CD34-CD29+CD44+CD90+ cell population). (B, C, D, E) In vitro differentiation of Mesenchymal Cells. The cells were shown to be multipotent stem-cells by differentiation capacity along osteogenic, chondrogenic and adipogenic lineages.(B): Cells were incubated in lineage-specific induction media and then analyzed by histochemical and cytological staining for (C) Adipocytes (Oil Red-O staining), (D) Chondrocytes (Alcian blue staining) and (E) Osteoblasts (Alizarin Red staining). Characterization of rat BMSC derived extracellular vesicles. (F) AFM topography image of the EV preparations. Scale bars are 1 µm. (G) CONAN assay. (H) Size distribution and diameter of EV samples. (I) Representative western blots showing the expression of: GM130, ANX XI, TSG 101, ANX V, TERT and CD29 (Integrin beta1) in rBMSC and rBMSC-derived EVs. Uncropped gel from different gels. Experiments were performed 3 times with similar results.
Fig 5: CD9 depletion alters EGFR- and PDGF-dependent FAK activation and reduced ITGB1 expression. a Western blot analysis of the expression of ITGB1/ß1 integrin in control (scramble shRNA) and CD9-depleted (Cd9 shRNA) PEC and (b) quantification. Tubulin was used as a loading control. c Representative images of immunofluorescent stainings of ITGB1 (red) and PODXL/podocalyxin (green) in glomeruli from iPec-Cd9wt/wt and iPec-Cd9lox/lox mice after 14 days of the NTN model. Scale bar, 50 µm. Nuclei were stained with DAPI (blue). d Western blot analysis of the expression of phospho-PDGFR (Y1009), PDGFR, phospho-FAK Y397, and FAK in control (scramble shRNA) and CD9-depleted (Cd9 shRNA) PEC after time sequential stimulation with PDGF-BB and (e–g) quantifications. Tubulin was used as a loading control. h Western blot analysis of the expression of phospho-EGFR Y1068, EGFR, phospho-FAK Y397, and FAK in control (scramble shRNA) and CD9-depleted (Cd9 shRNA) PEC after time sequential stimulation with HB-EGF and (i–k) quantifications. Tubulin was used as a loading control. e–g, i–k The data represent mean +/- s.e.m. of n = 4 experiments. *P < 0.05 scramble shRNA vs. Cd9 shRNA using two-way ANOVA test. Source data are provided as a source data file
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