Fig. 7.
Desmoglein-2/desmocollin-2 knockdown results in mis-localization of c-Src and CAR from the Sertoli cell surface, and increased CAR internalization. Sertoli cells (0.03–0.05 × 106 cells/cm2) were transfected on day 4 (A–D) or day 3 (E) with one of the following: non-targeting (Ctrl), desmoglein-2- (Dsg-2), desmocollin-2- (Dsc-2) or a mixture of desmoglein-2/desmocollin-2- (Dsg-2/Dsc-2) specific siRNA duplexes, and cells were harvested two days thereafter (A–D). (A, B) Redistribution of c-Src and CAR from the cell-cell interface as shown by immunofluorescent staining. Transfected Sertoli cells (0.03–0.05 × 106 cells/cm2) tracked with siGLO transfection indicator (red) were immunostained for c-Src (A) or CAR (B; green). Nuclei were visualized with DAPI (blue). Micrographs are presented as merged images. Arrowheads (A, b and B, b–d) show loss of c-Src and CAR from the cell-cell interface, respectively. Results are representative of at least three independent Sertoli cell cultures in which at least 50 images were collected using a 20X objective and analyzed in Photoshop. Bar in A, a (23μm) also applies to b. Bar in B, a (23 μm) also applies to b–d. (C) Increase in the rate of CAR endocytosis following desmoglein-2/desmocollin-2 silencing by RNAi. Transfected Sertoli cells (0.5 × 106 cells/cm2) were assessed for changes in the kinetics of CAR endocytosis by an endocytosis assay. Following isolation of biotinylated proteins, immunoblotting was used to quantify internalized CAR. Controls included (i) total biotinylated cell surface proteins without stripping (total, positive control) [note: lysates used for isolation of biotinylated proteins were diluted 10-fold, but no dilution was made in the actin immunoblot], (ii) lack of biotinylation (no biotin, negative control) and (iii) stripping of biotin without endocytosis (stripped, negative control). (D) A plot summarizing results in C after normalizing against actin. CAR internalization is expressed as a percentage of total cell surface CAR. Each data point represents mean ±SD (n = 3). *, P < 0.05; **, P < 0.01 (Student’s t-test). (E) Effects of desmoglein-2, desmocollin-2 and desmoglein-2/desmocollin-2 RNAi on TER. TER measurements across the Sertoli cell epithelium (1.0 × 106 cells/cm2) were taken daily at specified time-points, including both before and after transfection on day 3. Results are representative of three independent TER experiments with each experiment yielding similar results (mean ±SD, each experiment had triplicate data points). *, P < 0.05; **, P < 0.01 (Dunnett’s test against Ctrl subsequent to one way ANOVA).