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. Author manuscript; available in PMC: 2011 Jun 1.
Published in final edited form as: Int J Biochem Cell Biol. 2010 Feb 25;42(6):975–986. doi: 10.1016/j.biocel.2010.02.010

Fig. 8.

Fig. 8

Relative cell viability following RNAi and validation of the use of non-targeting siRNA. Sertoli cells were transfected on day 3 (A–B) or day 4 (C) with one of the following: nuclease-free water (vehicle Ctrl), non-targeting (Ctrl) siRNA, 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, C). (A) Sertoli cell viability after transfection with control (Ctrl), desmoglein-2 (Dsg-2), desmocollin-2 (Dsc-2) or desmoglein-2/desmocollin-2 (Dsg-2/Dsc-2) duplexes. Sertoli cells cultured in 96-well plates were transfected under the same conditions as described for TER experiments (200 nM siRNA for each group, i.e. Ctrl, desmoglein-2 RNAi and desmocollin-2 RNAi, and 100 nM siRNA each for desmoglein-2/desmocollin-2 RNAi). Relative cell viability, as reflected by absorbance at 450 nm, was measured 48 hours after transfection. Bar = mean ± SD (n = 4). (B) 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. Each data point represents mean ±SD (n = 3). *, P < 0.05; **, P < 0.01. (C) Transfected Sertoli cells (0.03–0.05 × 106 cells/cm2) tracked with siGLO transfection indicator (red) were immunostained for ZO-1 (a, c, e) and CAR (b, d, f, green). Arrowheads (e, f) indicate mislocalization of ZO-1 and CAR following desmoglein-2/desmocollin-2 knockdown.