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. 2022 Nov 24;12(12):1749. doi: 10.3390/biom12121749

Figure 5.

Figure 5

Activin A-mediated reduction in BG shedding is SMAD3- but not SMAD2-dependent. Epithelial 12Z cells were plated in 6-well plates and transfected with SMAD2 siRNA, SMAD3 siRNA, or negative control siRNA. RT-qPCR of the gene silencing efficiency for each siRNA was performed to ascertain the silencing of SMAD2 and SMAD3 (A). SMAD2/3 double-silenced and single-silenced 12Z cells were subsequently stimulated with activin A (25 ng/mL) for 48 h and supernatants were analyzed by sBG ELISAs. SMAD2/SMAD3 double gene knockdown significantly abrogated the activin A-dependent reduction in BG shedding (B). Single gene knockdown revealed that only the silencing of SMAD3 and not of the SMAD2 gene blocked the activin A-mediated effects (C). These results were further corroborated using the specific inhibitor of SMAD3, SIS3 (5 μM) (D). Untreated cells and cells treated with Neg. siRNA or DMSO (0.01%) were used as controls. Each bar represents the mean ± SEM of 3 independent experiments performed in duplicate. Dunnett’s test was used for statistical analysis; ** p < 0.01; *** p < 0.001; # p < 0.0001; siRNA, small interfering RNA; Neg. siRNA, negative control siRNA; sBG, soluble betaglycan; Act A, activin A.