Figure 2.

Altered TGF-β signaling following SMAD4 knockout in CP-B cells. (A-D) Fold change in mRNA expression of TGF-β signaling targets (RT² Profiler PCR Arrays) in SMAD4 wild-type cells (CP-B Parental and CP-B Cas9) (A, C) and SMAD4 knockout cells (CP-B Ex1 and CP-B Ex2) (B, D) upon treatment with 10 ng/mL TGF-β1 for 16 hours without serum (A, B) or in the presence of serum (C, D) compared to respective vehicle treated cells. Thresholds for fold change ≥1.5 and ≤0.7 were chosen for up-regulated and down-regulated genes, respectively. Reciprocal fold change values are shown (y=0 or fold change 1 (no change) and y=-2 or fold change 0.5). The arithmetic mean of housekeeping genes (ACTB, B2M, GAPDH, HPRT1 and RPLPO) was used to calculate C_T values for each gene. N=1 experiment per each cell line (SMAD4 wild-type = mean fold change ± SD of CP-B Parental and Cas9 cells; SMAD4 knockout = mean fold change ± SD of Ex1 and Ex2 clones; or SMAD4 knockout = fold change of Ex2 for graph D). (E) 4 way Venn diagram representing the set of differentially regulated genes following treatment with 10 ng/mL TGF-β1 for 16 hours compared to vehicle treated cells in the presence or absence of serum in SMAD4 wild-type and SMAD4 knockout cells. Gene names in red and blue colour represent up- and down-regulated genes, respectively.