Rac1 cdc42

IPA-3 and U0126 were purchased from Selleck Chemicals (Shanghai, China). Antibodies and their sources are as follows: antibodies against p-PAK1 (T423, #2601), PAK1 (#2602), PAK2 (#4825), PAK3 (#2609), Rac1/Cdc42 (#4651), Raf1 (#9422), p-Raf1 (S338, #9427), MEK1 (#2352), p-MEK1 (S298, #9128), ERK1/2 (#4695), p-ERK1/2 (T202/Y204, #4370), MMP-2 (#4022) and MMP-9 (#3852) were obtained from Cell Signaling Technology (Beverly, MA). Primary antibodies against p-PAK2 (S141, #SAB4504634) and Actin (#4700) were purchased from Sigma-Aldrich (Shanghai, China).

H1299 cells were plated in RPMI media containing 10% FBS at a density of 5.0 × 10⁴/mL into each 100 × 20 mm tissue culture dish. The cells were incubated (37°C/5% CO₂) overnight to allow them to adhere to the plates. The next day, the media were removed and replaced with experimental media containing PCAIs (0 – 5 μM) or 5 μM NSL-100 and then incubated for 24 h. The cells were washed with PBS, lysed with RIPA buffer and the amount of protein in lysates was determined using Pierce BCA Protein Assay kit (Thermo Scientific, Rockford, IL). Lysate volumes containing 50 μg of protein were boiled in Laemmli sample buffer and subjected to SDS-PAGE. Proteins were then transferred onto PVDF membrane and immunoblotted using antibodies against Rac1, Cdc42 and RhoA (67B9) that were purchased from Cell Signaling Technology (Danvers, MA). Bound antibodies were visualized using horseradish peroxidase-linked anti-rabbit IgG (Santa-Cruz Biotechnology, sc-2004) and ECL reagents (Bio-Rad) according to the manufacturer's recommendation.

The polyclonal antibody against TIAM1 was purchased from Bethyl Laboratories, Inc (clone A300-099A; Montgomery, TX, USA). The monoclonal antibody against β-actin was purchased from Sigma-Aldrich (Darmstadt, Germany). Antibodies against Nanog (D73G4, #4903), Oct4 (#75463, D7O5Z), ALDH (D9Q8E, #54135), phospho-Rac1/Cdc42 (Ser71, #2461), and Rac1/Cdc42 (#4651) were purchased from Cell Signaling Technology (Danvers, MA, USA).
TIAM1 expression was transiently downregulated using two predesigned Silencer Select siRNAs directed against TIAM1 (#s14138 and s14139) (Thermo Fisher Scientific), and a non-targeting siRNA was used as a negative control. CRC cells were transfected with the annealed siRNA for 24 h using Lipofectamine RNAimax (Thermo Fisher Scientific), and TIAM1 expression was assessed by qPCR, 24 h following initial transfection. Tiam1 expression was examined by western blotting and subsequent experiments were conducted 48 h following the transfection.