Ml141

Five male BALB/c mice were i.p. injected with 100 μl ML-141 (selective inhibitor of Cdc42, 1 mg/ml in 30% dimethylsulfoxide-saline, Tocris Bioscience, Bristol, UK) daily for 3 weeks.^{37 (link), 43 (link), 44 (link)} Five blank control mice and five DMSO control mice were i.p. injected with 100 μl saline and 30% dimethylsulfoxide-saline respectively everyday for 3 weeks. Urine of all mice was collected weekly before killed.

Anti-Legionella antibody was from Public Health Ontario and anti-VipA antibody was generously provided by Dr. H Shuman (University of Chicago, USA). pSrc (Y416), total Src, total Akt antibodies were from Cell Signaling (Danvers, MA, USA) and the pAkt (S743) antibody was from ThermoFisher (Life technologies, Carlsbad, CA, USA). Anti-calnexin antibody was from BD biosciences (Mississauga, ON, Canada). FuGENE (HD) was from Promega Biosciences (Madison, WI, USA).
The following inhibitors were used in this study: PP2 (25 μM, Tocris) (Hanke et al., 1996 (link)), Ly294002 (100 μM, Sigma) (Vlahos et al., 1994 (link)), membrane permeable C3 transferase (0.5 μg/mL, Cytoskeleton Inc.) (Ridley and Hall, 1992 (link)), ML141 (20 μM, Tocris) (Surviladze et al., 2010 ), Blebbistatin (200 μM, Sigma) (Straight et al., 2003 (link)), Nsc23766 (50 μM, Tocris) (Gao et al., 2004 (link)), ROCK (1 μM, Millipore) (Narumiya et al., 2000 (link)), SMIFH2 (25 μM, Millipore) (Rizvi et al., 2009 (link)), CK-666 (80 μM, Sigma) (Nolen et al., 2009 (link)).

Anti-α2-integrin (sc-74466), anti-β-Catenin (sc-59737) and anti-RhoGDI (sc-373724) antibodies were from Santa Cruz Biotechnology. Anti-β1-integrin (AB1952), anti-active-β1-integrin (12G10), anti-α3-integrin (MAB2290), anti-p-Src (Y418; 07-909), anti-Src (GD11), anti-GAPDH (MAB374) and anti-GFP (MAB1083) antibodies were from Merck. Anti-p-Shp2 (Y542; 3751) and Shp2 (3752) antibodies were from cell signalling. Anti-E-cadherin (ab1416) and anti-laminin-β1 (ab44941) antibodies were from Abcam. Anti-vinculin (hVIN-1), anti-phosphotyrosine (4G10) and anti-HSC-70 (N69) antibodies were from Sigma-Aldrich. Anti-α4 integrin (MAB1354) was from R&D Systems. Anti-α5-integrin (eBioSAM-1) antibody was from eBioscience. Anti-p-RhoGDI (Y156; OAA100735) antibody was from Aviva Systems Biology. Anti-laminin-α3 (MAB21441) was from Novus Biologicals. Anti-Cdc42 (ACD03) antibody was from cytoskeleton. Anti-mouse horseradish peroxidase (HRP) and anti-rabbit HRP were from Dako. Anti-mouse Alexa 488 and Alexa 568, anti-rabbit Alexa 488 and Alexa 568, Sulfo-NHS-LC-biotin and phalloidin Alexa 647 were all obtained from Thermofisher. BTT-3033, PP2 and ML141 were from Tocris/Biotechne (Bristol, UK); shp099 was from Millipore. α2-integrin and control shRNA vectors were from Sigma-Aldrich. Calyculin A and protease inhibitor cocktail 1 were obtained from Calbiochem. Puromycin was obtained from GE Healthcare.

Human bone marrow MSCs were purchased from Cyagen Biosciences Inc. (Guangzhou, China). The cells were identified by detecting cell surface markers and the MSC multipotent potential for differentiation toward the adipogenic, osteogenic, and chondrogenic lineages; cells were maintained as described previously [19 (link)]. Cells were passaged every 3–4 days using 0.25% trypsin–EDTA (Gibco) when they reached approximately 80% confluence and were used for the experimental protocols between passages 5 and 10 [20 (link)].
For the Ang II treatments, MSCs were serum starved for 6–8 h, and different concentrations of Ang II were added for 24 h or as indicated in the figures. To inhibit the activities of AT1R, AT2R, FAK, RhoA, Rac1, and Cdc42, cells were pretreated with AT1R antagonist Losartan (5 μM; Sigma-Aldrich, St. Louis, MO, USA), AT2R antagonist PD-123319 (5 μM; Tocris Biosciences, Bristol, UK), FAK inhibitor PF-573228 (5 μg/ml; Tocris Biosciences), RhoA inhibitor C3 transferase (5 μg/ml; Cytoskeleton, Denver, CO, USA), Rac1 inhibitor NSC23766 (50 μM; Tocris Biosciences) [21 (link)], or Cdc42 inhibitor ML141 (5 μg/ml; Tocris Biosciences) for 30 min.

The geranylgeranyl transferase I inhibitor GGTI-286, the farnesyl transferase inhibitor FTI-276, the cell permeable IkB kinase inhibitor peptide and its inactive control peptide, the NF-kB inhibitor SN50, the SN50M inactive control peptide, the Rac inhibitor NSC23766, the Rho-associated protein kinase (ROCK) inhibitors, Y- 27362 and H1152, and fluvastatin were purchased from Calbiochem (San Diego, CA, USA). Atorvastatin, mevalonate, squalene, geranylgeranyl pyrophosphate (GGPP), farnesyl pyrophosphate (FPP), Bay 11–7082 were purchased from Sigma-Aldrich (St. Louis, MO, USA). The Cdc42 inhibitor, ML141, was purchased by TOCRIS (Bristol, UK). Human recombinant DKK-1 was from R&D Systems (Minneapolis, MN, USA). Antibody against RAP1A was purchased from Santa Cruz Biotechnology Inc. (Dallas, Texas, USA).