Experimental procedures supplemental information
Drugs and others reagents
Collagen types I and IV from human placenta, ammonium pyrrolidinecarbodithioate (PDTC) and anti-actin antibody were purchased from the Sigma Chemical Company (St. Louis, MO, USA). Primary antibodies against IκBα, Bax, PARP, p65/NFκB, H3, Bcl-xL, Bcl-2, c-IAP2, x-IAP and secondary antibody anti-goat were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibodies against ILK, GSK-3β, P-GSK-3β, AKT, P-AKT and Bim were purchased from Cell Signaling Technology (Danvers, MA, USA). Secondary antibody anti-rabbit were purchased from Dako (Barcelona, Spain). Anti-1integrin and anti-1 active conformation antibodies and anti-1 activator were purchased from Chemicon (Temecula, CA, USA). All antibody dilutions were among 1:500 and 1:5000. Specific Signal-SilencerTM AKT siRNA, targets AKT1 and AKT2 and the GSK-3 fusion protein were purchased from Cell Signaling Technology (Danvers, MA, USA). SilencerTM negative control#1 siRNA was from Ambion, Inc. (Austin, TX, USA), and ILK specific siRNA was from Bionova Científica (Barcelona, Spain). ReliaBLOT® Block and the ReliaBLOT® HRP Conjugated were from Bethyl Laboratories, Inc. (Montgomery, USA). All reagents were prepared in DMSO so that the final concentration was < 0.1%, except collagen types I and IV, which were dissolved in 10 mMacetic acid. The 3xNFκB-TK-Luc reporter plasmid, which contains a three tandem repeat of the NFκB-binding motif of the H-2k gene upstream of the thymidine kinase minimal promoter has been described elsewhere [1] and was a gift from Dr. Manuel Fresno (Centro de Biología Molecular Severo Ochoa-UAM, Spain). The plasmid encoding a phosphorylation-resistant IκBα mutant, which differs from IκBα by Ser-to-Ala mutations at residues 32 and 36 (IκBαSer32,36/Ala) was kindly provided by Dr. A. Ortiz (Fundación Jiménez Díaz-UAM, Spain). Other antibodies and reagents were from Sigma Chemical Company (St. Louis, MO, USA).
Human Mesangial Cells (HMC) culture
HMC were cultured according to previously described procedures. The identity of the cells was confirmed by morphologic and functional criteria, as previously described[2]. The culture medium was Roswell Park Memorial Institute 1640 supplemented with 10% fetal bovine serum (FBS), 20mM L-glutamine and antibiotics (penicillin 100U/ml and streptomycin 100 mg/ml). Confluent cells were used between 5-12 passages. When cells reached confluence, they were sub-cultured at a ratio of 1:4 in RPMI 1640 supplemented with 10% FCS and antibiotics.
Western blot analysis
Cells samples were lisated in a solution containing 150mM NaCl, 10mM Tris-HCl (pH 7.4), 5mM ethylene diamine tetra acetate, 1% deoxycholic acid, 0.1% sodium dodecyl sulphate, 1% Triton X-100 and protease inhibitors 1mM phenylmethylsulphonylfluoride, 10 mg/ml aprotinin, 2 mg/ml leupeptin and the phosphatase inhibitor 0.2mM NaVO4. Cell proteins (30–40 g) were run in an 8–10% SDS–polyacrylamide gel, transferred onto a nitrocellulose membrane (Trans-Blot Transfer Medium, Bio-Rad, CA, USA) and incubated overnight at 4ºC with specific antibodies as previously described[3]. This incubation was followed by a second incubation with a peroxidase-conjugated secondary antibody and immunoreactive products were detected by chemiluminescence using the enhanced chemiluminescence Western Blotting Detection Reagents (Amersham Biosciences, UK) following the protocol provided by the manufacturer.
Immunofluorescence analysis.
After treatments cells were fixed with 4% paraformaldehyde in PBS, 10 min at RT, rinsed, and permeabilized with 0.5% Triton X-100 in PBS (10 min), cells were then incubated for 1 h with 5% donkey serum in PBS to block nonspecific binding. Afterwards cells were incubated overnight at 4ºC with anti-p65/NFκB antibody (1:100) and then rinsed with PBS. Finally, cells were incubated with FITC-conjugated anti-rabbit secondary antibody,for 1 h in the darkness. Slides were then washed and mounted with FluorSaveTM Reagent (Calbiochem, La Jolla, CA, USA). Dual-colour detection was performed by confocal laser scan microscopy LEICA TCS-SL (Heidelberg, Germany).
References
1- Yano O, Kanellopoulos J, Kieran M, Le Bail O, Israel A, Kourilsky P (1987) Purification of KBF1, a common factor binding to both H-2 and beta 2-microglobulin enhancers. EMBO J 6:3317-3324
2- Garcia-Escribano C, Diez-Marques ML, Gonzalez-Rubio M, Rodriguez-Puyol M, Rodriguez-Puyol D (1993) Somatostatin antagonizes angiotensin II effects on mesangial cell contraction and glomerular filtration Kidney Int43:324–333
3- Calleros L, Lasa M, Rodríguez-Alvarez FJ, Toro MJ, Chiloeches A (2006) RhoA and p38 MAPK mediate apoptosis induced by cellular cholesterol depletion. Apoptosis 11(7):1161-1173
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