1. Glucose mM Laminin β1 Actin 5612182430 Insulin Laminin β1 Actin 010pM100pM1nM S 1A. Dose-dependent increase in high glucose (HG)- and high insulin (HI)-induced laminin  1 synthesis. Quiescent MCT cells were incubated with or without different concentrations of HG or HI for 5 min and immunoblotting with laminin  1 antibody was performed on cell lysates. The blots were reprobed with an anti-actin antibody to assess loading. Representative blots from 2 independent experiments are shown.

2. S 1B. HG, HI and HG+HI stimulate laminin  1 synthesis for up to 48 hours. MCT cells were incubated with HG, HI or HG+HI for 60 min or for up to 72 hours. Immunoblotting with laminin  1 antibody was performed on cell lysates. The blots were reprobed with an anti-actin antibody to assess loading. Representative blots from 3 experiments are shown. Composite data from 3 experiments are shown in a graph; † p<0.01, * p<0.05 vs control by ANOVA. 01212244872 Glucose (hr) Laminin β1 Actin 01212244872 0.00 0.25 0.50 0.75 1.00 * * * Laminin/Actin Time in hours 01212244872 Insulin (hr) Laminin β1 Actin 012122448 72 0.0 0.2 0.4 0.6 0.8 1.0 1.2 * Laminin/Actin Time in hours 01212244872 Glucose+Insulin (hr) Laminin β1 Actin 01212244872 0.0 0.2 0.4 0.6 0.8 † † † Laminin/Actin Time in hours

3. HG, HI and their combination (HG+HI) do not induce synthesis of type IV collagen and fibronectin following incubation for up to 60 min. Western blotting was performed on cell lysates using an anti-collagen type IV antibody and anti-fibronectin antibody. The lower panels show blots reprobed with anti-actin antibody to assess loading. Representative blots from 2 experiments are shown for type IV collagen. Histogram shows composite data from 3 experiments for fibronectin and the changes were not found to be significant. S 1C. Type IV Collagen Actin 0510153060 Glucose min Type IV Collagen Actin 0510153060 Glucose+Insulin min Type IV Collagen Actin 0510153060 Insulin min Glucose min Fibronectin Actin 0510153060 0510153060 0.0 0.2 0.4 0.6 Time in minutes Fibronectin/Actin 0510153060 0.0 0.2 0.4 0.6 Time in minutes Fibronectin/Actin Glucose+Insulin min Actin Fibronectin 0510153060 Actin Insulin min Fibronectin 0510153060 0510153060 0.0 0.2 0.4 0.6 Time in minutes Fibronectin/Actin

4. S 1D. HG, HI and their combination (HG+HI) increase synthesis of laminin β1 as evident by 35 S labelling studies. Quiescent MCT cells were pre-incubated with [ 35 S]-methionine for 2 hours prior to incubation with HG or HI. Equal amounts of protein from each group was immunoprecipitated using anti-laminin  1 antibody. The protein coupled to protein A agarose beads were separated by boiling with sample buffer lacking bromophenol blue and centrifuged. The supernatants were spotted on 3mm filter paper for determining radioactivity. Composite data from 3 experiments are shown in a graph; ‡ p<0.001, † p<0.01, * p<0.05 vs control by ANOVA. † Glucose (min) 0510153060 0 40 80 120 [ 35 S] labelled laminin β1 (% of control) * 0510153060 0 50 100 150 [ 35 S] labelled laminin β1 (% of control) * * Insulin (min) [ 35 S] labelled laminin β1 (% of control) 0510153060 0 40 80 120 ‡ ‡ Glucose+Insulin (min)

5. S 2A. HG, HI and HG+HI induced laminin  1 synthesis in glomerular epithelial cells. Glomerular epithelial cells were treated with HG, HI and HG+HI for the time duration as shown in figure. Immunoblotting with laminin  1 antibody was performed on cell lysates. The lower panels in each figure show blots reprobed with anti-actin antibody to assess loading. Representative blots from 3 experiments are shown. † p<0.01, * p<0.05 vs control by ANOVA. 0510153060 Laminin β1 Glucose min Actin * 0510153060 0.0 0.2 0.4 0.6 † Laminin/Actin Time in minutes 0510153060 Laminin β1 Insulin min Actin 0510153060 0.00 0.25 0.50 0.75 1.00 * * Laminin/Actin Time in minutes 0510153060 Laminin β1 Glucose+Insulin min Actin 0510153060 0.0 0.2 0.4 0.6 0.8 1.0 * * * Laminin/Actin Time in minutes

6. Laminin β1 synthesis, induced by the three conditions in glomerular epithelial cells, was inhibited by cycloheximide but not by actinomycin D. MCT cells were pre-incubated with either actinomycin D or cycloheximide prior to incubation with or without HG, HI or HG+HI. Actinomycin D did not inhibit laminin  1 synthesis but cycloheximide did in cells treated with HG, HI and HG+HI. Loading was assessed by immunoblotting with actin antibody. ‡ p<0.001, † p<0.01, * p<0.05 by ANOVA. S 2B. 0.0 0.2 0.4 0.6 0.8 * † Laminin/Actin Glucose+Insulin – + – + – –+ –– + + + –– – – –+ Laminin β1 Actin Cycloheximide(10μm) Actinomycin(10μm) 0.00 0.10 0.20 0.30 0.40 0.50 ** Laminin/Actin Glucose – + – + – –+ –– + + + –– – – –+ Laminin β1 Actin Cycloheximide(10μm) Actinomycin(10μm) 0.0 0.2 0.4 0.6 0.8 †‡ Laminin/Actin Insulin – + – + – –+ –– + + + –– – – –+ Laminin β1 Actin Cycloheximide(10μm) Actinomycin(10μm)

7. S 3. Cycloheximide induced p38 MAPKinase phosphorylation but not HG, HI or HG+HI. Cells were pre-incubated with cycloheximide, followed by treatment with or without HG, HI or HG+HI. Cycloheximide induced p38 MAPkinase phosphorylation but not HG, HI, or both together. Representative blots from 3 independent experiments are presented. Cycloheximide(10μm) Glucose p38 MAPK + – +– + – – + P. p38 MAPK Insulin p38 MAPK P. p38 MAPK + – +– + – – + Cycloheximide(10μm) + – +– + – – + Glucose+Insulin p38 MAPK P. p38 MAPK Cycloheximide(10μm)

8. S 4. LY294002 and rapamycin block HG-, HI- and HG+HI-induced laminin  1 synthesis. Quiescent MCT cells were incubated with or without 25  M LY294002, an inhibitor of PI3-kinase (A), or 22nM rapamycin, an inhibitor of mTOR (B), for 1 hour prior to treating the cells with HG, HI or HG+HI for 5 min. Immunoblotting with laminin  1 antibody was performed on cell lysates. The blots were reprobed with an anti-actin antibody assess loading. Representative blots from 3 independent experiments are shown. AB LY 294002(25µM) Glucose Laminin Actin +– + – +––+ + – +– + – –+ Insulin LY 294002(25µM) Laminin Actin +–+– +––+ LY 294002(25µM) Laminin Actin Glucose+Insulin Rapamycin(22nM) Glucose Laminin Actin + – + – + – –+ +– + – + – – + Insulin Rapamycin(22nM) Laminin Actin + – + – + – – + Rapamycin(22nM) Laminin Actin Glucose+Insulin

9. S 5. A B DN-PI3-K Glucose Vector – + – + – –+ –– + + + P. Akt Insulin DN-PI-3K Vector – + – + – –+ –– + + + P. Akt Glucose+Insulin DN-PI-3K Vector – + – + – – + – – + + + P. Akt DN-mTOR Glucose Vector – + – + – – + – – + + + P.p70S6K Insulin DN-mTOR Vector – + – + – –+ –– + + + P.p70S6K Glucose+Insulin DN-mTOR Vector – + – + – –+ –– + + + P.p70S6K Expression of dominant negative PI3-kinase and kinase-dead mTOR constructs block phosphorylation of their downstream targets. These constructs do not carry a tag. Success of mutant transfection was demonstrated functionally by showing that HG-, HI- and HG+HI-induced increment in phosphorylation of Akt and p70S6Kinase, downstream substrates for PI3-kinase and mTOR, respectively, was blocked.