P-Akt Thr308. T-Akt *** *** Anti-α3 IgG Ctrl

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1 P-Akt Thr38 P-Akt Thr38 Relative pakt (Thr38) expression (normalized to total Akt) Anti-α3 IgG Anti-α3 IgG V Fig or 1 integrin blockade effects on Akt Thr38 phosphorylation. Western blotting analysis of phosphorylated P- and Thr38 expression in human fetal islet cells following 24 hours of cell culture on collagen I/IV upon anti-α3, 1 or treatment or without treatment []. Representative blots are shown. Data are normalized to the untreated controls and expressed as M+SEM (n=3 experiments/group). p<1.

2 Cell adhesion (Fold Change) A Anti-α3 IgG Cell spreading (Fold Change) Anti-α3 IgG V IgM IgM Fig 2 : 3 or 1 integrin blockade decreases INS-1 cell adhesion and spreading on collagens I and IV. INS-1 cell adhesion (A) and spreading () after 3 hours of culture on collagen I or IV and treated with anti-a3, anti- 1, IgG, IgM or without treatment (). Data are normalized to the untreated controls. M±SEM, n=5-6 experiments/group. p<1, p<1 vs. or untreated controls.

3 A V immunopositive cells (%) Anti-α3 IgG Insulin immunopositive cells (%) Anti-α3 IgG IgM IgM Fig 3 : 3 or 1 integrin blockade decreases and insulin expression in INS-1 cells on collagens I and IV. Quantitative analysis of (A) and () insulin expression following anti-α3, anti- 1, IgG or IgM treatment or without treatment [] and 24 hours of cell culture on collagen I or IV. Data are expressed as M±SEM (n=5-6 experiments/group). p<5, p<1, p<1 vs. or untreated controls.

4 A Relative phosphorylated ERK1/2 expression (normalized to total ERK) U126 P- ERK T- ERK V U126 P- ERK T- ERK C Relative cleaved caspase 3 expression (normalized to total caspase 3) Relative expression (normalized to ) U126 U126 C-Casp 3 T-Casp U126 U126 C-Casp 3 T-Casp 3 Fig. 4 : MEK inhibitor effects on INS-1 cell signaling and expression. Western blot analyses of (A) ERK1/2 phosphorylation, () caspase3 cleavage and (C) expression following 24 hour culture on collagen I or IV and treatment with the MEK inhibitor, U126. Representative blots are shown. Data are normalized to untreated controls and expressed as M+SEM (n=3 experiments/group). p<5, p<1.

5 P-AktThr 38 P-Akt Thr38 Relative pakt (Thr38) expression (normalized to total Akt) Anti-α3 IgG IgM P-AktThr 38 Anti-α3 IgG IgM P-AktThr 38 V Fig or 1 integrin blockade effects on Akt Thr38 phosphorylation. Western blotting analysis of phosphorylated P- and Thr38 expression in INS-1 cells following 24 hours of cell culture on collagen I/IV upon anti-α3, 1 or treatment or without treatment []. Representative blots are shown. Data are normalized to the untreated controls and expressed as M+SEM (n=3 experiments/group). p<5, p<1.

6 Relative phosphorylated Akt expression (normalized to total Akt) A P- AktSer 473 T- Akt P- AktThr 38 T- Akt V P- AktSer 473 T- Akt P- AktThr 38 T- Akt Relative phosphorylated GSK3-β protein expression (normalized to total GSK3-β) Relative cleaved caspase 3 protein expression (normalized to total caspase 3) C D V P- GSK3-β T- GSK3-β C-Casp 3 T-Casp P- GSK3-β T- GSK3-β C-Casp 3 T-Casp 3 XIAP XIAP E Relative XIAP expression (normalized to ) Relative expression (normalized to ) Fig. 6 : PI3K inhibitor effects on INS-1 cell signaling and expression. Western blot analyses of (A) phosphorylated (P) and total (T) Akt Ser473, phosphorylated (P) and total (T) Akt Thr38, () XIAP, (C) phosphorylated (P) and total (T) GSK3-, (D) cleaved (C) and total (T) caspase 3 (Casp 3) and (E) expression following 24 hours of culture on collagen I or IV and treatment with the PI3K inhibitor,. Representative blots are shown. Data are normalized to the untreated controls and expressed as M+SEM (n=3 experiments/group). p<5, p<1, p<1.

7 A P-FAK P-FAK P-ERK1/2 P-ERK1/2 T-FAK T-FAK T-ERK1/2 T-ERK1/2 Relative pfak expression (normalized to total FAK) Relative perk1/2 expression (normalized to total ERK1/2) C C-Casp3 C-Casp3 D Relative Caspase 3 Cleavage (normalized to total Caspase 3) T-Casp T-Casp3 Relative expression (normalized to ) V V Fig. 7 : α3β1 integrin blockade decreases FAK, ERK1/2 phosphorylation, reduces expression and increases caspase 3 cleavage. Western blotting analysis of phosphorylated [P] and total [T] FAK (A), P- and T-ERK1/2 (), cleaved [C] and total [T] caspase (Casp) 3 (C) and expression (D) following 24 hours of cell culture on collagen I/IV upon anti-α3 1, treatment or without treatment []. Representative blots are shown. Data are normalized to the untreated controls and expressed as M+SEM (n=3 experiments/group). p<1.

8 A P-AktSer 473 P-AktSer 473 P-Akt Thr38 P-Akt Thr38 Relative pakt Ser473 expression (normalized to total Akt) Relative pakt Thr38 expression (normalized to total Akt) C XIAP XIAP D P-GSK3β P-GSK3β T-GSK3 β T-GSK3β Relative XIAP expression (normalized to ) V Relative pgsk3β expression (normalized to total GSK3β) V Fig. 8 : α3β1 integrin blockade decreases Akt and GSK3β phosphorylation, reduces XIAP expression. Western blotting analysis of phosphorylated P- and Ser473 (A); P- and Thr38 (); XIAP (C) and P- and T-GSK3β (D) expression following 24 hours of cell culture on collagen I/IV upon anti-α3 1, treatment or without treatment []. Representative blots are shown. Data are normalized to the untreated controls and expressed as M+SEM (n=3 experiments/group). p<5, p<1, p<1.

9 Table 1: List of antibodies used for immunofluorescence and/or western blotting analysis Primary Antibody Dilution Company, Location Mouse anti-phosphorylated Akt (Ser 473) 1:2 a 2 or Rabbit anti-phosphorylated Akt (Thr 38) Rabbit anti-akt Rabbit anti-phosphorylated Thr22/Tyr24 ERK12 Rabbit anti-erk1/2 Rabbit anti-xiap Rabbit anti-cleaved Caspase 3 Rabbit anti-caspase 3 Rabbit anti-phosphorylated GSK3-β Rabbit anti-gsk3-β Rabbit anti- Mouse anti- Mouse anti-human Insulin Mouse anti-rdu Rabbit anti-ki67 1:2 1:2 a or 2 1:2 a or 2 1:2 a or 1 1:8 1:1 1:1 1:2 1:2 1:1 a or 5 1:2 1:1 a 1:5 a 1:2 Gift from Dr. Wright, Vanderbilt University, USA D iosciences, Mississauga, ON, Canada Sigma, Saint Louis, Missouri, USA Sigma, Saint Louis, Missouri, USA Abcam, Cambridge, MA, USA a dilution factor used for immunofluorescence