Fig. 3

Suppression of TGF-β signaling by BG is dependent on the presence of its GAG chains. A, B Western blot and signal quantification of SMAD2/3 phosphorylation in SKOV-3 BGKO cells expressing either control vector (BGKO-Cntl), FL-BG (BGKO-FL), or ∆GAG-BG (BGKO-∆GAG) treated with increasing doses of A TGF-β1 or B TGF-β2 (25 pM to 100 pM). All signaling quantifications were performed by normalization of phospho-SMAD2/3 to the total SMAD2/3 signal. Phospho-SMAD signal normalized to SMAD signal is plotted by Mean ± SEM, (n = 3 combined trials). *p < 0.05, unpaired t-test between FL-BG and ∆GAG-BG within the same TGF-β treatment dose. (TGF-β1 at 25 pM, p = 0.0238, 100 pM, p = 0.0214, FL-BG vs. ∆GAG-BG. TGF-β2 at 25 M, p = 0.0167, 50 pM TGF-β2, p = 0.0185, FL-BG vs. ∆GAG-BG.) C, D Western blot and signal quantification of SMAD2/3 phosphorylation in HEYA8 cells expressing either control, FL-BG, or ∆GAG-BG, treated with increasing doses of C TGF-β1 or D TGF-β2 (25 pM to 100 pM). All signaling quantifications were performed by normalization of phospho-SMAD2/3 to the total SMAD2/3 signal. Phospho-SMAD signal normalized to SMAD signal is plotted by Mean ± SEM, (n = 3 combined trials). *p < 0.05, **p < 0.01, unpaired t-test between FL-BG and ∆GAG-BG within the same TGF-β treatment dose. (TGF-β1 at 25 pM, p = 0.0093, 50 pM, p = 0.0.193, 100 pM p = 0.0174, FL-BG vs. ∆GAG-BG. TGF-β2 at 100 pM, p = 0.0445, FL-BG vs. ∆GAG-BG.) E Representative immunofluorescence images of SMAD2 or nuclei (Hoechst) in response to 25 pM of TGF-β1 or TGF-β2, in HEYA8 cells. Scale Bar = 50 μm. Quantification of nuclear accumulation of SMAD2 was analyzed using cell profiler. The ratio of nuclear SMAD2 compared to total cellular SMAD2 is presented. Mean ± SEM, (n = 7 replicates). ****p < 0.0001, One-way ANOVA followed by unpaired t-test. (p < 0.0001, TGF-β1, and p = 0.0007, TGF-β2, FL-BG vs ∆GAG-BG.) F, G Western blot of phospho-SMAD2/3 in FL-BG and ∆GAG-BG expressing HEYA8 cells treated with 25 pM F TGF-β1 or G TGF-β2 either alone or in combination with ( +) 200 pg/mL to (+ +) 400 pg/mL of recombinant sol-BG. Phospho-SMAD2/3 signal normalized to actin signal is plotted by Mean ± SEM, (n = 2). *p < 0.05; **p < 0.05, unpaired t-test between TGF-β treated compared to TGF-β + sol-BG combination. (p = 0.0217, FL-BG TGF-β2 treated vs. TGF-β2 + 400 pg/mL sol-BG, and p = 0.0302, ∆GAG-BG TGF-β2 treated vs. TGF-β2 + 200 pg/mL sol-BG, p = 0.0171, ∆GAG-BG TGF-β2 treated vs. TGF-β2 + 400 pg/mL sol-BG)