TGF-β production and expression of mesenchymal markers in A549 cells.

<p>(<b>A</b>) Quantification of TGF-β in supernatants from cells cultured for 48 h in NG (white bar), HG (black bar) or OG (gray bar). (<b>B</b>) Western blot of cell lysates loads analyzing expression levels of N-cad, (first lane) and Vimentin (second lane) in cells cultured in NG (white bar), HG (black bar) or OG (gray bar) conditions with or without 2 ng/mL TGF-β. Signal intensities were normalized, with GAPDH as loading control, and relative intensities of N-cad (<b>C</b>) and Vimentin (<b>D</b>) are shown. The results are representative of 3 independent experiments. Quantitative analyses are shown as mean ± standard deviation. P values were calculated using the Student's t test. *<i>P≤0.01</i>; **P<i>≤0.005</i>.</p

Analysis of cell morphology and motility.

<p>Cell morphology (<b>A</b>); cell motility (<b>B</b>) and cell circularity (<b>C</b>) of A549 cells treated in NG, HG or OG conditions with (right panel) or without TGF-β (left panel). Representative photos are presented. Tracks of 50 random individual cells on gold solution (<b>D</b>) were measured using the Scion Image program represented as squared pixels, and are shown as mean ± SD. NG (white bar), HG (black bar) or OG (gray bar). *P≤0,005.</p

Effect of hyperglycemia onfFN biosynthesis.

<p>Western blot of A549 cell lysates cultured for 48 h in NG (white bar), HG (black bar) or OG (gray bar) medium with (+) or without (−) TGF-β, showing expression of total FN (first lane) and onfFN (second lane) (<b>A</b>). Signal intensities were normalized, with GAPDH as loading control, and relative intensities of total FN (<b>B</b>) and onfFN (<b>C</b>) are shown. (<b>D</b>) Western blot of A549 total FN (first lane) and onfFN (second lane) immunoprecipitated from cell lysates by FDC-6 mAbs, submitted (+) or not (−) to the remotion of <i>O</i>-glycosylation. Human plasma FN (pFN, 0.5 µg) was used as control. qRT-PCR analysis of gene that codifies IIICS domain of onfFN (<b>E</b>) and GalNacT6 (<b>F</b>) respectively. Graph shows one of three independent experiments as mean ± SD. * <i>P≤0.005</i>. Effect of anti-TGF-β blocking antibody in the expression of total FN (first line) and onfFN (second line) (<b>G</b>).</p