Fig. 2.

NSCLC cells secret SRGN heavily linked with CS-GAG side chains. a-b CM derived from designated cells was treated with 100 mU of enzymes as indicated for 24 h at 37 °C, followed by western blot analysis against anti-SRGN antibody. c Absolute amounts of GAGs and GAG-derived disaccharides in the CM of H1299/Mock versus H1299/SRGN cells and H460/Sh-CTRL versus H460/Sh-SRGN cells were quantitated. An aliquot of CM that was measured to contain 250 μg protein was used for GAG extraction. Total GAG amount was assessed by blyscan glycosaminoglycan assay kit. Crude GAGs were digested by ChaseABC (100 mU) and Heparanase I + III (100 mU) for 24 h at 37 °C, and subjected to LC-MS/MS analysis of CS and HS disaccharides. Absolute quantification was conducted by constructing standard curves for the individual disaccharide standard products. The amounts of GAGs and disaccharides in relation to altered expression of SRGN were obtained by normalizing the amounts of GAGs and individual disaccharides in H1299/SRGN and H460/sh-CTRL to those in H1299/Mock and H460/sh-SRGN cells, respectively. d RT-PCR analysis of the expression of SRGN, HS and CS synthesis genes in H1299 and H460 cells. Relative expression is shown by comparing the expression of each individual gene to that of GAPDH. e CM derived from H1299/SRGN cells was treated with or without enzymes as indicated for 24 h at 37 °C, followed by western blot analysis. Secretory proteins carrying compact HS and CS chains were assessed by anti-HS (10E4) and anti-CS (CS56) antibodies, respectively. SRGN core protein carrying ∆HS, ∆C4S and ∆C6S stubs was determined by anti-∆HS (3G10), anti-∆C4S (BE123) and anti-∆C6S (MK302) antibodies