Fig 9. Epithelial secretome induces fibroblast activation and collagen via TGF-β.
Condition medium of HaCaT cells treated with areca nut with or without ALK5 inhibitor; SB 431542 (-/UN; condition media of untreated HaCaT cells,-/5H, areca nut treated HaCaT cells,-/ALK5; ALK5 inhibitor treated HaCaT cells and-/5H+ALK5 inhibitor treated HaCaT cells was used to treat serum deprived hGF cells for 48 hours. Simultaneous direct treatment of areca nut with or without ALK5 inhibitor (SB 431542) (UN/-, 5H/-, ALK5/-, 5H+ALK5/-) was given to another set of serum deprived hGF cells for the same duration. Fibroblast activation and total collagen was assessed by αSMA stress fiber formation by immunocytochemistry and direct red 80 staining respectively. a] Condition media of areca nut treated HaCaT cells (-/5H) induced αSMA stress fibres significantly more as compared to untreated (-/UN) and direct treatment of hGF cells with areca nut (5H/-). It got compromised with ALK5 inhibitor, SB 431542 (-/ALK5). Direct treatment of TGF-β with (T+ALK/-) or without ALK5 inhibitor (T/-) was used as positive control for the experiment (image magnification 63X). b] Representative images for total collagen staining by direct red 80 (image magnification 10X) expressed in hGF cells upon respective treatments depicted above each panel. The treatments are as described in 4a. Note the significant increase in the collagen staining when hGF cells were treated directly with TGF-β or conditioned media of HaCaT cells treated with areca nut. Both these regulations were compromised in the presence of ALK5 inhibitor (SB 431542). c] Bar diagram showing quantitation of direct red staining for total collagen measured as O.D per 105 cells of treatments described in 4b.