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. 2004 Aug;165(2):659–669. doi: 10.1016/s0002-9440(10)63330-5

Figure 9.

Figure 9

Thy-1 is mechanistically important for differential regulation of TGF-β activation. A: Thy-1-transfected RFL-6 CD90 cells express Thy-1 on the cell surface, whereas empty vector-transfected RFL-6 EV cells do not. RFL-6 EV (top right) and RFL-6 CD90 cells (bottom left) were harvested and incubated with fluorescein isothiocyanate-conjugated anti-CD90.2 antibody. Expression of Thy-1 on the cell surface was determined by flow cytometry. RFL-6 EV cells treated with fluorescein isothiocyanate-conjugated mIgG1κ (top left) were used as a control. B: RFL-6 EV and RFL-6 CD90 cells were cultured in six-well plates until 70 to 80% confluent. Cells were made quiescent in F12K media with 0.1% FBS for 24 hours and in serum-free media for 2 hours. Cells were treated with increasing concentrations of IL-4, PDGF-BB, IL-1β, or BLM for 24 hours. Conditioned media were harvested and assayed for active TGF-β activity and total TGF-β activity. Cell number in each well was determined by cell counting. Values of TGF-β activity were normalized for cell number. Results are shown as mean ± SD of triplicate wells. *, P < 0.01 for basal active TGF-β activity versus 5 ng/ml IL-4-, 20 ng/ml PDGF-BB-, 10 ng/ml IL-1β-, or 0.5 μg/ml BLM-induced active TGF-β activity in Thy-1 (−) cells. C: Expression of Thy-1 in RFL-6 CD90 cells does not abrogate increased phosphorylation levels of Smad3 in response to exogenous TGF-β1. RFL-6 EV and RFL-6 CD90 cells were treated with 8 pmol/L TGF-β1 for 1 to 24 hours as indicated. Levels of phosphorylated Smad3 and total Smad3 were determined by immunoblot. Phosphorylated Smad3 levels were normalized to total Smad3. Results are the means of three independent experiments ± SD. *, P < 0.01 for basal control versus TGF-β1 at 1 hour in RFL-6 CD90 cells or RFL-6 EV cells.