CystC and Δ14CystC inhibit actin cytoskeletal rearrangements stimulated by TGF-β in NMuMG cells. (a) NMuMG cells were infected with ecotropic retrovirus encoding either green fluorescent protein (GFP; control), CystC, or Δ14CystC, and subsequently were isolated by fluorescence-activated cell sorting for GFP expression to yield stable polyclonal populations of control, CystC-expressing, and Δ14CystC-expressing cells as indicated (upper panel). The expression and secretion of recombinant CystC proteins by infected NMuMG cells were monitored by immunoblotting conditioned medium with anti-CystC antibodies (lower panel). (b) Control, CystC-expressing, and Δ14CystC-expressing NMuMG cells were incubated in the absence or presence of transforming growth factor-β1 (TGF-β1; 5 ng/ml) for 8 hours, whereupon alterations in actin cytoskeletal architecture were revealed by direct rhodamine–phalloidin immunofluorescence. Shown are representative images from a single experiment that was repeated twice with identical results. (c) NMuMG cells were stimulated with TGF-β1 (5 ng/ml) for 0 to 24 hours in the presence of either glutathione S-transferase (GST), GST–CystC, or GST–Δ14CystC (each at 10 μg/ml) as indicated. Afterward, altered actin cytoskeletal architecture was revealed by direct rhodamine–phalloidin immunofluorescence. Shown are representative images from a single experiment that was repeated once with identical results.