Fig. 1.

Physiological regulation of exogenously introduced Gli2. (A) NIH 3T3 cells were transiently transfected for expression of GFP-Gli2 and stained 48 h later. Overexpressed Gli2 is localized in the cytoplasm, at one end of the primary cilium (visualized with anti-detyrosinated tubulin), and in the nucleus (visualized with DAPI). (Scale bar, 5 μm.) (B) NIH 3T3 cells transfected for expression of Gli2 show that high levels produce unregulated reporter expression irrespective of ShhN stimulation, whereas lower levels can contribute to increased reporter expression with regulation by ShhN. Gli-luciferase reporter and control SV40-Renilla luciferase were cotransfected with the indicated proportion of Gli2 DNA (% of total DNA, wt/wt). Following transfection, cells were grown to confluency, incubated with Shhn, and assayed for reporter activity. Error bars indicate SD. (C) A Hh-responsive NIH 3T3/HA-Gli2 clone. An NIH 3T3 cell clone with a stably integrated construct for expression of HA-Gli2 was established and chosen for its low level of HA-Gli2 expression and assayed with Gli-luciferase reporter for response to ShhN stimulation. This clone displays Hh-responsive, cyclopamine-suppressible pathway activity. Error bars indicate SD. (D) 3T3/HA-Gli2 cells were incubated in the absence or presence of ShhN with vehicle (Veh), cyclopamine (Cyc), or SAG1, and then lysed and analyzed by IP-Western with anti-HA antibodies. Markers of molecular mass are indicated on the Left, and migration of full-length Gli2 (HA-Gli2, arrow) and its repressor form (HA-Gli2R, arrowhead) on the Right.