Fig. 7.

The zinc coordination center of Shh is required for long-range signaling. a Experimental setup and immunofluorescent staining of Shh-expressing Hek293t aggregates. Hek293t cells were transfected with Shh or Shh-C199*, washed off the culture dish with PBS and placed on a rotating platform in DMEM without serum for 2 days, followed by live staining with 5E1. Shades of brown represent differences in transfection efficiency. b Experimental setup: Hek293t cells were transfected with Shh-constructs, washed off the culture dish with PBS, and mixed with mESC Ptch1^LacZ/+;Shh^−/− reporter cells in a 1:4 ratio. Chimeric aggregates were then formed on a rotating platform in the absence of serum. The Ptch1:LacZ expression was measured after 3 days and normalized to total protein content. c Ptch1:LacZ expression in chimeric aggregates in response to the indicated versions of Shh expressed by Hek293t cells. Box-and-Whisker plot, n = 2. *p < 0.05, **p < 0.01, ****p < 0.0001. d 5E1 live staining of Shh in the chimeric aggregates described in B and C. e Experimental setup: Hek293t cells were transfected with Shh-constructs, washed off the culture dish with PBS, and incubated on a rotating platform. Separate mESC Ptch1^LacZ/+;Shh^−/− reporter aggregates were added to the dish in a 1:4 ratio and Ptch1:LacZ expression in mESCs was measured after 3 days. f Ptch1:LacZ expression normalized to total protein in mESC aggregates co-cultured with Shh-expressing Hek293t aggregates. Box-and-Whisker plot, n = 4, ****p < 0.0001