Hh promotes the production of PI(4)P.

(A) A WT disc was stained for Smo. Dashed yellow line indicates the A/P boundary defined by Ci staining. Green box indicates the region for density analysis that is shown on the right, with a yellow line indicating the A/P boundary. (B) A WT disc was stained for Ci and PI(4)P. Arrow indicates PI(4)P accumulation in A compartment cells near the A/P boundary, and arrowhead indicates PI(4)P accumulation in P compartment cells. Dashed yellow lines indicate the A/P boundary defined by Ci staining. Green box indicates the region for density analysis using ImageJ software (NIH, version 1.48v) that is shown on the right with a yellow line indicating the A/P boundary. All wing imaginal discs shown in this study were dissected from third instar larvae with different genotypes and are shown with anterior on the left and ventral on the top. (C) Effect of Hh treatment on PIP content. S2 or NIH3T3 cells were stimulated with either 60% Hh-conditioned medium or Shh protein (5nM) before lipid extraction. A Thermo TSQ Vantage triple-quad mass spectrometer coupled with a Shimadzu HPLC as the front-end separation was used for the MRM measurement. * p < 0.01 versus time 0 (Student’s t test). (D) Left panel: S2 cells were treated with Stt4 dsRNA or Sac1 dsRNA, followed by treatment with control medium, 30% Hh-conditioned medium (Hh+), or 60% Hh-conditioned medium (Hh++). PI(4)P was detected by ELISA. * p < 0.01 versus control (first column); † p < 0.01 versus high level of Hh treatment (third column). Right panel: S2 cells were transfected with UAST-Ptc construct or treated with Ptc dsRNA and PI(4)P detected by ELISA. p < 0.01 versus control (first column). (E) S2 cells were treated with the indicated dsRNA followed by treatment with different amounts of Hh-conditioned medium and assayed for ptc-luc reporter activity. * p < 0.01 versus control (first column). † p < 0.01 versus high level of Hh (third column). †† p < 0.05 versus high level of Hh (third column). The efficiency of RNAi is shown in S3C and S3D Fig. (F) S2 cells were transfected with indicated constructs, treated with 60% Hh-conditioned medium, and assayed for ptc-luc activity. * p < 0.05 versus control. † p < 0.001 versus Hh alone. In these experiments, S2 cells stably expressing tub-Ci were used as they have full responsiveness to Hh stimulation. The expression of HA-Inp54p, Inp54pD281A, and IpgD are shown in the right panel by direct western blot with the anti-HA antibody, using lysates from cells expressing the indicated HA-tagged constructs. GFP was used as a transfection and loading control. The underlying data of panels C–F can be found in S1 Data.