DEV030015 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure 1 -

  Fig. S1. Knockdown of CanA1 by RNAi does not affect Hh-induced Smo accumulation. (A) A wild-type wing. (B) A wing from flies expressing UAS-CanA1RNAi by ap-Gal4. (C-C′′) A wing disc expressing UAS-CanA1RNAi by ap-Gal4 was immunostained with SmoN and β-gal antibodies. Knockdown of CanA1 by RNAi had no influence on Smo accumulation and did not modulate Hh signaling in wing disc.

• Supplemental Figure S2 -

  Fig. S2. (A) The efficiency of knocking down individual phosphatase in Fig. 2A was estimated by RNAi targeting each of the overexpressed HA-tagged phosphatase. (B) The efficiency of Cos2 RNAi used for Fig. 2I was examined by probing the cell lysates with anti-Cos2 antibody. β-Tubulin was used as the loading control.

• Supplemental Figure S3 -

  Fig. S3. Smo and Cos2 domains that are responsible for PP4 interaction in S2 cells. (A) Mapping PP4 interaction domain in SmoCT. S2 cells were transfected with HA-PP4 plus various Flag-tagged Smo deletion mutants. Cell extracts were immunoprecipitated with Flag antibody followed by western blot with HA antibody. Cell lysates were also subjected to direct western blot with anti-Flag or anti-HA antibodies to monitor the protein levels. Asterisks indicate the Flag-tagged Smo proteins. SmoCT lacking amino acids 626-678 does not interact with PP4. (B) Summary of Cos2-PP4 interaction. With the similar approach for mapping PP4 binding domain in SmoCT, S2 cells were co-transfected with Flag-tagged PP4 and HA-tagged Cos2 variants, followed by IP with HA antibody and western blot with Flag antibody to detect the Cos2-bound PP4 (data not shown).

• Supplemental Figure S4 -

  Fig. S4. PP4 has little effect on Myc-SmoΔ626-678 phosphorylation. S2 cells were co-transfected with UAS-Myc-SmoΔ626-678 and UAS-GFP, with or without UAS-HA-PP4, and treated with Hh-conditioned medium or control medium. Cell extracts were immunoprecipitated with anti-Myc followed by western blot with anti-Myc antibody to examine the phosphorylation levels of Myc-Smo. The expression of GFP was used as an internal control. Cell lysates were probed with HA antibody to show the expression of HA-PP4. The overexpression of PP4 has little effect on the phosphorylation of Smo lacking amino acids 626-678.

• Supplemental Figure S5 -

  Fig. S5. Knockdown of phosphatases by RNAi in regulating Smo cell-surface accumulation. S2 cells were transfected with CFP-Smo or CFP-SmoSD123, in combination with indicated phosphatase dsRNA or HA-tagged phosphatase. Transfected cells were then treated with Hh-conditioned medium or control medium. To visualize the cell-surface-localized Smo, transfected cells were immunostained with anti-SmoN antibody before cell permeabilization. The total amount of expressed Smo was indicated by CFP signal. The efficiency of knockdown individual phosphatase was estimated by RNAi targeting each of the overexpressed HA-tagged phosphotase (not shown). (C, compare with A) PP4R dsRNA treatment did not promote Smo cell-surface accumulation. (D, compare with B) PP4R RNAi elevated the Hh-induced Smo cell-surface accumulation. (E and F, compare with A and B, respectively) Wdb RNAi had no effect on Hh-induced Smo cell-surface accumulation. (G,H) Expressing HA-PP4 downregulated Hh-induced Smo cell-surface accumulation, whereas expressing HA-Mts did not. (J, compare with I) Hh promoted further accumulation of SmoSD123 on the cell surface. (K,L) Mts RNAi and Wdb RNAi did not elevate the cell-surface accumulation of SmoSD123. (M) PP4R RNAi induced further cell-surface accumulation of SmoSD123. (N) Ratio of cell surface level (SmoN signal) to total level (CFP signal) of Smo. Each data set was based on 15 individual cells. WT, CFP-Smo.

• Supplemental Figure S6 -

  Fig. S6. Ci^FL and ptc-lacZ expression are inhibited by inactivating Mts. (A-A′′′) A wing disc expressing UAS-P35 by MS1096 Gal4 was immunostained with Ci, β-gal and SmoN antibodies. (B-B′′′) A wing disc co-expressing UAS-DN-Mts with UAS-P35 (to block apoptosis due to loss of Mts activity) by MS1096 Gal4 was immunostained with Ci, β-gal and SmoN antibodies. Arrow in B indicates the remarkably decreased Ci^FL. Arrow in B′ indicates the attenuated ptc-lacZ expression. The expression of DN-Mts has little effect on Smo accumulation. Note that the MS1096 Gal4 is expressed at lower level in the ventral region than in the dorsal region of the wing imaginal disc.

• Supplemental Figure S7 -

  Fig. S7. Overexpressing PP4 does not affect the levels of Ci^FL. A wing disc expressing two copies of UAS-HA-PP4 by ap-Gal4 was immunostained with anti-Ci and anti-HA antibodies. The overexpression of PP4 in dorsal compartment cells had no effect on Ci^FL, compared with Ci^FL staining in ventral compartment cells.

• Supplemental Figure S8 -

  Fig. S8. Gain- and loss-of-function of Mts or Wdb in regulating Ci and Smo in the wing. (A-B′′) Wing discs expressing UAS-Wdb or UAS-Mts by ap-Gal4 were stained with Ci and SmoN antibodies to show the elevation of Ci^FL and the little, if any, changes in Smo accumulation. Arrows in A and B indicate the elevation of Ci^FL. (C-C′′) A wing disc expressing UAS-WdbRNAi by ap-Gal4 was stained with Ci and SmoN antibodies. Arrow in C indicates the decreased levels of Ci^FL in dorsal UAS-WdbRNAi-expressing cells. (D-D′′) A wing disc expressing UAS-MtsRNAi by act>CD2>Gal4 at 20°C was stained with Ci and SmoN antibodies. Arrow in D indicates the reduced Ci^FL staining in Mts RNAi cells. The expression of GFP marks the cells expressing UAS-MtsRNAi. (E-G) Wing discs expressing UAS-Mts or UAS-Wdb alone, or simultaneously expressing UAS-Mts and UAS-Wdb by ap-Gal4 were stained with anti-Ptc antibody. Arrow in G indicates that co-expressing Mts with Wdb induces higher level of ectopic Ptc expression. Co-expressing Mts with Wdb does not affect Smo accumulation in wing discs (not shown).