SUPPLEMENTARY INFORMATION

Supplementary Figure Legends

Figure S1. Itch promotes endogenous Gli1 ubiquitination. (a-b) NIH3T3 cells were transfected with Itch siRNA (siItch) or scrambled (siCTR) oligos and after 24h with HA-Ub expression plasmid. Endogenous Gli1 ubiquitination was determined by anti-HA immunoblotting of immunoprecipitated Gli1 (a). Western blot of endogenous Gli1 and Itch are shown (b). The decreased ubiquitination of Gli1 and steady-state levels of Gli1 and Itch after siRNA were quantified and are representative of four independent experiments.

*siItch vs siCTR p<0.01; **siItch vs siCTR p<0.05. (c) Endogenous Gli1 was immunoprecipitated from NIH3T3 cells expressing the indicated proteins and treated with MG132 proteasome inhibitor prior to lysis. Western blot was performed with HA antibody to detected conjugated HA-Ub. The same membrane was reprobed with an anti-Gli1 antibody. Bottom panel, Itch protein levels in total cell lysates. (d) Itch ubiquitinates Gli1 in vitro. In vitro-translated 35S-labelled Gli1 was tested as described in Methods, in the absence or presence of recombinant Itch, for the indicated times. 35S-labelled Gli1 was revealed by SDS PAGE and fluorography. The graph represents the amount of Gli1 from each time point normalized to the amount at the initiation of reaction.

Figure S2. Gli1 stability is regulated via degron DN and DC-independent pathway. (a) Schematic representation of Gli1 protein. ZNF: DNA binding domain; DC: ßTrCP binding site; DN: destruction signal independent of DC; TAD: trans-activation domain. (b-c) Western blot of Gli1wt and Gli1 mutants following ectopic Itch expression in HEK293 cells. (d) Myc-Itch and HA-Gli1 constructs were co-transfected in HEK293 cells and the lysates immunoprecipitated with an anti-Myc antibody. The interaction of Gli1 with Itch was detected with a HA antibody. The blot was reprobed with anti-Myc. Bottom panel, Gli1 protein levels in total cell lysates.

Figure S3. Gli1 ubiquitination and function is regulated via degron DN and DC-independent pathway. (a) Gli1WT or Gli1∆398∆DC mutant were immunoprecipitated with rabbit Gli1 antibody from HEK293 cells expressing the indicated proteins, followed by Western blot with HA antibody to detected conjugated HA-Ub. Arrows indicate the Gli1WT or Gli1∆398∆DC immunoprecipitation. Bottom panel, Itch protein levels in total cell lysates. (b) Luciferase activity in HEK293 cells transfected with 12X Gli-Luc and Gli1WT or Gli1 indicated mutants alone or with Itch. *p<0.05. (c) Itch targets C-terminus end of Gli1 for ubiquitination. Flag-Gli1424-755 or Flag-Gli1424-1106 were immunoprecipitated with a Flag antibody from HEK293 cells expressing HA-Ub in presence or in absence of Myc-Itch, followed by western blot with HA antibody to detected conjugated HA-Ub. The blot was reprobed with anti-Flag. Bottom panel, Itch protein levels in total cell lysates. (d) Flag-Gli1424-1106 wild type (WT) or mutated in PPXY and pSP motifs (TM) were immunoprecipitated with a Flag antibody from HEK293 cells expressing HA-Ub in presence or in absence of Myc-Itch. Ubiquitination assay was performed as described in (c). (e) Gli1N-VP16 (2-424 aa) or Gal4-Gli1C-400 (755-1106 aa) were immunoprecipitated with VP16 or Gal4 antibodies from HEK293 cells transfected with HA-Ub in presence or absence of Myc-Itch. Ubiquitination assay was determineted as described in (c).

Figure S4. MappingGli1/Numb interacting region. (a) Schematic representation of Gli1 and Gli1 mutants. (b) GST-Numb was bound to glutathione-Sepharose beads and used for in vitro pull-down assays. Gli1wt and several mutants were in vitro translated and then incubated with free GST or GST-Numb. Protein complexes were detected by fluorography. (c) Gli1 wild type and Flag-Numb or Flag-Numb∆174-421 mutant were in vitrotranslated, incubated in binding buffer and immunoprecipitated with free or peptide-saturated Flag antibody. The interaction with Gli1 was detected by immunoblotting.

Figure S5. Itchsynergizes with Numb to induce Gli1 ubiquitination.GFP-Gli1was immunoprecipitated with a GFP antibody from HEK293 cells transfected with the indicated plasmids, followed by western blot with HA antibody to detected conjugated HA-Ub. The blot was reprobed with anti-GFP. Bottom panel, Itch, Numb and Numb∆PTB protein levels in total cell lysates.

Figure S6. (a) Effect of Gli1TM mutant on adhesion-independent growth: representative pictures. D283 cells were transfected with the indicated vectors and soft agar colony formation assay was performed. (b) Effect of Gli1TM and Gli1∆Dc mutants on cell transformation. RK3E cells were transfected with the indicated vectors, grown for three weeks and stained with crystal violet and the number of transformed foci were counted. * p<0.01.

Figure S7. (a) Levels of endogenous c-Jun protein, in Daoy cells, in presence of increasing amount of Gli1. (b) Endogenous c-Jun protein levels from Daoy cells transfected with Itch siRNA (siItch) or scrambled (siCTR) oligos. (c) Luciferase activity in Daoy cells transfected with MMP1-luc (grey bars) or 12X Gli-luc (black bars) and the indicated plasmids.*p<0.01

Supplementary Methods

In vitro ubiquitination assay was performed as described(Zhao et al., 2008), by incubating in vitro translated proteins, at 30 C with 50 mM Tris-HCl pH 7.5, 5 mM MgCl2, 200 microM okadaic acid, 2 mM ATP, 0.6 mM DTT, 1 mM ubiquitin aldehyde, E1, E2 and Ubiquitin. Polyubiquitinated products were subjected to SDS-PAGE and fluorography.

Supplementary References

Zhao X, Heng JI, Guardavaccaro D, Jiang R, Pagano M, Guillemot F, et al. (2008). The HECT-domain

ubiquitin ligase Huwe1 controls neural differentiation and proliferation by destabilizing the N-Myc

oncoprotein. Nat Cell Biol.10: 643-653