Supplementary methods Immunocytochemistry was performed on adherent cells grown overnight on coverslips, fixed in 4% (w/v) paraformaldehyde, blocked for 90 min with 2% (v/v) horse serum and incubated with primary antibody for 1 h at room temperature. Alexa555 Cy3 fluorophore conjugated anti-rabbit (A-31572, Invitrogen, Victoria, AUS) was diluted at 1:500 and incubated for 1 h in the dark at room temprature. Coverslips were mounted onto slides using Vectashield mounting medium with 6- diamidino-2-phenylindole (DAPI) counterstain (H-1500, Vector Laboratories, California, USA). Slides were stored at -20 o C. IF was performed as described for immunocytochemistry, however frozen tissue sections were cut on Superfrost coated slides. Western Blotting Cell pellets were re-suspended in RIPA buffer (50 mm Tris, 150 mm NaCl, 0.1% SDS, 0.5% Na.Deoxychlorate, 1% Triton 100, 1mM PMSF, Complete Mini, EDTA- Free protease inhibitor tablets(11836170001, Roche, Basel, Switzerland)) and incubated on ice for 10 min after which the lysates were cleared by centrifugation at top speed for 10 min. Protein quantitation was carried out using the Bio-Rad Protein Assay Reagent (500-0006, Bio-Rad California, USA). 40ug of protein was denatured in LDS sample buffer (NP0007, Invitrogen) as per protocol. Proteins were separated by SDS-PAGE on a 4-12% Bis-Tris Gel (NP0323, Invitrogen) in MOPS running buffer (1 M MOPS, 0.5 M EDTA, 2 M Sodium Acetate) then transferred to PVDF membrane (IPVH00010, Millipore, NSW, AUS). Non-specific binding was blocked in 5% skim milk powder TBS-.Tween (0.1%), followed by incubation with primary antibody overnight at 4 C. All subsequent washes were made in TBS-T(0.1%).
Following appropriate horseradish peroxidase-conjugated secondary antibody (NA931V/NA934V GE Healthcare NSW, AUS) incubation the membrane was washed before detection with enhanced chemiluminescence (ECL; NEL104/105 PerkinElmer, MA USA). Mammosphere assays Briefly M6 cells with and without SHH transduction were trypsinized and filtered to obtain a single cell suspension. Cells were thoroughly washed and resuspended in mammosphere media. A minimum of 1000 cells were counted. The number of single cells was >99% in all experiments. Cells were grown in a serum-free M6 growth medium, supplemented with B27 (17504-044, Invitrogen) and 20 ng/ml bfgf (354060 BD Biosciences, CA, USA), and 4 ug/ml heparin (H0777 Sigma-Aldrich). Single cells were plated in ultralow attachment 6 well plates (Corning) at a density of 20,000 viable cells/well in primary culture and 1000 cells/well in subsequent passages. To generate secondary mammospheres and subsequent tertiary mammospheres, primary mammospheres were collected by gentle centrifugation (800 g, 10 seconds) after 10 days. Cells were centrifuged and the pellet gently resuspended in the residual medium to prevent dissociation of mammospheres. After washing, primary mammospheres were non-enzymatically dissociated then neutralized with mammosphere medium. The cells obtained from dissociation were sieved through a 40-um sieve and analyzed microscopically for single cellularity. Single cells were then plated in ultralow attachment 6 well plates (Corning) at a 1000 cells/well for secondary mammospheres.
Supplementary Table 1. Antibodies used for immunohistochemistry. Antibody Species Dilution Antigen Retrieval Hh Ligand H-160 (sc-9024, Santa Cruz) PTCH (ab27529, Abcam) GLI1 H-300 (sc-20687, Santa Cruz) LYVE-1 R&D) (BAF2125, Phospo-Histone H3 (Ser10) (Cell signaling 9701) Polyclonal Polyclonal Polyclonal Goat Polyclonal polyclonal 1:80 20 minutes boiling waterbath (Dako retrieval solution S2367) 1:50 20 minutes boiling waterbath (Dako retrieval solution S2367) 1:100 30 s at maximum a pressure cooker (DAKO) in DAKO ph 6.1 solution (s1699) 1:100 20 minutes boiling waterbath (Dako retieval solution S1699) 1:100 2 minutes at maximum a pressure cooker in DAKO ph 6.1 solution (s1699) Ki67 (SP6), Neomarkers, USA; Cyclin A (6E6) Novocastra, UK Cyclin B1 (7A9) Novocastra, UK Cyclin E (13A3), Novocastra, Newcastle, UK monoclonal Mouse monoclonal Mouse monoclonal Mouse monoclonal 1:200 Leica/Vision Biosystem Bondmax automated system using ER2 (high ph) antigen retrieval solution 1:100 2 minutes at maximum a pressure cooker in DAKO ph 6.1 solution (s1699) 1:40 2 minutes at maximum a pressure cooker in DAKO ph 6.1 solution (s2367) 1:40 2 minutes at maximum a pressure cooker in DAKO ph 6.1 solution (s1699) Primer/Probe sets
Supplementary table 2: primers used for Roche LightCycler480 Gene Primer Fwd 5 Primer Rev 5 UPL Cat No SHH CAA ATT ACA ACC CCG ACA TC GCA TTT AAC TTG TCT TTG CAC CT 32 04687655001 Ptch1 Ptch2 Gli1 Gli2 Hhip Pecam 1 -actin GGC CTG GCA GAG GAC TTA C GGA AGC ACC TTT TGA GTG GA 10 04685091001 GTC CAC CTA GTG CTC CCA AC CTC AGC TCC TGA GCC ACA TT 40 04687990001 GGA CCC ACT CCA ATG AGA AG CAT GCA CTG TCT TCA CGT GTT 33 04687663001 AGA ACC GCA CTC ACT CCA AT AGC TGG GGT CTG TGT ACC TC 64 04688635001 GTG TTC GGA GAT CGC AAT G TTT TCT TGC CAT TGC TTG GT 67 04688660001 AGC CAG TAG CAT CAT GGT CA AGC AGG ACA GGT CCA ACA AC 25 04686993001 GGA TGC AGA AGG AGA TTA CTG C CCA CCG ATC CAC ACA GAG TA 63 04688627001
Supplementary table 3: program used for Roche LightCycler480 Target temp Acquisition mode Hold Ramp rate ( C/s) Sec Target (per C) Step size ( C) Pre-Incubation 94 None 7 min 4.8 0 0 Amplification Analysis mode: Quantification Cycles: 45 Target temp Acquisition mode Hold Ramp rate ( C/s) Sec Target (per C) Step size ( C) 94 None 15 sec 4.8 0 0 60 None 30 sec 2.5 50 0.5 72 Single 15 sec 1.5 0 0 Cooling 40 30 sec 2.5 0 0 0
A Normal duct Hyperplasia Mammary intraepithelial neoplasia Invasive carcinoma B C3 (1)/Tag lesions 250 Hh ligand H score 200 150 100 50 n=10 ** n=15 n=15 n=9 0 normal hyperplasia MIN Ca Supplementary Figure 4. (A) IHC for HH in mammary glands from C(#)-Tag transgenic mice. (B) Quantitation of Hh IHC.
Supplementary Figure 3. (A) Western blot for GLI1 (left) and Flag epitope (right) in HCT-116 cells transfected with Gli1Flag construct (transfection performed in triplicate). HCT-116 are endogenously negative for Gli1 expression. Flag +ve known to contain a Flag tagged protein of 60 kda. (B) Immunofluorescence of M6 cells for GLI1 antigen. M6 cells show GLI1 in both the cytoplasm and in the nucleus. All images 400x, bar = 20 m. (C) Immunohistochemistry of FFPE mouse embryo (E14,5) for GLI1 with matched concentration IgG negative control. Perichondrium (arrow) is positive for nuclear GLI1. Images at 200x, bar = 50 m.
Supplementary Figure 2. (A) Western blot for PTCH in HCT-116 cells transfected with mouse Ptch (transfection performed in triplicate). HCT-116 cells are endogenously negative for Ptch expression. (B) Immunofluorescence of mouse embryo (E13.5) and M6 mouse mammary carcinoma cell line for PTCH antigen. Image of developing bone around growth plate (transverse section) shows cells of the perichondrium (arrow) positive for PTCH. Image of cerebellar anlage shows external granular cells (asterix) positive for PTCH. M6 cells show correct subcellular localisation of PTCH at the plasma membrane. All images at 400x, bar = 20 m. (C) Immunohistochemistry of FFPE mouse embryo (E13.5) for PTCH antigen with matched concentration of IgG negative control. Images show developing bone around growth plate (transverse section) shows cells of the perichondrium (arrow) positive for PTCH. Images at 200x, bar = 50 m.
Supplementary Figure 1. (A) Western blot for Hh ligand in M6 cells transfected with a sirna directed against Hh ligand for 48 h or 72 h (samples transfected in triplicate). M6 cells endogenously express Hh ligand. A549 cells do not endogenously express Hh ligand and served as a negative control. (B) Immunofuorescence of M6 cells and M6 cells infected to stably express Hh ligand. Images 400x, bar = 20 m. (C) Immunofuorescence (top) and immunohistochemistry (bottom) of mouse neural tube for Hh ligand. Both show Hh ligand around cells of the floor plate (arrows), with matched concentration IgG negative control for the immunohistochemistry analysis. All images of ventral portion of developing neural tube (transverse sections). Immunofuorescent images 400x, bar = 20 m, Immunohistochemical images 200x, bar = 50 m.