All chemicals, which are not listed in Table 3 were purchased from Merck KGaA, Darmstadt.
Table 3: Utilized reagents and chemicals
Reagents and chemicals Supplier
50 bp, 100 bp plus, 1 kb DNA ladder Thermo Fisher Scientific Inc., Waltham, USA
Acetic acid Carl Roth GmbH & Co.KG, Karlsruhe
Agarose Bio-Budget Technologies GmbH, Krefeld
Ampicillin Carl Roth GmbH & Co. KG, Karlsruhe
BD BactoTM Tryptone BD Biosciences GmbH, Franklin Lakes, USA
Boric acid Carl Roth GmbH & Co.KG, Karlsruhe Bovine serum albumin (BSA) Carl Roth GmbH & Co.KG, Karlsruhe
Chloroform Carl Roth GmbH & Co.KG, Karlsruhe
Citric acid Carl Roth GmbH & Co.KG, Karlsruhe
Collagen from rat tail Merck KGaA, Darmstadt
Collagenase H Roche Diagnostic GmbH, Basel, Switzerland
Contrast agent Imeron 300 Bracco Imaging Deutschland GmbH, Konstanz
Cresol Red Merck KGaA, Darmstadt
Deoxyribonucleotide triphosphates (dNTPs) Roche Diagnostic GmbH, Basel, Switzerland Dimethyl sulfoxide (DMSO) Thermo Fisher Scientific Inc., Waltham, USA Dithiothreitol (DTT) Thermo Fisher Scientific Inc., Waltham, USA DNA Gel Loading Dye (6X) Thermo Fisher Scientific Inc., Waltham, USA DNase/RNase-free H2O Thermo Fisher Scientific Inc., Waltham, USA
Eosin Y Merck KGaA, Darmstadt
Ethanol (EtOH), 99 % Fisher Scientific, Schwerte
Ethidium bromide (0.07 %) Inno-train Diagnostik GmbH, Kronberg im Taunus
Ethylendiaminetetraacetic acid (EDTA) MP Biomedicals, Santa Ana, USA
EtOH, 99 %, denatured CVH Chemie-Vertrieb GmbH & Co. Hannover KG, Hannover
Glycergel mounting medium Agilent Technologies Inc., Santa Clara, USA
Haematoxylin, Mayer’s Merck KGaA, Darmstadt
Hydrogen peroxide (H2O2, 35 %) Carl Roth GmbH & Co. KG, Karlsruhe Isoflurane (FORENE®) Abbott Laboratories, North Chicago, USA
Isopropyl alcohol Carl Roth GmbH & Co. KG, Karlsruhe
Kanamycin Carl Roth GmbH & Co. KG, Karlsruhe
Liquid Barrier Marker Carl Roth GmbH & Co. KG, Karlsruhe
Methanol Carl Roth GmbH & Co. KG, Karlsruhe
NuPAGE MES SDS Running Buffer (20X) Thermo Fisher Scientific Inc., Waltham, USA
Paraformaldehyde Carl Roth GmbH & Co. KG, Karlsruhe
Pertex mounting medium Medite GmbH, Orlando
Phosphatase inhibitor cocktail tablets
(PhosSTOPTM) Roche Diagnostic GmbH, Basel, Switzerland
Phosphate buffered saline (PBS) tablets Thermo Fisher Scientific Inc., Waltham, USA
Potassium aluminum sulfate Merck KGaA, Darmstadt
Powdered milk Carl Roth GmbH & Co. KG, Karlsruhe
Propidium Iodide (PI) Miltenyi Biotec GmbH, Bergisch Gladbach Protease inhibitor cocktail tablets (cOmpleteTM,
mini) Roche Diagnostic GmbH, Basel, Switzerland
Random hexamers Thermo Fisher Scientific Inc., Waltham, USA
S.O.C Medium Thermo Fisher Scientific Inc., Waltham, USA
SeeBlue® Plus2 Pre-Stained Protein Standard Thermo Fisher Scientific Inc., Waltham, USA Sodiumdodecylsulfate (SDS) Carl Roth GmbH & Co. KG, Karlsruhe Trichloro acetaldehyde hydrate Merck KGaA, Darmstadt
TritonTM X-100 Merck KGaA, Darmstadt
Trypan blue Merck KGaA, Darmstadt
Tween® 20 Scharlau Chemie S.A., Barcelona, Spain
Tween® 80 Merck KGaA, Darmstadt
Xylene Fisher Scientific, Schwerte
Yeast extract Merck KGaA, Darmstadt
3.3.1 Signaling pathway inhibitors
Table 4: Utilized small molecule inhibitors for in vitro experiments Inhibitor Solvent Incubation
concentration Supplier
Cyclopamine EtOH 1 – 10 µM Sigma-Aldrich Inc., St. Louis, USA (now Merck KGaA, Darmstadt)
Everolimus EtOH 50 nM Sigma-Aldrich Inc., St. Louis, USA (now Merck KGaA, Darmstadt)
HhAntag DMSO 3.1 - 50 µM Genentech, San Francisco, USA
MK-2206 DMSO 5 µM Selleckchem, Houston, USA
Pictilisib DMSO 0.6 - 10 µM Selleckchem, Houston, USA and Genentech, San Francisco, USA
PI-103 DMSO 3 µM Alexis Biochemicals, San Diego, USA
Rapamycin DMSO 100 nM Merck KGaA, Darmstadt
Smoothened agonist (SAG)
DMSO 100 nM Cayman Chemical Company, Ann Arbor, USA
Sonidegib DMSO 3.1 - 50 µM Active Biochem LTD, Kowloon, Hong Kong and Novartis AG, Basel, Switzerland
UO126 DMSO 2 – 25 µM Cell Signaling Technology, Danvers, USA Vismodegib DMSO 3.1 - 50 µM Selleckchem, Houston, USA and Genentech, San
Francisco, USA
Table 5: Utilized small molecule inhibitors for in vivo experiments Inhibitor Daily dose [µg/g
body weight]
Stock concentration [mg/mL] *
Supplier
HhAntag 100 10 Genentech, San Francisco, USA
Pictilisib 75 37.5 Genentech, San Francisco, USA
Sonidegib 80 10 Novartis AG, Basel, Switzerland
Vismodegib 100 10 Genentech, San Francisco, USA
*All drugs for in vivo experiments were resuspended in vehicle as described in section 4.4.5.
3.3.2 Enzymes Table 6: Utilized enzymes
Enzymes Supplier
BamHI Thermo Fisher Scientific Inc., Waltham, USA
Collagenase H Roche Diagnostic GmbH, Basel, Switzerland
EcoRI Thermo Fisher Scientific Inc., Waltham, USA
Hind III Thermo Fisher Scientific Inc., Waltham, USA
HpaI Thermo Fisher Scientific Inc., Waltham, USA
KpnI Thermo Fisher Scientific Inc., Waltham, USA
NheI Thermo Fisher Scientific Inc., Waltham, USA
NotI Thermo Fisher Scientific Inc., Waltham, USA
Proteinase K Carl Roth GmbH & Co. KG, Karlsruhe
RNase A Carl Roth GmbH & Co.KG, Karlsruhe
SuperScriptTM II Reverse Transcriptase Thermo Fisher Scientific Inc., Waltham, USA Taq Polymerase (MolTaq basis) Molzym GmbH & Co. KG, Bremen
3.3.3 Kits and ready-to-use reagents
Unless stated otherwise all kits and ready-to-use reagents listed in Table 7 were used according to the manufacturer’s instructions.
Table 7: Utilized kits and ready-to-use reagents
Reaction system Supplier
FITC Annexin V Apoptosis Detection Kit I BD Biosciences Corp., Franklin Lakes, USA Cell Proliferation ELISA, BrdU
(chemiluminescent) Roche Diagnostic GmbH, Basel, Switzerland
Dako REALTM EnVisionTM Detection System Agilent Technologies Inc., Santa Clara, USA Dual-Luciferase® Reporter Assay System Promega Inc., Fitchburg, USA
NeonTM Transfection System Kit Thermo Fisher Scientific Inc., Waltham, USA Pierce BCA Protein Assay Kit Thermo Fisher Scientific Inc., Waltham, USA Pierce ECL Plus Western Blot substrate Thermo Fisher Scientific Inc., Waltham, USA Platinum SYBR Green qPCR Super Mix Thermo Fisher Scientific Inc., Waltham, USA PureLink® HiPure Plasmid Midiprep kit Thermo Fisher Scientific Inc., Waltham, USA QuantiTect SYBR Green PCR kit Qiagen, Venlo, Netherlands
TRIzolTM Reagent Thermo Fisher Scientific Inc., Waltham, USA Water soluble tetrazolium salt 1 (WST-1) reagent Roche Diagnostic GmbH, Basel, Switzerland