Calf bovine serum

Hydroxypropyl-β-cyclodextrin (HP-β-CD) MW 1380 Da (SR: 0.6), dexamethasone (DMS), thiourea, fluorescein diacetate (FDA), Ellman’s reagent (2,2′-dinitro-5,5′-dithiobenzoic acid), l-cysteine hydrochloride monohydrate, tris(hydroxymethyl)aminomethane, Sephadex G-15, Type II porcine gastric mucin, Dulbecco’s Modified Eagle Medium (DMEM), calf bovine serum, a mixture of antibiotics consisting of an aqueous solution of penicillin (10,000 U/mL) and streptomycin (10,000 µg/mL), 10 mM phosphate buffer pH 7.3 without Ca²⁺ and Mg²⁺ (PBS_A), and a trypsin-EDTA buffer solution containing 0.25% trypsin were all purchased from Sigma-Aldrich (Darmstadt, Germany). Fibroblast BALB/3T3 clone A31 cells (CCL-163) were obtained from American Type Culture Collection.

Primary mouse embryonic fibroblast cells (NIH/3T3) were seeded in six-well plates (ThermoFisher Scientific, Waltham, MA, USA) and 35 mm tissue culture dishes (Azzota Scientific, DE, USA) for fluorescent intensity quantification and AFM indentation experiments, respectively, using Dulbecco’s Modified Eagle’s Medium (ATCC, Rockville, MD, USA), together with 10% (V/V) Calf Bovine Serum (Sigma, St. Louis, MO, USA) and 1% (V/V) penicillin-streptomycin (Gibco, Grand Island, NY, USA). The cell culture vessels were maintained in the incubator at the temperature of 37 ${}^{\circ }$ and humidified atmosphere of 5% CO ${}_2$ . The cultured cells were ready after 24 h.
To investigate the different cytoskeletal states of F-actin and microtubules, the cells were treated with latrunculin B (George Town, Cayman Islands) and nocodazole (Belgium, USA), respectively. Living 3T3 cells were divided into two groups for the actin and microtubule treatments, respectively. The cellular F-actin were inhibited using latrunculin B at the final concentration of 0 nM (control), 10 nM, 30 nM, 40 nM, 60 nM, 75 nM, and 100 nM in the aforementioned cell culture medium. The cellular microtubules were treated with nocodazole at the final concentration of 0 nM (control), 10 nM, 30 nM, 50 nM, 75 nM, 100 nM, and 200 nM in cell culture medium. The cells were treated for 30 min in the incubator before the AFM measurements.

Doxycycline (Dox)‐inducible GLI1‐expressing HaCaT keratinocytes and mouse BCC cell line ASZ00122 were grown as described previously.23, 24 Induction of GLI1 expression in HaCaT keratinocytes was done as reported in Refs. 25, 26 Murine NIH/3 T3 cells (AMS Biotechnology Ltd, Abingdon, UK) transduced with pBabe‐puro‐GLI1 or empty control vector were grown in Dulbecco's modified Eagle medium (DMEM) (Sigma, St. Louis, MO) supplemented with 10% calf bovine serum (Sigma) and penicillin–streptomycin (Sigma). Chemicals and reagents used for cell treatments are listed in Supporting Information, Table S1. Recombinant human and mouse IL6 and Dox were used at a concentration of 50 ng/ml, unless indicated otherwise. For three‐dimensional (3D) cultures, 1 × 10⁴ human HaCaT keratinocytes were seeded in 12‐well plates (Greiner Bio‐one, Kremsmünster, Austria), cultured and analyzed in a blinded fashion as described previously.25