Begm bullet kit

MCF10A cells were obtained from American Type Culture Collection (ATCC) and the laboratory of Dr Frank McCormick (UCSF) and cultured in Dulbecco's modified Eagle's medium and F12 (DMEM:F12) containing 5% Horse serum, 50 U ml⁻¹ penicillin-streptomycin, 10 μg ml⁻¹ insulin, 0.5 μg ml⁻¹ hydrocortisone, 20 ng ml⁻¹ EGF, 10 μg ml⁻¹ cholera toxin. NBHE cells were obtained from Lonza and cultured in BEGM BulletKit (Lonza). Cells were lysed in either M-PER Mammalian Protein Extraction Reagent (Thermo) with 4 μg ml⁻¹ aprotinin, 4 μg ml⁻¹ leupeptin, 4 μg ml⁻¹ pepstatin, 1 mM PMSF, 1 mM Na3VO4, and 5 mM NaF or NP40 buffer containing 50 mM Tris pH 7.5, 1 mM EDTA, 150 mM NaCl, 1% Nonidet P-40, 4 μg ml⁻¹ aprotinin, 4 μg ml⁻¹ leupeptin, 4 μg ml⁻¹ pepstatin, 1 mM PMSF, 1 mM Na3VO4, and 5 mM NaF. Nuclear and cytoplasmic fractions were prepared using NE-PER extraction reagents (Thermo). Western blot analysis was carried out according to standard methods. Protein concentrations of cell extracts were determined by the Bradford assay. The following commercial antibodies were used as probes: FLAG (Abcam; 1:1,000), GLS (Abcam, AB156876; 1:1,000 for Fig. 1d; Abcam, AB60709; 1:1,000 for Fig. 3b), ASCT2 (Cell Signaling, 1:1,000), MYC (Cell Signaling; 1:1,000), β-Tubulin (Sigma; 1:5,000).

Human embryonic kidney (HEK) 293, HepG2, Hep3B, Sk‐Hep1 and THLE2 were obtained from American Type Culture Collection (ATCC). Huh7 and SNU423 were purchased from Korea Cell Line Banks (KCLB). According to the manufacturer's instructions, the growth culture medium was prepared by mixing DMEM or EMEM with 10% FBS and additives (Gibco). For the cultivation of THLE2 cells, the BEGM Bullet Kit (CC‐3170) from Lonza was used. The Bullet Kit contains BEBM basal medium and supplements. The final growth medium consists of the following: BEBM supplemented with 10% FCS, bovine pituitary gland extract, hydrocortisone, epidermal growth factor (EGF), insulin, triiodothyronine, transferrin, retinoic acid, 5 ng/mL human recombinant EGF (Gibco) and 70 ng/mL ο‐phosphorylethanolamine (Sigma‐Aldrich). All cells applied in this study were cultured at 37℃ in a humidified 5% CO₂ atmosphere. Polyclonal antibodies against the epitope tags (HA and Myc), KLF4, USP11, ubiquitin and β‐actin were obtained from Santa Cruz Biotechnology, Inc Anti‐Flag antibody, anti‐Flag‐M2 affinity gel, cycloheximide (CHX), sorafenib, palmitic and oleic acid were purchased from Sigma‐Aldrich. Lipid contents of cells were measured with commercial Triglyceride assay kit (Abcam).

Primary normal human bronchial epithelial cells (NHBE) and asthmatic bronchial epithelial cells (DHBE) were purchased from Lonza Walkersville, Inc. The cell culture was performed following the manufacturer’s protocol. Cells were grown in Bronchial Epithelial Cell Basal Media (BEGM Bulletkit™; Lonza, Clonetics, San Diego, CA) supplemented with 2 ml BPE, 0.5 ml hydrocortisone, 0.5 ml hEGF, 0.5 ml epinephrine, 0.5 ml transferrin, 0.5 ml insulin, 0.5 ml retinoic acid, 0.5 ml triiodothyronine, and 0.5 ml GA-1000. Cells were maintained at 37°C in 5% CO₂ incubator, and passaged with ReagentPack™ (Lonza; Clonetics, San Diego, CA, USA) containing trypsin-EDTA, trypsin neutralizing solution, and HEPES buffered solution.

HBECs were isolated by initial short‐term pronase (Roche, Basel, Switzerland) and DNase (Sigma‐Aldrich) digestion of bronchoscopy biopsies obtained from nonasthmatic donors from the Department of Pulmonology, Jagiellonian University Medical College (Krakow, Poland). In total, HBECs from two nonasthmatic donors were obtained (one male and one female Caucasian of age 26 and 30, respectively). Both donors underwent diagnostic bronchoscopy, and chronic airway disease was ruled out during further analyses. Frozen HBECs of passage 0 were thawed and cultured in BEGM Bulletkit (Lonza, Switzerland) media containing bovine pituitary extract, insulin, hydrocortisone, gentamicin and amphotericin‐B, retinoic acid, transferrin, triiodothyronine, epinephrine, and human epidermal growth factor as recommended by the manufacturer, and cells were transferred to a cell culture incubator (at 37°C under 5% CO₂) for further experiments. This study was approved by the Ethics Committee of the Jagiellonian University Medical College.

Dopamine hydrochloride was purchased from Alfa Aesar (Gdansk, Poland). Doxorubicin hydrochloride (DOX·HCl) was purchased from LC Laboratories (Boston, MA, USA). PAMAM dendrimers G 3.0, phosphate buffer saline (PBS), Menadione, sodium tetraphenylborate, tris (hydroxymethyl) aminomethane, O-[N-(3-Maleimidopropionyl)aminoethyl]-O′-[3-(N-succinimidyloxy)-3-oxopropyl]heptacosaethylene glycol (molecular weight of 1570.76 g/mol), fetal bovine serum (FBS), penicillin, streptomycin, fibronectin, bovine collagen type I, bovine serum albumin, 0.1% poly-l-lysine, Muse^® Oxidative Stress Assay and MUSE^® Annexin V and Dead Cell assay were purchased from (Merck Darmstadt, Germany). Bronchial Epithelial Basal Medium (BEBM) and BEGM Bullet Kit were purchased from Lonza (Basel, Switzerland). WST-1 Cell Proliferation Reagent was purchased from Takara Bio (Shiga, Japan). Calcein AM, ethidium homodimer-1, Hoechst 33342, Minimum Essential Medium Eagle (MEM), dulbecco’s phosphate buffered saline (DPBS), 4% formaldehyde solution, sodium pyruvate, sodium pyruvate were purchased from Thermo Fisher Scientific (Waltham, MA, USA).