Penicillin streptomycin

Human OvCa A2780 and SKOV3 cell lines were obtained from ECACC and ATCC, respectively, and authenticated by Genetic Testing Biotechnology Corporation (Suzhou, China) by detecting short tandem repeat (STR) markers. All cell lines were grown in RPMI-1640 medium supplemented with 10% fetal bovine serum (Gibco, USA) and 1% penicillin/streptomycin (TransGen Biotech, China) in incubators with 5% CO₂.
A2780 UBE2E2-knockout cells were created using CRISPR/Cas9 technology. Plasmids expressing Cas9, GFP, puromycin resistance markers, and single guide RNAs (sgRNA) targeting exon 2 of human UBE2E2 (the sequences are shown in Fig. 2A) were cotransfected into A2780 cells. The transfected cells were enriched by selection with puromycin (2 µg/ml) and FACS, and clones were selected by dilution. The loss of UBE2E2 expression was verified by qRT-PCR and western blot analysis.
MG132 and t-butylhydroquinone (tBHQ) (MedChem Express, USA) were dissolved in dimethyl sulfoxide (DMSO) and maintained at −20 °C.

Male Wistar rats were obtained from the Experimental Animal Center of Jilin University, Changchun, China (permit number: SYXK 2018-0001). Primary MSCs were isolated from the bone marrow of young (1–2-month-old) and old (15–18-month-old) male Wistar rats using the whole bone marrow adherent method (Ma et al., 2017 (link)). First, a 10 ml syringe was used to wash the marrow cavity, and MSCs were aseptically isolated from the femurs. MSCs were then harvested and plated into 10 cm culture dishes in complete medium containing 89% Dulbecco’s modified Eagle medium with nutrient mixture F-12 (Gibco; Life Technologies, Grand Island, NY, United States) supplemented with 10% fetal bovine serum (Gibco; Life Technologies, Grand Island, NY, United States) and 1% penicillin streptomycin (TransGen Biotech; Beijing, China). After incubation for 24 h at 37°C with 5% CO₂, non-adherent cells were removed and the culture medium was replaced every 3 days. When the cells reached approximately 80% confluency, MSCs were digested using 0.25% trypsin-EDTA (Gibco; Life Technologies, Grand Island, NY, United States) and passaged at a 1:3 ratio.

Human Jurkat and HEK 293 T cells were purchased from the American Type Culture Collection (ATCC). The fibroblast-like synoviocyte (FLS) cell line MH7A was prepared from intra-articular soft tissues of the knee joints of RA patients and obtained from Prof. Mei Liu (Nanjing Normal University, Jiangsu, China). Both HEK 293 T cells and MH7A cells were maintained in Dulbecco’s modified Eagle’s medium (Gibco Laboratories, NY, USA) supplemented with 10% fetal bovine serum (FBS, Biological Industries, KBH, Israel). Jurkat cells were cultured in RPMI 1640 medium (GIBCO Laboratories, NY, USA) with 10% FBS. The cell medium was supplemented with penicillin/streptomycin (TransGen, Beijing, China). All the cells were cultured at 37 °C in a humidified atmosphere of 5% CO₂. Transfections were performed according to the manufacturer’s instructions with Lipofectamine 2000 (Invitrogen, CA, USA) or DharmaFECT Duo transfection reagent (ThermoScientific, MN, USA).

All lung cancer paraffin sections and lung cancer tissues were obtained from the First Affiliated Hospital of Dalian Medical University. The lung cancer tissues were kept in liquid nitrogen for protein extraction. Human NSCLC A549, H1299, H358 cell lines were purchased from American Type Culture Collection (ATCC, Manassas, VA). A549, H1299, H358 cells were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum (TransGen) and 1% penicillin-streptomycin (TransGen) at 37°C in humidified air containing 5% CO₂.

The clinically isolated strain UPEC307 was collected from a urinary tract infection patient and used in all infection experiments. UPEC307 was cultured in Luria Bertani (LB) broth at 37 °C with shaking at 180 r/min until the mid-log phase, at which point bacteria were collected and the culture concentration was determined by spectrophotometry.
T24 cells (HTB-4) were purchased from ATCC and grown in a DMEM (Sigma, St. Louis, MO, USA) medium supplemented with 10% fetal bovine serum (Procell, Wuhan, China) and 1% penicillin/streptomycin (TransGen Biotech, Beijing, China). Cells were cultured at 37 °C under 5% CO₂. Dictamnine was purchased from Chengdu Herbpurify Co., Ltd. (Chengdu, China), dissolved in dimethyl sulfoxide (DMSO, Solarbio Life Sciences, Beijing, China), and filtered with a 0.2 µm filter (Pall, New York, NY, USA) to remove bacteria.

All animal experiments were approved by the Animal Care Commission of the College of Veterinary Medicine of Northwest A&F University (Permit Number: NWAFAC1019). Six tissue samples, i.e., heart, liver, spleen, lung, kidney, longissimus dorsi muscle, were collected from sixty-day-old fetuses (n = 3) and two-year-old adults (n = 3) of the Chinese Qinchuan (QC) beef cattle breed. All samples were provided by Shanxi Kingbull Livestock Co., Ltd., Baoji city, China. Primary bovine myoblasts (PBMs) were isolated from fetal longissimus dorsi muscle following the established protocols [19 (link)]. Myoblasts were cultured in growth medium (GM) consisting of high-glucose Dulbecco’s modified Eagle’s medium (DMEM, Gibco) with 1% penicillin-streptomycin (HyClone) and 20% fetal bovine serum (TransGen, Beijing, China). Myoblast differentiation was stimulated in DMEM containing 2% horse serum (HyClone) and 1% penicillin-streptomycin (differentiation medium, DM). Cells were incubated at 37 °C with 5% CO₂.