A3160802

The six human cell lines were purchased from ATCC. Cells were grown in culture medium supplemented with 10% fetal bovine serum (Gibco Life Technologies A31608-02, Thermo Fisher Scientific, Waltham, MA, USA) at 37 °C, 5% CO₂, and 5% O₂. HCT116, U2OS, and RKO cell lines were grown in Dulbecco’s Modified Eagle’s Medium (DMEM, Gibco Life Technologies 31966021, Thermo Fisher Scientific, Waltham, MA, USA), MRC5-N cell line was grown in Minimum Essential Medium Eagle (MEM-aplha, Gibco Life Technologies 22561021, Thermo Fisher Scientific, Waltham, MA, USA), RPE-1 cell line was grown in Roswell Park Memorial Institute Media (RPMI, Gibco Life Technologies 61870044, Thermo Fisher Scientific, Waltham, MA, USA), and K562 in Iscove Modified Dulbecco Media (IMDM, Gibco Life Technologies 21980032, Thermo Fisher Scientific, Waltham, MA, USA) supplemented with decomplemented serum. Aphidicolin (Sigma-Aldrich AO781-1MG, Sigma-Aldrich, St. Louis, MO, USA) stock solution was diluted in DMSO (Sigma-Aldrich D8418-250mL, Sigma-Aldrich, St. Louis, MO, USA) and kept at −20 °C for a maximum of 2 months after first thawing. Cells were synchronized in G1/S with 0.5 mM L-Mimosine (Sigma-Aldrich M0253, Sigma-Aldrich, St. Louis, MO, USA) for 24 h.

DMEM/F12, cat. C470п (PanEco, Moscow, Russia); Fetal Bovine Serum, qualified, One Shot format, Brazil, Gibco, cat. A3160802 (Thermo Fisher Scientific, Waltham, MA, USA); GlutaMAX Supplement, Gibco, cat. 35050061 (Thermo Fisher Scientific, Waltham, MA, USA); ITS, cat. Φ065 (PanEco, Moscow, Russia); EGF cat. CB-1101001 (PanEco, Moscow, Russia); FGF-2, cat. CB-1102021 (PanEco, Moscow, Russia); Dispase II, Sigma-Aldrich, cat. D4693 (Merck Life Science, Amsterdam, The Netherlands); Antibiotic-Antimycotic (100X), Gibco, cat. 15240062 (Thermo Fisher Scientific, Waltham, MA, USA).

Cells were grown at 37° C with 5% CO2 using DMEM (Gibco, C11965500BT) with 10% FBS (Gibco, A3160802) and 1% Penicillin/Streptomycin (Gibco, 15140122). SNRPB knockdown lentivirus (Lv-shSNRPB1/Lv-shSNRPB2) and the negative control (Lv-shNC) were purchased from GenePharma Co. Ltd. Cells were transfected and screened using puromycin (Sigma-Aldrich, P9620) for 4 days. Target sequences were listed below, Lv-shSNRPB1: ccACAAGGAAGAGGTACTGTT, Lv-shSNRPB2: ccTCCCAAAGATACTGGTATT, Lv-shNC: TTCTCCGAACGTGTCACGT.

The human colon cancer cells (HCT116 and Caco-2) were purchased from Beijing Beina Chuanglian Biotechnology Institute (Beijing, China). HCT116 cells were cultured in RPMI-1640 (SH30809.01B, Hyclone, Logan, UT, USA). Caco-2 cells were cultured in DMEM (SH30243.01, Hyclone, Logan, UT, USA). Both cell lines were cultured in a medium containing 10% (v/v) FBS (A31608-02, Gibco, Carlsbad, CA, USA) and antibiotics (100 IU/mL penicillin and 100 IU/mL streptomycin) at 37 °C with 5% CO₂. 1 × 10⁵/well HCT116 cells were inoculated into 24-well plates for luciferase reporter assay. 1 × 10⁶/well Caco-2 cells were inoculated into 6-well plates, and then 6 μg miRNA overexpressed plasmid was transfected into Caco-2 cells which were reached to 90~95% confluence 24 h later. After 24 h or 48 h transfection, cells were collected for Real-time PCR and Western blot analysis.

The mice secretin tumor cell line (STC-1) was purchased from Beijing Beina Chuanglian Biotechnology Institute (Beijing, China). The cells were cultured in DMEM (SH30243.01, Hyclone, Logan, UT, USA) containing 10% (v/v) FBS (A31608-02, Gibco, Carlsbad, CA, USA) and antibiotics (100 IU/mL penicillin and 100 IU/mL streptomycin) at 37 °C with 5% CO₂. The medium was changed every 2 days. Cells were trypsinized with 0.25% trypsin-EDTA (Sigma-Aldrich, St. Louis, MO, USA) when they reached 80–90% confluence. Each experiment was repeated at least three times (n ≥ 3).

The following cell lines, namely HEK293T, PC-3, and DU145, were procured from the Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China): HEK293T and DU145 cells were cultivated in Dulbecco’s modified eagle medium (DMEM; SH30022.01, Gibco, USA), whereas PC-3 cells were maintained in RPMI-1640 medium (C11875500BT, Gibco, USA). The incubation conditions were maintained at 37 °C with a 5% CO₂ atmosphere. The culture media were supplemented with 10% fetal bovine serum (FBS; A3160802, Gibco, USA) and 1% penicillin–streptomycin solution (15140-122, Gibco, USA). For transfection experiments, the HEK293T, PC-3, and DU145 cells were transfected with the designated plasmids utilizing the polyethyleneimine (PEI; 23966-1, Polysciences, USA) technique. With regard to cellular treatment, PC-3 cells underwent serum starvation in RPMI-1640 medium overnight, followed by treatment with JNK-IN-8 (1 μmol/L and 2 μmol/L, HY-13319, MCE, USA) and bafetinib (5 μmol/L, HY-50868, MCE, USA) for specified durations. All cultured cells were subjected to routine mycoplasma contamination screening via DNA detection method.

hBMSCs were purchased from American Type Culture Collection (ATCC, Manassas, USA). mBMSCs were obtained from Cyagen Biosciences Company (Guangzhou, China). These cells were cultured with a Mesenchymal Stem Cell Growth Kit for Bone Marrow-derived MSCs (ATCC PCS-500-041) (supplemented with rh FGF basic: 125 pg/mL, rh IGF-1: 15 ng/mL, 7% fetal bovine serum (FBS) and L-Alanyl-L-Glutamine: 2.4 mM). Hypoxic preconditioning was induced in an oxygen control incubator (Smartor 118, Zhejiang, China) filled with 5% CO₂ and 90–94% N₂. The oxygen concentrations and hypoxic incubation times were indicated as shown. Cells cultured under normoxic conditions (21% O₂) served as a control.
The human keratinocyte (HaCaT) cell line was purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China) (originally from American Type Culture Collection (Manassas, USA)) and was cultured in Minimum Essential Medium (MEM; Procell Life Science & Technology, PM150410) containing 10% FBS (Gibco, A3160802) and 1% penicillin and streptomycin (Gibco). HaCaT cells were treated with high glucose (HG) (25 mM glucose) and 1% O₂ hypoxia.