Rpmi 1640

Acrylamide, bromophenol blue, 1.5 M Tris-HCL, 1.0 M Tris-HCL, sodium dodecyl sulphate (SDS) and tetramethylene diamine (TEMED) was purchased from Servicebio (Wuhan, China). RPMI-1640 and DMEM were purchased from Servicebio (Wuhan, China). Fetal bovine serum was purchased from QMSuero (Wuhan, China). Antibiotics was purchased from Biosharp (Hefei, China). Antibodies for DBF4 was purchased from Immunoway (Jiangsu, China). ERBB2 and p-ERBB2^Y1221/1222 were purchased from Abclonal (Wuhan, China). STAT3 and p-STAT3 were purchased from Abmart (Shanghai, China). PI3K, p-PI3K, AKT and p-AKT were purchased from Cell Signaling Technology. P38 MAPK, p-P38 MAPK, N-CADHERIN, VIMENTIN and SNAIL were purchased from Proteintech (Wuhan, China). MMP9 and GAPDH were purchased from Servicebio. Dacomitinib (a specific, irreversible inhibitor of the ERBB family that targets EGFR, ERBB2, and ERBB4) were purchased from MedChemExpress (Shanghai, China). Lipo8000™ transfection reagent was purchased from FuShen (Shanghai, China). Kcockdown and overexpression plasmids for lentivirus production was synthesized by Miaoling Biology (Wuhan, China).

Progesterone, ursolic acid, apigenin, luteolin and miconazole (≥ 95%) were purchased from Aladdin. Rosmarinic acid (≥ 95%) was purchased from Shanghai Puyu Kemao Co., Ltd. (Shanghai, China) and Dimethyl sulfoxide (DMSO) (≥ 99.8%) from Shandong Yousuo Chemical Technology Co., Ltd. (Linyi, Shandong, China).SDS-PAGE gel preparation kits, Prestained Protein Marker II (10–200 kDa), RPMI-1640, Radio Immunoprecipitation Assay (RIPA) lysate, Cocktail protease inhibitors, phenylmethylsulfonyl fluoride (PMSF), phosphorylation protease inhibitors, bicinchoninic acid (BCA) kit, Coomassie brilliant blue, loading buffer, Trizol, SweScript RT II First Strand cDNA Synthesis Kit and 2 × SYBR Green qPCR Master Mix were all purchased from Servicebio (Wuhan, Hubei, China). Fetal Bovine Serum (FBS) was purchased from Zhejiang Tianhang Biotechnology Co., Ltd. (Huzhou, Zhejiang, China). Potato Dextrose Agar (PDA) was purchased from Qingdao Hope Bio-Technology Co., Ltd. (Qingdao, Shandong, China) Clinical strains 1078, 1079, 130, and 1413 were shared by Wuhan No. 1 Hospital (Wuhan, Hubei, China). Trichophyton mentagrophytes ATCC9533 was purchased from Shanghai Bioresource Collection Center (Shanghai, China).

AGS, SGC7901, SNU1 and MKN45 cell lines were bought from American Type Culture Collection (ATCC, Manassas, VA, USA). The AGS cell line was cultured in Dulbecco’s Modified Eagle Medium (DMEM) /F-12 (ref: G4610, Servicebio, Wuhan, China) while the SGC7901, SNU1 and MKN45 cell lines were cultured in RPMI1640 (ref: G4530, Servicebio, Wuhan, China), along with 10% fetal bovine serum (FBS, ref: 10099141C, ThermoFisher, Waltham, MA, USA) and 1% antibiotics (Penicillin and Streptomycin) (ref: G4003, Servicebio, Wuhan, China). Cells were incubated at 37 °C with 5% CO₂.

RWPE-1, a normal prostate epithelial cell line, was cultured in DMEM (G4510; Servicebio, Wuhan, China) and PCa cell lines (LNCaP, DU-145, and PC3) were cultured in RPMI-1640 (G4530; Servicebio, Wuhan, China). All cell lines were obtained from the Second Hospital of Lanzhou University and incubated at 37°C and 5% CO₂ in a humidified incubator. All mediums were supplemented with 10% fetal bovine serum (FBS; 1276-025; Gibic, Beijing, China). Transfection was performed using Homo-CDCA8 (shRNA-CDCA8) and shRNA-negative control (shRNA-NC) lentivirus (GebePharma; Shanghai, China). LNCaP and DU-145 were cultured in six-well plates and transfected with lentivirus and Polybrene enhancer for 16 h. After 72 h, 2 μl 0.1 μg/μl puromycin was added to select stable cell lines. The shRNA single-strand sequences were as follows: Sh-CDCA8: 5’-TTGACTCAAGGGTCTTCAA-3’; Sh-NC: 5’-ACTACCGTTGTTATAGGTG-3’.

Human OC cell lines SKOV3, A2780, HEY, and HO8910 were obtained from the Obstetrics and Gynecology Hospital Affiliated to Fudan University and grown at 37°C and 5% CO₂ in RPMI-1640 (Servicebio, Wuhan, China) medium with UltraGRO™ cell culture supplement (AventaCell, Atlanta, USA) and penicillin-streptomycin (Yuchunbio, Shanghai, China). For 5-Azacytidine (5-AzaC) treatment, the growth medium was supplemented with 10 μmol/L or 100μmol/L 5-AzaC (MCE, USA) for 12 h or 24 h.

Human A549 cells (Cat#CCL-185™, ATCC, Manassas, VA, USA) and H1299 cells (Cat#CL-0165, Procell, Wuhan, China) were maintained at 37°C in the humidified incubator with 95% relative humidity and 5% carbon dioxide. The medium for cell culture was Kaighn’s Modification of Ham’s F-12 Medium (F-12K medium, for A549) (Cat#30–2004, ATCC) or RPMI-1640 (Cat#G4538, Servicebio, Wuhan, China), supplemented with 10% fetal bovine serum (FBS) (Cat#G8001, Servicebio) and 1% antibiotics (Cat# PB180120, penicillin/streptomycin, Procell). HEK293T cells (Cat#CL-0005, Procell) were maintained using standard protocols (DMEM+10% FBS+1% penicillin/streptomycin) provided by Procell.
Pemetrexed (PEM, Cat#SML1490) was obtained from Sigma (St. Louis, MO, USA). PEM-resistant A549 (A549/PEM) cells were established by exposing the parental cell line to a single high concentration of PEM (the 50% inhibitory concentration (IC50) of PEM for A549 cells) over 6 months [28 (link)]. The A549/PEM cells were confirmed to get stable resistance by calculating the PEM resistant index (RI) using the method: RI = the IC50 value of A549/PEM cells/the IC50 value of A549 cells. When the RI > 5, the cells were identified to have the phenotype of PEM resistance. After cultivation in a low concentration (0.5 μM) PEM medium, A549/PEM cells were used for subsequent experiments.

Human THP-1 cells (CL-0233) were kindly provided by Procell Life Science&Technology (Wuhan, China) and subjected to STR authentication in Procell Life Science&Technology. THP-1 cells were cultured with RPMI-1640 (G4530-500ML, Servicebio, China) supplemented with 10% Fetal Bovine Serum (FBS) (FSP500, ExCell Bio, China) and 1% Penicillin-Streptomycin (P/S) (15,140,122, GIBCO, USA). The cells were inoculated in 6-well plates at a density of 5 * 10^5/ml, and the cells were inoculated with phorbol 12-myristate 13-acetate (PMA) (100 ng/ml; Hy-18,739, MedChemExpress, USA) was cultured in the culture medium for 48 h to differentiate into macrophages. THP-1 macrophages differentiated from PMA were treated with 80 µg/ml ox-LDL (Yiyuan Biotechnology, China) for 24 h. Foam cell development was determined using Oil Red O staining. An incubator with 5% CO₂ and 37 °C was used to culture cells.