Sw480

CRC cell lines of SW480, HCT116, SW620, and DLD1 were supplied by Cell Bank, Chinese Academy of Sciences. Human normal colonic epithelial cells NCM460 were purchased from the American Type Culture Collection (Manassas, Virginia, USA). SW480 and SW620 cells were cultured in L‐15 (11415064, Gibco) medium containing 1% penicillin/streptomycin and 10% fetal bovine serum (Gibco, USA) at 37°C in an incubator. HCT116 cells were cultured in McCoy's 5a medium (12330031, Gibco) supplemented with 1% penicillin/streptomycin and 10% fetal bovine serum at 37°C in a 5% CO₂ incubator. DLD1 cells were cultured in RPMI 1640 medium (11875093, Gibco) supplemented with 1% penicillin/streptomycin and 10% fetal bovine serum at 37°C in a 5% CO₂ incubator. NCM460 cells were cultured in Dulbecco's modified Eagle's medium (KeyGEN BioTECH, Jiangsu, China) with 10% fetal bovine serum (Gibco, USA) at 37°C in a 5% CO₂ incubator.
For experimental purposes, cells were transfected with overexpression vectors to upregulate target gene expression, siRNA to knockdown specific gene expression, block oligonucleotides to inhibit mRNA translation, and a negative control to ensure the specificity of the observed effects. All transfections were performed using the Lipo6000 transfection reagent, following the manufacturer's protocols for transient transfection.

Two human colorectal cancer cell lines, SW480 and HCT116, and a colon fibroblast cell line, CCD-18Co, were obtained from the American Type Culture Collection. SW480 cells were maintained in Leibovitz's L-15 medium (Gibco) with 10% fetal bovine serum (FBS) (Hyclone, Logan, UT, USA), 2 mM L-glutamine, 0.1 mg/mL streptomycin, and 100U/mL penicillin at 37 °C in a standard humidity incubator. HCT116 cells were cultured in RPMI 1640 medium (Invitrogen) with 10% FBS and maintained in a humidified incubator at 37 °C with 5% CO₂. CCD-18Co cells were cultured in Dulbecco's Modified Eagle Medium (DMEM) with high glucose (Life Technology, NY, USA), supplemented with 100 mg/ml streptomycin, 100 units/ml penicillin, and 10% fetal bovine serum. The cells were maintained at 37 °C with 5% CO₂ and ≥ 90% humidity.
Genchem Biotechnology Co. Ltd. (Shanghai) provided lentiviral-based small hairpin RNA (shRNA) targeting HSF4 and a control lentivirus with scrambled shRNA. The sequences used were si-HSF4 #1: 5’-GCAAGCUGAUCCAGUGUCUTT-3', si-HSF4 #2: 5’-CGCCAACUCAACAUGUACGTT-3', and scrambled: 5’-UUCUCCGAACGUGUCACGU-3'. Experiments found that si-HSF4 #2 was more effective than si-HSF4 #1; therefore, only si-HSF4 #2 was used for shRNA constructs. SW480 and HCT116 cells were infected with these lentiviral particles and maintained in L-15 or RPMI 1640 medium containing 2 μg/mL or 1 μg/mL puromycin, respectively. After two weeks of selection, western blot analysis was performed to measure HSF4 expression, confirming the creation of stable cell lines.

The human CRC cell lines SW620 (RRID: CVCL_0547), SW480 (RRID: CVCL_0546), HCT116 (RRID: CVCL_0291), Caco-2 (RRID: CVCL_0025), LoVo (RRID: CVCL_0399), and the normal colon epithelial cell line NCM460 (RRID: CVCL_0460) were all purchased from the Cell Bank of the Chinese Academy of Sciences. Upon obtaining the cell lines, immediate validation procedures were performed, including recent analysis involving short tandem repeat (STR) analysis, mycoplasma testing, and cell viability assessment. In brief, the validation of all cell lines was conducted using the LONZA MycoAlertTM mycoplasma detection kit (Lonza, #LT07-418), with all results yielding negative outcomes. Additionally, none of the cell lines used were found in the commonly misidentified cell line database maintained by the International Cell Line Authentication Committee.
All cell lines were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM; Gibco, #11965092) or Roswell Park Memorial Institute 1640 (RPMI 1640; Gibco, # 11875119) supplemented with 10% fetal bovine serum (FBS; Gibco, #10099-141) in a humidified incubator at 37 °C with 5% CO₂.
To establish EFTUD2-overexpressing SW480 cell lines, a lentiviral vector carrying EFTUD2 (pcDNA3.1-Flag-EFTUD2-GFP/Puro) was transfected into the cells, while an empty vector (pcDNA3.1-Flag) was used as a negative control. The cloning primers used for EFTUD2 overexpression were as follows:

Forward: 5′-TAACCTCTGAAAGAGGAACTTGGTTAGGTACCATGTGTCAGACACTTGCTCAGTCT-3′,

Reverse: 5′-TCGTACACCTTGGAAGCCATGGTGGCTAGCGATGCTCTCGCCTGCTCAGCT-3′.

Similarly, to knock down EFTUD2 expression, lentiviral-mediated EFTUD2-shRNA was transfected into the HCT116 and Caco-2 cell lines using specific fragments, with a control group established for comparative purposes. The sequence of EFTUD2 shRNAs were as follows:

shRNA#1: 5′-CCCATTATTAAGCCAGTGAAA-3′.

shRNA#2: 5′-GCCTCTCACAGAACCCATTAT-3′.

Following infection, stable cell lines were generated by passaging cells for 72 h in a medium containing 5 µg/mL puromycin (Sigma, Missouri, USA, #S250) until all uninfected cells were eliminated. The transfection efficiency was assessed using both Western blotting and RT-qPCR.