TCGA-GTEx Lung Cancer Cohort
We downloaded The Cancer Genome Atlas (TCGA) - The Genotype-Tissue Expression (GTEx) cohort data from the website of University of California Santa Cruz Xena Browser (https://xenabrowser.net)[14, 15]. Data collation using Excel, we abandoned the recurrent tumor, primary lung adenocarcinoma, primary lung squamous cell carcinoma and solid tissue normal remained for further analysis.
Gene enrichment analysis
ASF1B co-expression genes were extracted from cBioPortal (http://www.cbioportal.org/) with TCGA LUAD cohort, which has |r| ≥ 0.35. These co-expressed genes were further carried out Gene Enrichment analysis based upon Kyoto Encyclopedia of Genes (KEGG) in DAVID (https://david.ncifcrf.gov/summary.jsp). Gene sets, which p<0.05 were identified as enriched. GSEA was applied to further explored the pathways that correlate to ASF1B expression. Gene sets meets P<0.05 and FDR < 0.25 were considered remarkable enriched.
The non-neoplastic lung epithelial cell 16HBE, BEAS-A2 and six NSCLC cell lines (A549, H460, H1299, H520, H1975 and SPC-A1) were obtained from the ATCC (American type culture collection). Cell lines were cultured in RPMI-1640 medium (Thermo Fisher Scientific, USA) containing 10% fetal bovine serum (Gibco, USA). The culture dishes were placed in the incubator at 37°C, 5% CO2.
Cell infection and transfection
The siRNA targeting ASF1B and control vector were provided by RiboBio (Guangzhou, China) for si-RNA-mediated gene knockdown. The siRNA transfection was performed ASF1B using Lipofectamine 3000 (Thermo Fisher Scientific, Inc.) according to the manufacturer’s instructions.
Total RNA was extracted using Trizol RNA reagent (Themo Fisher Scientific, USA). Reverse transcription was performed using the PrimeScript™ RT reagent Kit with gDNA Eraser (Takara, Japan). qRT-PCR was conducted using SYBR Green™ Premix Ex Taq™ II (Takara, Japan) in Applied Biosystems 7500 (AB, USA).
Western blot analysis
Western blotting was performed according to the standard protocol with the following antibodies:α-tublin, β-actin, Slug, CDK2/4, CCND1, P27 (CST), Snail, Slug, E-cadherin, Vimentin (Abcam).
Cell counting kit-8 (CCK-8) and colony formation assays
Cells were planted into 96-well plates with 1000 cells per well. CCK-8 reagents (Djingo, Japan) were added at every at 24h for five days. The optical density was estimated at 450 nm wavelength. Cells were planted in in 6-well plates (500/well) and incubation for 2 weeks at 37 °C, 5% CO2. Colonies were washed triple with PBS and stained with crystal violet for 15min.
Wound Healing assays
1 × 106 non-small cell lung cancer cells were routinely seeded and inoculated in a 6-well plate. When the density reached 100%, scratching was conducted with 200ul sterile tips perpendicularly to the cell plane. Taking photos under the microscope at the time pace: 0 h, 24 h.
Migration assays were performed using transwell chambers (Corning USA). For migration assays, cells (2×105 cells) were seeded with serum-free medium onto the top chamber, and the bottom chamber was filled with 10% FBS medium. After 18h, all the chambers collected and fixated with methanol for 30 min, stained with 0.01% crystal violet for 15 min.
For data processing, GraphPad Prism version 8.0 was used. Values in this analysis are described in the form of mean SD (Standard Deviation) unless otherwise specified. Independent survey contrasts between two groups When the two groups' SDs were equal, the Student's t-test (two-tailed) was used, and when the SDs were different, the Student's t-test with Welch's correlation was used. If the variances were equivalent in multigroup samples statistics, one-way ANOVA was used; if not, Welch's ANOVA was used. Both ANOVAs were subjected to Bonferroni post hoc examinations. The TCGA LUAD Cohort samples were split into two categories based on median values. A statistically significant difference was described as a difference of *P<0.05, ** denotes P< 0.01, *** denotes P < 0.0001.