Bioinformatics analysis
LUAD gene expression data-set (GSE148036) was downloaded from the Gene Expression Omnibus database (https://www. ncbi. nlm. nih. gov/geo/query/acc. cgi?acc = GSE148036) for gene difference and GSEA analyses. Moreover, the Ualcan database (http://ual can. path. uab. edu) was employed to analyze the relationships among gene expression, clinical stages, and survival rates of NSCLC patients.
Immunohistochemical staining
Immunohistochemical staining was performed on several tissue microarrays (TMAs) with a thickness of 4 µm (HLugA150CS03, HLugA030PG02, HLugS030PG02, OD-CT-RsLug03-002, Outdo Biotech, Shanghai, China). The slices were repaired in citrate buffer at a high temperature and pressure for 3 minutes. Subsequently, the primary antibody of ESCO2 (1:200, ab86003; Abcam) was added into the slices, followed by incubation at 4℃ overnight. Immunoperoxidase labeling was performed with an ABCD Kit(MTB, Fuzhou, China). Finally, DAB chromogen was added into the samples and the nuclei were dyed with hematoxylin.
Based on the percentage and the intensity of cell staining, ESCO2 expression levels were allocated into four categories, namely negative (no staining,-), weakly positive (only partially colored and light yellow granules,+), moderately positive (yellow granules, 2+), and strongly positive (dark yellow or yellowish brown granules, 3+). Since no standard to judge the positive immunohistochemical expression of ESCO2 is currently available and most of the lung cancer specimens (96/131) were categorized as level 2 + or more, the expression levels of 2 + and 3 + were defined as positive, while the remaining two were defined as negative.
Plasmid transfection and interference
EX-NEG-M14 plasmids were obtained from GeneCopoeia Co. Ltd.. si-ESCO2 and negative control si-NC were purchased from Anhui General Bio Co. Ltd., and Lipofectamine 3000 (Invitrogen) was used to transfect cells.
MTT and clone formation experiments
After 24 h of transfection, a total of 3000 cells were cultured in 10% serum 1640 medium, and 20 µL of MTT solution (5 mg/ml Thiazolyl Blue) was added to each plate. The absorbance numbers at 490 nm at 24 h, 48 h, 72 h, and 96 h were detected and recorded by an automatic microplate reader. Another 800 cells contained in 4 ml of 10% serum 1640 medium were cultured in a 6-cm dish for 10 days. All cells were fixed in ice methanol and dyed with hematoxylin after the experiments.
Transwell assay
After 48 hours of transfection, cells were inoculated in the upper chamber at a density of 5 × 105 cells/200 µL, and 600 µL of 20% serum medium was added to the lower chamber. The cells were harvested after 24 h of inoculation and fixed in ice-methanol for 15 minutes, followed by hematoxylin staining. Ten fields of view were randomly selected, and the number of cells migrating to the lower chamber in each of the selected fields was counted.
Western blot assay
The extracted proteins were separated by SDS-PAGE, transferred to a polyvinylidene difluoride (PVDF) membrane, and incubated with the primary antibodies(Table S1). On the next day, the samples were incubated with the horseradish peroxidase (HRP), which was coupled with anti-rabbit/mouse secondary antibody, at 37°C for 2 h. Enhanced chemiluminescence (ECL) detection reagents were used to visualize the results.
qRT-PCR assay
TRIzol and chloroform were used to extract total RNA from the treated A549 cells, Reverse transcription was carried out by a PrimeScript RT kit (TAKARA). A 20µL SYBR Green PCR SuperMix system (Transgen, Beijing, China) was used for DNA amplification, and the amplification cycle was 50 ℃, 2 min; 95 ℃, 10 min; 95 ℃, 40 s for 40 cycles, and finally end by 60 ℃, 60 s. β-actin was used as the control group, and the data were analyzed by 2 − ΔΔCT.
Immunoprecipitation
The immunoprecipitation experiments were performed with the Immunoprecipitation Kit (Proteintech, USA) following the manufacturer’s protocol. The experiment results were detected by Western blot.
Statistical analysis
Data analysis was performed on GraphPad Prism 8. 0. The chi-square test was used to analyze the correlation between clinicopathological factors and protein expression levels. The t-test was used to detect the gray value in the Western blot assay. p < 0. 05 was considered statistically significant.