Hpa007196

We used the lung cancer database in the Kaplan-Meier plotter (http://kmplot.com/analysis/index.php? p=service&cancer=lung) to analyze the association between prognosis and ALKBH5 and FTO mRNA expression in NSCLC cohorts. Data were downloaded on December 10, 2020. Kaplan-Meier curves for overall survival (OS) were generated and stratified according to the median expression of each mRNA. To assess the mRNA expression of ALKBH5 and FTO, data from the Cancer Genome Atlas (TCGA) (NSCLC, Provisional) were downloaded from cBioPortal (http://www.cbioportal.org/) on November 11, 2019. Expression data were obtained in the form of RNA-seq by Expectation Maximization (RSEM).
Cells grown on coverslips were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100. After blocking with 5% bovine serum albumin in PBS (-) at room temperature for 1 h, the cells were probed with primary antibodies against ALKBH5 (HPA007196, Atlas Antibodies) and then incubated with a Goat anti-Rabbit IgG (H + L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 546 (#A-11010, Thermo Fisher Scientific, Waltham, MA, USA). Nuclei were stained with ProLong® Gold Antifade Reagent with DAPI (#8961, CST), after which the cells were imaged via fluorescence microscopy using z-stack image reconstructions (BZ-9000; Keyence, Osaka, Japan).

Cells grown on coverslips were xed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100. After blocking with 5% bovine serum albumin in PBS (-) at room temperature for 1 h, the cells were probed with primary antibodies against ALKBH5 (HPA007196, Atlas Antibodies) and then incubated with a Goat anti-Rabbit IgG (H + L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 546 (#A-11010, Thermo Fisher Scienti c, Waltham, MA, USA). Nuclei were stained with ProLong® Gold Antifade Reagent with DAPI (#8961, CST), after which the cells were imaged via uorescence microscopy using z-stack image reconstructions (BZ-9000; Keyence, Osaka, Japan).

Resected NSCLC samples from Hamamatsu University School of Medicine and Seirei Mikatahara General Hospital were collected and named as the HUSM cohort. Tissue microarray (TMA) sections were analyzed using immunohistochemistry (IHC) as previously described [44] . Cores of insufficient quality or quantity were excluded from analysis. Antibodies for ALKBH5 (HPA007196, Atlas Antibodies, Stockholm, Sweden) and FTO (Ab124892, Abcam, Cambridge, UK) were diluted at 1:400, whereas those specific for EGFR E746-A750 deletion (#2085, D6B6, Cell Signaling Technology diluted at 1:100, followed by incubation at room temperature for 0.5 h. Protein expression levels were then assessed using the H-score, which was calculated by multiplying the percentage of stained tumor area (0%-100%) by the staining intensity (scored on a scale of 0-3) to yield a value ranging from 0 to 300.