Additional file 3 of The m6A demethylase ALKBH5 promotes tumor progression by inhibiting RIG-I expression and interferon alpha production through the IKKε/TBK1/IRF3 pathway in head and neck squamous cell carcinoma
Creators: name: Jin, Shufang
name: Li, Mingyu
name: Chang, Hanyue
name: Wang, Ruijie
name: Zhang, Zhiyuan
name: Zhang, Jianjun
name: He, Yue
name: Ma, Hailong
Publisher: name: figshare
Year: 2022
Subjects: subject: Biophysics
subject: Biochemistry
subject: Medicine
subject: Cell Biology
subject: Genetics
subject: FOS: Biological sciences
subject: Molecular Biology
subject: Pharmacology
subject: Immunology
subject: FOS: Clinical medicine
subject: Cancer
subject: Infectious Diseases
subject: FOS: Health sciences
subject: Virology

description: Additional file 3: Supplementary Figure S1. The correlation between ALKBH5 expression and clinicopathological features in 138 HNSCC patients. (a-d) The correlation between the IRS of ALKBH5 and age, sex, pathological grade and lymph node status was analyzed in 138 HNSCC patients. (e) ROC curves were generated according to the IRS of ALKBH5 in HNSCC and normal controls. (f) The pancancer expression profile of ALKBH5 according to the GEPIA dataset. Figure S2. The correlation between FTO expression and clinicopathological characteristics in 138 HNSCC patients. (a) Representative images of immunohistochemical staining for FTO protein on a tissue microarray composed of 138 HNSCC tissues and 20 normal epithelial tissues. Scale bars: 100 μm. (b) The expression level of FTO was analyzed in HNSCC and the normal control. (c) ROC curves were generated according to the IRS of FTO in HNSCC and normal controls. (d-h) The correlation between the IRS of ALKBH5 and age, sex, pathological grade, lymph node status and TNM stage was analyzed in 138 HNSCC patients. (i) The Kaplan–Meier method with a two-tailed log-rank test was used to plot survival curves in The Cancer Genome Atlas (TCGA) HNSCC dataset with high and low FTO expression. The log-rank test was used to compare the survival rate. (j) The expression profile of FTO in pancancer tissues according to the GEPIA dataset. *P &lt; 0.05, **P &lt; 0.01. Figure S3. ALKBH5 mRNA was detected after siRNA transfection for 24 hours. Figure S4. The EdU assay was performed after siRNA transfection for 48 hours. Figure S5. The cell cycle distribution was analyzed using flow cytometry (PI staining) after transfection for 48 hours. Figure S6. Apoptotic cells were detected using flow cytometry (Annexin V/PI staining) after transfection for 48 hours. Figure S7. The migration and invasion capacities were detected using Transwell inserts with and without Matrigel, respectively, after transfection for 48 hours. Figure S8. Venn diagrams show differentially expressed genes with &gt;2-fold alterations after ALKBH5 knockdown. Figure S9. Volcano plots showing the m6A enrichment and mRNA expression levels of genes in ALKBH5-deficient cells compared to the control. Figure S10. Relative DDX58 levels were detected by qRT-PCR after ALKBH5 silencing for 24 h. Figure S11. The IFNα concentration in the supernatant was detected after knockdown or overexpression of both ALKBH5 and RIG-I for 48 h. Figure S12. The tumors in a xenograft model after ectopic expression of ALKBH5 with or without RIG-I expression are shown. Figure S13. Bubble chart showing the KEGG enrichment analysis of the immunoprecipitated proteins from ChIRP coupled with mass spectrometry. Figure S14. HNRNPC knockdown efficiency of three siRNAs, detected using immunoblotting. Figure S15. IFNα concentration measured using an enzyme-linked immunosorbent assay (ELISA) after RIG-I overexpression or silencing for 48 h. Figure S16. The IC50 of IFNα determined after 72 hours in HNSCC cells. Figure S17. The representative image of ALKBH5 overexpression and mIFNα treatment in SCC7-bearing C3H mice. Figure S18. The infiltration of immune cells in tumor microenvironment was analyzed after mIFNα or PBS treatment in SCC7-bearing xenograft. Figure S19. The percentage of M2 macrophage was analyzed in SCC7-bearing C3H mice by flow cytometry.