Additional file 1 of TNFAIP2 confers cisplatin resistance in head and neck squamous cell carcinoma via KEAP1/NRF2 signaling

Additional file 1. Figure S1. (a) TNFAIP2 mRNA expression between HNSCC and normal tissues from the TCGA. (b) Kaplan‒Meier survival curve of HNSCC patients with low versus high TNFAIP2 expression from the TCGA. (c) Kaplan‒Meier survival curve of HNSCC patients with low versus high TNFAIP2 expression from the GEO (GSE65858). (d) TNFAIP2 protein expression in HNSCC patients with or without TPF chemotherapy. (e) Correlations between TNFAIP2 mRNA expression and Taxol and 5-fluorouracil IC50 in 10 HNSCC cell lines. (f) Western blot analysis of the efficiency of TNFAIP2 overexpression/knockdown in HNSCC cell lines. (g) CCK-8 assays of cell viability in TNFAIP2-overexpressing HNSCC cell lines with or without cisplatin treatment. (h-i) Colony formation and qualification in TNFAIP2-overexpressing FADU (h) and CAL33 (i) cells with or without cisplatin treatment. (j-k) Cisplatin IC50 evaluation in TNFAIP2-knockdown FADU (j) and CAL33 (k). (l) Tumor weight of each group after the indicated treatments. Data are presented as the mean ± SEM. n.s., not significant; *** P < 0.001. Figure S2. (a) Flow cytometry analyses of cell cycle percentage in TNFAIP2-overexpressing HNSCC cell lines. (b) RT‒qPCR analysis of cisplatin resistance-associated genes in TNFAIP2-overexpressing HNSCC cell lines. (c-d) Flow cytometry analyses of ROS in TNFAIP2-knockdown FADU (c) and CAL33 cells (d). (e) Analyses of GSH/GSSG and SOD in TNFAIP2-knockdown HNSCC cell lines. (f-g) Flow cytometry analyses of cisplatin-induced apoptosis in TNFAIP2-overexpressing FADU (f) and CAL33 (g) cells. (h-i) Cisplatin IC50 evaluations in TNFAIP2-knockdown FADU (h) and CAL33 (i) cells with or without the JNK pathway inhibitor SP600125 (20 µmol/L, 2 h). Data are presented as the mean ± SEM. n.s., not significant; *** P < 0.001. Figure S3. (a) Relative luciferase activity in TNFAIP2-knockdown HNSCC cell lines transfected with ARE dual-luciferase reporter plasmids. (b) Relative luciferase activity in TNFAIP2-overexpressing HNSCC cell lines transfected with ARE dual-luciferase reporter plasmids. (c-d) The mRNA (c) and protein (d) levels of NRF2 target genes in TNFAIP2-knockdown HNSCC cell lines. (e-f) The mRNA (e) and protein (f) levels of NRF2 in TNFAIP2-knockdown HNSCC cell lines. Data are presented as the mean ± SEM. n.s., not significant; *** P < 0.001. AREs, antioxidant response elements. Figure S4. (a) Twelve unique peptides of KEAP1 identified by LC‒MS. (b) Relative luciferase activity in TNFAIP2-overexpressing HNSCC cell lines transfected with ARE dual-luciferase reporter plasmids with or without KEAP1 knockdown. (c-d) The mRNA (c) and protein (d) levels of KEAP1 and p62 in TNFAIP2-knockdown HNSCC cell lines. (e) Co-IP assay of the TNFAIP2 and NRF2 interaction in HNSCC cell lines. Data are presented as the mean ± SEM. n.s., not significant; *** P < 0.001. Figure S5. (a) Relative luciferase activity in TNFAIP2-knockdown HNSCC cell lines transfected with ARE luciferase reporter plasmids and rescued by wild-type TNFAIP2 or the N328-520 fragment. (b) RT‒qPCR analysis of NRF2 target genes in TNFAIP2-knockdown HNSCC cell lines rescued by wild-type TNFAIP2 or the N328-520 fragment. (c) Flow cytometry analyses of ROS in TNFAIP2-knockdown HNSCC cell lines rescued by transfection of TNFAIP2 with DLG deletion. (d) Relative luciferase activity in TNFAIP2-knockdown HNSCC cell lines transfected with ARE luciferase reporter plasmids and rescued by transfection of TNFAIP2 with DLG deletion. (e) RT‒qPCR analysis of NRF2 target genes in TNFAIP2-knockdown HNSCC cell lines rescued by transfection of TNFAIP2 with DLG deletion. (f-g) Cisplatin IC50 evaluations in TNFAIP2-knockdown FADU (f) and CAL33 cells (g) rescued by transfection of TNFAIP2 with DLG deletion. Data are presented as the mean ± SEM. n.s., not significant; *** P < 0.001. Table S1. Demographic and clinical characteristics of HNSCC patients. Table S2. Univariate and multivariate Cox analyses of HNSCC patients. Table S3. Target sequences of siRNAs in this study. Table S4. Plasmids used in this study. Table S5. Primary and secondary antibodies used in this study. Table S6. Primer for amplifying the human transcripts used in this study. Table S7. Critical commercial reagents used in this study.