Fig. 1: Erlotinib-induced gene expression in A549 cells is differentially suppressed by various TNF inhibitors.

a EGFRwt lung cancer adenocarcinoma line A549 was treated for 24 h with vehicle or 1 µM erlotinib, with/without 100 µg/mL Enbrel (Enb/etanercept), 10 µM thalidomide (Tha), 10 µM prednisolone (PRDN). RNAseq was performed on 24 RNA samples in three independent experiments as in the Methods. Volcano plot shows the differentially expressed genes affected by erlotinib, identified by EdgeR. b Violin plot shows the genes with Gene Set Enrichment Analysis (GSEA) gene ranking metric z-normalized scores in the comparison of erlotinib and control > 1.96 (p < 0.05) and false discovery rate (FDR) in the Benjamini-Hochberg method < 0.05. These erlotinib-upregulated genes would have 31%, 42%, and 66% blocked (z-score < 1.96) by Enbrel, thalidomide, and prednisolone, respectively. c Among genes in the TNF signaling pathway (Reactome Pathway Database), erlotinib-induced 18 leading-edge genes with z-score > 1.96 were blocked by the 3 TNF inhibitors at different strengths, via comparing erlotinib-induced GSEA metric scores between absence and presence of each inhibitor. d, e Real-time PCR was performed to validate the erlotinib-induced TNF mRNA can be blocked by these three drugs in EGFRwt and EGFR mutant lung cancer cells. f, g TNF protein levels were examined by Enzyme-Linked Immunosorbent Assay (ELISA). h, i Erlotinib-increased NF-κB activity was blocked by these three drugs in luciferase reporter assays. j–o Western Blot (WB) shows the IκBa degradation was blocked by these three drugs. Specifically, prednisolone was able to increase IκBa expression. Data were quantified by ImageJ. p–q Prednisolone-increased IκBa mRNA levels were determined by real-time PCR. r–w Non-canonical NF-κB pathway components NIK/MAP3K14 and RELB was also induced by erlotinib and blocked by prednisolone in mRNA and protein levels. 100 nM erlotinib is used for HCC827. Treatments last for 6 (j–q) or 24 h (others). Real-time PCR were performed in 3 independent experiments, showing each value and mean ± Standard Error of the Mean (SEM). WB images are one representative from at least three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, by two-way analysis of variance (ANOVA), with Bonferroni-adjusted. The statistical analysis above was performed on Graphpad Prism 9.0.0. Source data are provided as a Source Data file. p = 0.0001,0.001, 2E−6;8E−7,3E−10,1E−9;1E–4,6E−4,2E−5;0.006,0.001,0.001;0.04,0.03,0.01;0.002,1E−7,3E−7 (d–i). 5E−6,1E−5;2E−5,1E−5;5E−5,3E−6;2E−7,3E−8;1E−5,2E−6;4E−6,1E−7 (p–u).