Supplementary Figure legends

Supplementary Figure S1. AhR protein as an unfavorable prognostic factor and therapeutic target for NSCLC. A to C. TCGA analysis of AhR at the mRNA level in relation to NSCLC stage, lymph node infiltration, metastasis, disease relapse and patient survival. D. qPCR analysis of cyp1a1, cyp1b1, IDO1, IDO2 and TDO in AhRlow PC-9 and AhRhigh H1975 cells. E. PC-9, SPC-A-1 and H1975 cells were treated with 0, 1 and 10 µmol/L α-NF for 24 h, and cell viability was measured by MTT assay. F. PC-9 and H1975 cells were treated with increasing concentrations of TCDD (1, 5 and 10 nmol/L), β-NF (1, 5 and 10 µmol/L) and Kyn (10, 50 and 100 µmol/L) for different time intervals. A measure of cell proliferation and viability was determined by the OD value at 490 nm. G. H1975 cells were treated with 1 µmol/L β-NF or 10 µmol/L Kyn and were grown in soft agar for 3 weeks. Representative images of soft agar colony formation are shown. H. PC-9 and H3122 cells were treated as indicated. Phosphorylation of Akt and Erk and cell apoptosis were determined by immunoblotting.

Supplementary Figure S2. Inhibition of AhR sensitizes NSCLC cells to EGFR TKIs. A. Effect of AhR silencing on H1975 cell viability. B. Western blot analysis of cell apoptosis in H1975 cells expressing Dox-inducible AhR shRNAs.

Supplementary Figure S3. Effect of AhR overexpression on cell proliferation and sensitivity to chemotherapeutics. A. AhR WT or AhR CA mutant was stably overexpressed in PC-9 cells. Cell viability was determined by measuring the OD490 value at the indicated time intervals. B. PC-9 AhR WT and PC-9 GFP control cells were treated with cisplatin or paclitaxel and evaluated for cell viability by MTT assay.

Supplementary Figure S4. Activation of AhR activates Src independently of its transcriptional activity. A. H1975 cells were treated with 10 nmol/L TCDD for 15 min or 30 min. Transcription levels of cyp1a1 and cyp1b1 were determined by qPCR analysis. B. Representative immunofluorescence images of AhR protein localization upon TCDD treatment. As a positive control for AhR nuclear translocation, cells were treated with TCDD for 12 h. Scale bar = 200 µm. C. H3122 and HCC78 cells were infected with Src Y527F or mCherry lentivirus, treated with 1 µmol/L crizotinib for 24 h, and evaluated for apoptosis and phosphorylation of Src, Akt and Erk. D. Cells stably carrying mCherry or Src Y527F were treated with DMSO or 1 µmol/L corresponding TKIs for 24 h and evaluated for the magnitude of TKI-induced growth inhibition.

Supplementary Figure S5. AhR activates Src through Jak2 kinase. A. Clustering analysis of the putative SH2 binding motif in protein adaptors. B. Quantitative analysis of phospho-protein kinase array profiling of AhR-ligand-treated and non-AhR-ligand-treated H1975 cells. C. Western blot analysis of Jak2 phosphorylation in H1975 cells treated with AhR ligands. D. Jak2 expression in PC-9 AhR WT and H1975 cells was knocked down by shRNAs. The resulting cells were treated with 10 nmol/L TCDD for 30 min and subjected to Western blotting. E. The PC-9 AhR WT cells were treated with 1 µmol/L gefitinib, 1 µmol/L ruxolitinib or a combination of the two agents for 24 h and subjected to Western blot analysis for apoptosis and kinase phosphorylation.

Supplementary Figure S6. AhR WT as a protein adaptor to initiate Src phosphorylation. A. H1975 cells were treated with 10 nmol/L TCDD for the indicated time intervals. Equal amounts of EGFR and α-tubulin were used as loading controls for cell membrane proteins and cytosolic proteins, respectively. B. Representative flourescence images of AhR WT, AhR CA and AhR CA ΔNLS localization. The AhR WT, AhR CA and AhR CA ΔNLS cDNA were in-frame fused to C-terminal GFP and introduced into HEK293 cells. The distribution of AhR WT, AhR CA and AhR CA ΔNLS protein was visualized by detecting GFP flourescence. Scale bar = 200 µm. C. The Flag-tagged AhR CA or AhR CA ΔNLS and HA-tagged Src plasmids were expressed in HEK293 cells. Immunoprecipitation was performed with anti-Flag tag and anti-HA tag antibodies, respectively. D. H1975 cells and PC-9 AhR WT cells were treated with 10 nmol/L TCDD for 30 min followed by TCDD withdrawal for the indicated time intervals. Src phosphorylation after TCDD treatment and TCDD withdrawal was determined by immunoblotting.