Supplementary Figure 1:TPCA-1 Inhibits STAT3 Phosphorylation

SUPPLEMENTARY FIGURE LEGENDS

Supplementary figure 1:TPCA-1 inhibits STAT3 phosphorylation.

A, 293T cells were stimulated by TNF following incubation with TPCA-1 or BAY11-7082 for 2 hours. B, Levels of STAT3 phosphorylation in 293T incubated by cytokines before treatment with DMSO or BAY11-7082 were tested. C, Hela cells were stimulated with IFN or IFN before treated with DMSO or BAY11-7082 or TPCA-1. Total proteins were analyzed for p-STAT3, STAT3, and GAPDH.

Supplementary figure 2:Dosedependent effects of TPCA-1 on STAT3 activation.

A, Western blot analysis of extracts from 293T cells, pre-incubated with 0 - 2 μM TPCA-1 for 2 h before stimulation with IFN for 30 min.B, SK-BR-3 cells were treated with 0 - 2 M TPCA-1. Total proteins from these cells were analyzed.

Supplementary figure 3: EGFR silence inhibits STAT3 activation in MDA-MB-231 cells. MDA-MB-231 cells were infected with lentivirus containing EGFR shRNA. After selected with puromycin for 3 days, total proteins were analyzed for EGFR, p-STAT3 by western blotting.

Supplementary figure 4: TPCA-1 inhibits endogenous STAT3 activity with or without exogenous IL6 in NSCLC with EGFR mutation.

A,Socs3 mRNA levels from HCC827 and H1975 cells treated with DMSO or TPCA-1 for 24 h were examined by real-time PCR. B,HCC827 cells were pretreated with TPCA-1 or DMSO for 2 h and then stimulated with IL6 for 2 h. Extracts from these cells was analyzed for pYSTAT3.

Supplementary figure 5:TPCA-1 blocks cell cycle in G2/M in HCC827 cells.

A549 and HCC827 cells were treated with 2 M TPCA-1 for 24 h, and then harvested for evaluating the cell cycle distribution.

Supplementary figure 6:Abundance of IL6 and COX2 in NSCLC cells and TPCA-1 inhibits their transcription.

A549, H1299 and HCC827 were treated with TPCA-1 or DMSO for 24 h, and then total RNAs were extracted. Transcription of COX-2, IL6 and GAPDH was determined by semi-quantitative PCR.

Supplementary figure 7: Combination of TPCA-1 increases sensitivity to gefitinib in TKI insensitive NSCLC. H1650 cells were treated with gefitinib alone or combined with TPCA-1(1 μΜ) for 72 h. Cell viability were measured by MTT assay. Data are the averaged results from 4 independent experiments, and error bars mark SDs. (*, P < 0.05, **, P < 0.01, ***, P < 0.001).

Molecular modeling

Crystal structure of Stat3 was retrieved from Protein Data Bank (PDB ID: 1BG1). The three dimensions structure of BAY11-7082 and TPCA-1 are obtained from PUBCHEM (CID: 9903786 and CID: 5353431). The program AutoDock Vina (version 1.1.2) was used to perform the molecular docking. The dimensions of grid box were set to 60 × 42 × 46 Å3 which mainly contains hydrophobic binding and SH2 domain. These binding cavities were the regions targeted for molecular docking. The polar hydrogen was assigned on the STAT3, and the gasteiger charges were added on the ligands.