Supporting Information

Files in this Data Supplement:

Supporting Figure 4
Supporting Table 1
Supporting Figure 5
Supporting Figure 6
Supporting Figure 7
Supporting Figure 8
Supporting Methods

Fig. 4. Expression of nAChR subunits in the human lung adenocarcinoma cell line NCI-H441, squamous cell carcinoma cell line NCI-H226, and primary human microvascular lung endothelial cells (HMEC-L), as detected by RT-PCR.

Fig. 5. Nicotine inhibits apoptosis induced by chemotherapeutic drugs in asynchronous A549 cells. Asynchronous A549 cells were treated with 20 mM gemcitabine, cisplatin, or taxol; the presence of 1 mM nicotine inhibited apoptosis, as seen in a TUNEL assay.

Fig. 6. Western blotting analysis shows that siRNA to XIAP and survivin (Santa Cruz Biotechnology) specifically suppresses the levels of the corresponding proteins in A549 cells (refer to TUNEL assay shown in Fig. 2e).

Fig. 7. A combination of a different set of siRNAs to XIAP and survivin (Cell Signaling Technologies) could ablate the antiapoptotic effect of nicotine whereas a nontargeting control siRNA (Santa Cruz Biotechnology) had no effect. (a) Western blotting analysis showing that the siRNAs ablate the expression of XIAP and survivin in A549 cells in a specific manner. (b) A combination of XIAP siRNA and survivin siRNAs completely reversed the protective effect of nicotine on drug-induced apoptosis in A549 cells. Apoptosis was ascertained by using a TUNEL assay. A nontargeting siRNA sequence was used as a control for all experiments.

Fig. 8. (a) The antiapoptotic effects of nicotine could be abrogated by a combination of a3-nAChR siRNA and b2-nAChR siRNA in A549 cells, whereas the individual siRNAs for a3-nAChR and b2-nAChR had only a partial effect. The siRNAs were transfected by using Oligofectamine reagent in A549 cells at a concentration of 50 pmol per sample. Eighteen hours after transfection the cells were serum-starved for 24 h and thereafter treated with 20 mM cisplatin in the presence or absence of 1 mM nicotine as described in Materials and Methods. Apoptosis was ascertained by using a TUNEL assay. (b) The ability of a3-nAChR siRNA and b2-nAChR siRNA to ablate the levels of the corresponding proteins in A549 cells was assessed by Western blotting analysis. The transfection of a3-nAChR siRNA and b2-nAChR siRNA also ablated the levels of XIAP and survivin in A549 cells. The combination of a3-nAChR siRNA and b2-nAChR siRNA caused increased suppression of XIAP and survivin in A549 cells.

Fig. 9. Nicotine induces survivin expression. A Northern blot shows the induction of survivin gene upon nicotine stimulation. Total RNA (10 mg) was extracted from quiescent A549 cells or cells stimulated with 1 mM nicotine. Subsequently, the RNA was hybridized with ³²P-labeled survivin fragment or XIAP fragment or GAPDH probe, and message was detected by autoradiography.

Table 1. Differential expression of nAChR subunits in human NSCLC cell lines

X, subunit detected; ND, not determined.

All nAChR polyclonal antibodies, survivin polyclonal antibody, Stat3 and Stat1 monoclonal antibodies, p53 monoclonal antibody, and p73 polyclonal antibody were obtained from Santa Cruz Biotechnology. XIAP monoclonal antibody was obtained from Stressgen Biotechnologies. Monoclonal antibody to p21/Waf1/Cip1 was obtained from NeoMarkers. The a7-nAChR monoclonal antobody was obtained from Sigma-Aldrich. Polyclonal antobodies for XIAP, c-IAP-1, and c-IAP-2 were obtained as kind gifts from H. G. Wang (Moffitt Cancer Center, Tampa, FL). The experimental techniques are described in the text.