Antibodies and Reagents Polyclonal rabbit anti-TIG3 was previously described [23,55]. BrdU (B2531) and b-actin (A1978) antibodies were purchased from Sigma. Caspase 3 (9665), Caspase 9 (9502), and cleaved PARP (9541) antibodies were from Cell Signaling. Bcl-XL (610211), EEA1 (610456), rab11 (610656), LAMP1 (611042), GM130 (610822), calnexin (610524), and cyclin D1 (554180) antibodies were purchased from BD Transduction Laboratories (San Jose, CA). Bax (sc-493), p21 (sc-6246), cdk4 (sc-601), cdk6 (sc- 7181), cyclin E (sc-481), cdk2 (sc-163), cyclin A (sc-239), cyclin B1 (sc-245), and cdk1 (sc-54) antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA). b-tubulin (ab11311), clathrin heavy chain (ab21679), pericentrin (ab28144) and mannose-6- phosphate receptor (ab2733) antibodies were from Abcam (Cam- bridge, MA). Alexa Fluor 488-conjugated goat anti-rabbit (A- 11034), Alexa Fluor 555-conjugated goat anti-mouse (A-21424), and Hoechst 33258 (H3569) were from Invitrogen (Carlsbad, CA). Rabbit HRP-conjugated IgG (NA934) and mouse HRP-conjugat- ed IgG (NXA931) were from GE Healthcare (Piscataway, NJ). BrdU Incorporation and flow cytometry To monitor BrdU incorporation, SCC-13 cells on coverslips were infected with appropriate adenovirus and after 24 h incubated with 10 mM BrdU (BD Pharmingen, 550891) for 2 h at 37 C. Cells were then fixed in 4% paraformaldehyde for 30 min at room temperature and washed with PBS containing 1% Triton X-100. The slides were then incubated in 1 N HCl on ice for 10 min, in 2 N HCl at 25 C for 10 min and 37 C for 20 min. Cells were then incubated with 0.1 M borate buffer for 12 min at 25 C and washed with PBS containing 1% Triton X-100. The cells were References 1. Ou CC, Hsu SC, Hsieh YH, Tsou WL, Chuang TC, et al. 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For flow cytometry, SCC-13 cells were infected with tAd5-EV or tAd5-TIG3 and after 24 h floating and attached cells were fixed in methanol, incubated with 20 mg/ml RNase for 30 min at 37 C followed by 50 mg/ml propidium iodide for 1 h prior, and the cells were collected for flow cytometric analysis. Real Time-PCR SCC-13 cells were infected with tAd5-EV or tAd5-TIG3 and at 24–48 h harvested for isolation of mRNA using the illustra RNAspin Mini Isolation Kit (GE Healthcare, 25-0500-71). cDNA was synthesized from isolated RNA using SuperScript III Reverse Transcriptase (Invitrogen, 18080-044) following the manufactur- er’s protocol. RT-PCR was performed using LightCycler 480 SYBR Green I Master (Roche, 04707516001). RT–PCR primer sequences include: TIG3 (59-CAGTATTGTGAGCAGGAACT- GTGA, 59-TTGGCCTTTTCCACCTGTTTAC), human cyclo- philin (59-CATCTGCACTGCCAAGACTGA, 59-TTCATGCC- TTCTTTCACTTTGC), and human p21 (59-AAGACCATGT- GGACCTGTCACTGT, 59-AGGGCTTCCTCTTGGAGAAG- ATCA). 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PLoS ONE | www.plosone.org 8 August 2011 | Volume 6 | Issue 8 | e23230