Supplemental Information MATERIALS AND METHODS Reagents and Antibodies Gefitinib was obtained from AstraZeneca Pharmaceuticals (Wilmington, DE). FuGENE 6 was purchased from Roche (Basel, CH). Polyethyleneimine (PEI), polybrene, RNase A, propidium iodide, protease inhibitor cocktail and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were purchased from Sigma-Aldrich (St Louis, MO). The caspase-3 inhibitor Z-DEVD-FMK was purchased from R&D Systems Inc. (Minneapolis). All cell culture-related reagents were purchased from Invitrogen (Carlsbad, CA). The sources of primary antibodies were as follows: AGO2, Dicer and Drosha, were purchased from Abcam Ltd. (Cambridge, UK). MET was purchased from Santa Cruz Biotechnology (Santa. Cruz, CA). Cleaved caspase-3, EGFR and pEGFR were from Cell Signaling Technology (Beverly, MA). Anti-α-tubulin antibody was purchased from Sigma. All secondary antibodies were purchased from Jackson Immuno Research (West Grove, PA). Western Blot Cells were lysed in RIPA buffer containing protease inhibitor cocktail. Protein concentration was determined by the BCA assay (Pierce). The equal amount of total protein was resolved by SDS-PAGE and transferred to PVDF membranes (Millipore, Bedford, MA). Membranes were blocked with tris-buffered saline/0.1 % Tween 20 (TBST) containing 5 % skim milk and then incubated overnight at 4 °C with the indicated primary antibodies. Membranes were washed with TBST and incubated for 1 h at room temperature with the appropriate secondary antibodies conjugated to horseradish peroxidase. Membranes were then washed and bound antibodies were visualized using ECL reagents (PerkinElmer, MA) and autoradiography. Semi-quantitative analysis of band intensities was performed by densitometry using image analysis software ImageJ. RNA Isolation, Reverse Transcription and Real-time PCR Quantification Total RNA was isolated using Trizol reagent (Invitrogen) and reverse transcribed into cDNA using M-MLV reverse transcriptase (Invitrogen) according to the manufacturer's instructions. Real-time PCR was performed using the Roche LightCycler 480 (Roche). The relative mRNA levels of the target gene were normalized to the mean of internal GAPDH. The mature miRNA sequences were obtained from the Sanger Center miRNA Registry and the stem-loop RT primers were designed according to Chen and Ridzon et al.1 Expression levels of miRNA genes were based on the amount of the target message relative to that of the RNU6B transcript, used as a control to normalize the initial input of total RNA. The relative levels of gene expression were represented as ΔCt = Ct gene – Ct reference, and the fold change of gene expression was calculated by the 2−ΔΔCt Method. All primers sequences are shown in Supplemental Table 2. Cytotoxicity Assays MTT assay was used to evaluate relative cell viability. For in vitro studies with a caspase-3 inhibitor, cells were pre-incubated with 10 μM of Z-DEVD-FMK at 37°C for 90 min, followed by treatment with 20 μM gefitinib. Absorbance of the dye was measured at a wavelength of 570 nm with background subtraction at 630 nm. Lentivirus Infection and shRNA Knockdown The pLKO.1-puro-based lentiviral vectors: TRCN0000290426 (shDicer#1) and TRCN0000290489 (shDicer#2), and the pLKO.1-shscramble were purchased from the National RNAi Core Facility at Academia Sinica, Taipei, Taiwan. Recombinant lentiviruses were packaged according to the manufacturer's instruction. Cultured cells were incubated with lentivirus containing 8 μg/ml polybrene for 24 h, replaced fresh medium and incubated for another 48 h. For stable cell lines, cells were selected by puromycin. Cloning and Expression of Dicer in PC9 Cells The PCR products were digested with BamH I and EcoR I and cloned into pcDNA6.1 to generate pcDN6.1-Dicer plasmid. Empty pcDNA6.1 plasmid was used as control. Cells were transiently transfected using FuGENE 6 as suggested by the manufacturer. Stable clones were selected for blasticidin resistance using standard protocols. Gefitinib-induced Cell Death Aliquots of 1 × 106 cells were collected and washed once with PBS and then fixed with 70 % ethanol overnight. Fixed cells were then washed twice in PBS and stained with 1 µg/ml propidium iodide in PBS containing 10 µg/ml RNase A for 1 h. The DNA content was measured by flow cytometry. For all assays, 10,000 cells were counted. Statistical Analysis All statistical analyses were performed with SPSS, version 13.0 (SPSS, Chicago, IL). Statistical evaluation of the data was performed with a 2-tailed Student's t-test for simple comparison between two values when appropriate. Pearson chi-square tests and Student's t-tests were used to compare the clinicopathologic characteristics of tumors (and patients) with high expression and those with low expression of Dicer. P values of less than 0.05 were considered statistically significant. Reference: 1. Chen C, Ridzon DA, Broomer AJ, et al. Real-time quantification of microRNAs by stem-loop RT-PCR. Nucleic Acids Res. 2005;33(20):e179.