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.