Supplementary Materials and Methods

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1 Supplementary Materials and Methods Reagents and antibodies was purchased from iaffin GmbH & Co KG. Cisplatin (ristol-myers Squibb Co.) and etoposide (Sandoz Pharma Ltd.) were used. Antibodies recognizing EGFR, GFP, cleaved caspase-9, myc-tag and p14 ARF were purchased from Sigma-Aldrich Corp. Antibodies against p21 Cip1, -tubulin, histone H3, phosho-foxo3a (Ser253) and cleaved caspase-3 were purchased from Cell Signaling Technology, Inc. Anti-HSP60 antibody (Santa Cruz iotechnology, Inc.), anti- -actin antibody (Chemicon, Inc.), anti-p16 INK4a antibody (Epitomics, Inc.) and anti-p53 antibody (DO-1, Santa Cruz iotechnology, Inc.) were used. For secondary antibodies, HRP-conjugated anti-mouse IgG, anti-rabbit IgG, anti-goat IgG and Cy3-conjugated anti-goat IgG were purchased from Zymed Laboratories, Inc. FITC-conjugated anti-mouse IgG (Jackson ImmunoResearch Laboratories, Inc.) and Alexa 488-conjugated anti-mouse IgG (Invitrogen Life Technologies Corp.) were used. The p14 ARF and other HPLC grade peptides were synthesized by Sigma-Aldrich Corp. Plasmid construction and transfection cdna was prepared from normal human dermal fibroblasts (NHDF) cells using an RNeasy kit (Qiagen) and a Super Script III kit (Invitrogen Life Technologies Corp.). Human p14 ARF CDS (NM_ ) was amplified from NHDF cdna and cloned into pluescript SK + vector. To generate the MTS-p14 ARF wild type and mutant plasmid, p14 ARF genes were subcloned into a pcmv/myc/mito vector (Invitrogen Life Technologies Corp.) at the NotI single sites or XhoI - NotI sites. For p14 ARF -NLS plasmid, p14 ARF genes were subcloned into a pef/myc/nuc vector (Invitrogen Life Technologies Corp.) at the NotI sites. MTS-GFP-p14 ARF and GFP-p14 ARF -NLS plasmid were generated by cloning the p14 ARF coding region into the pcmv/myc/mito/gfp and pef/myc/nuc/gfp vector (Invitrogen Life Technologies Corp.) in the NotI sites. For GFP-p14 ARF plasmid, GFP and p14 ARF genes were subcloned into pcdna4 His-MaxA plasmid at PstI - NotI sites. Cells were transiently transfected with p14 ARF plasmids using Lipofectamine LTX and Lipofectamine Plus reagents (Invitrogen Life 1

2 Technologies Corp.) according to the manufacturer s instructions. Small interference RNA The p14 ARF sirna oligonucleotides were synthesized by Sigma-Aldrich Corp. The sequence of sirnas were as follows: p14 ARF sense GAACAUGGUGCGCAGGUUC(d TdT), p14 ARF antisense GAACCUGCGCACCAUGUUC(dTdT). The sigenome SMARTpool for EGFR sirna and the ON-TARGETpuls Non-Targeting Pool for control sirna were purchased from Thermo Scientific Dharmacon, Inc. Cells were cultured in 12-well plates and were transfected with sirnas using LipofectAMINE RNAi Max (Invitrogen Life Technologies Corp.) according to manufacturer s instructions. Immunoblotting Cells were lysed with 20 l of 2 SDS sample buffer (250 mm Tris / HCl ph 6.8, 4% SDS, 20% glycerol, 10% -ME, 1% bromophenol blue), and harvested and sonicated. After boiling for 2 min, proteins were resolved on 15% SDS-polyacrylamide gels and blotted onto PVDF membranes. Nonspecific binding sites were blocked with 5% skim milk powder in TST (50 mm Tris/HCl ph 7.2, 140 mm NaCl, 5% Tween-20) for 1 h. The primary antibodies in 5% skim milk/tst were added and incubated overnight at 4 C. Next, PVDF membranes were washed three times for 10 min each with TST. lots were incubated for 1 h with secondary antibodies conjugated with HRP in 5% skim milk/tst, and then washed three times for 10 min with TST. Chemiluminescence signals were detected by Super Signal West pico reagent (Thermo Scientific, Inc.) and x-ray film using X-OMAT 2000 processor (Kodak, Ltd.). Cell cycle analysis pfucci-g1-orange and pfucci-s/g2/m-green plasmids (ML International Corp.) were co-transfected into and cells. Cells were synchronized by nocodazole (0.4 g/ml, Sigma-Aldrich Corp.) for 12 h. After washing with medium, gefitinib (1 mol/l) was added to cells and cells were cultured for 22 h. The cell cycle phase-specific fluorescence protein was examined by fluorescence microscopy (Olympus, Inc.). 2

3 For flow cytometric analysis, cells were treated with gefitinib (1 mol/l) for 24 h and were washed with PS. The cells were resuspended in 10 g/ml propidium iodide solution. DNA content was analyzed using FACScan and Cell Quest software (ecton Dickinson, Inc.). 3

4 Supplementary Figure Legends Figure S1. Cell cycle regulation and gene expression by gefitinib in the NSCLC cells. A, the cell growth of gefitinib-sensitive cell lines (:, HCC827: ) and gefitinib-resistant cell lines (:, H1975: ) was measured after 72 h of treatment at the indicated gefitinib concentration. Cell growth of each line was assessed by evaluating absorbance at 450 nm using WST-8 reagent. The ratio of the viable cells in each line is shown relative to untreated controls., cells were treated with gefitinib for 24 h. The expression of cell cycle specific fluorescent protein is indicated. Scale bar = 100 m. C, cells were treated with gefitinib (1 mol/l) for 24 h. PI fluorescence of nuclei was analyzed by FACScan flow cytometer. D, CDKN1A (white bar) and CDKN2A (black bar) mrna levels were analyzed by qpcr. and cells were treated with gefitinib (1 mol/l) for the indicated times. The mrna levels were corrected for differences in 18S rrna. The values are represented as the ratio of untreated controls. Results are presented as means ± SD (n=3, *p<5). Figure S2. p16 INK4a and p14 ARF expressions in response to gefitinib. A, and cells were treated with or without gefitinib for 48 hours. The mrna levels of CDKN2A isoforms were detected by qpcr. and C, p14 ARF () and p16 INK4a (C) mrna levels were measured by qpcr. The mrna levels were corrected for differences in 18S rrna. The values are represented as the ratio of untreated controls. Results are presented as means ± SD (n=3, *p<5). D, after treatment with gefitinib for the indicated times, endogenous p14 ARF, p16 INK4a and p21 Cip1 (CDKN1A) expression were detected by western blot using anti-p14 ARF antibody. E, HCC827 and H1975 cells were stained with anti-p14 ARF and anti-hsp60 antibodies. The localization of endogenous proteins was examined by fluorescence microscopy. Nuclei were stained with Hoechst Scale bar = 10 m. Figure S3. Regulation of p14 ARF by chemotherapetic drugs 4

5 , (black bar) and (white bar) cells were treated with gefitinib (1 mol/l), cisplatin (8 mol/l) and etoposide (; 20 mol/l, ; 5 mol/l) for 48 hours. Cell growth was measured by WST-8 reagent and the ratio of viable cells is shown relative to untreated controls (no drug). Results are presented as means ± SD (n=3, *p<5)., s were analyzed by qpcr. (black bar) and (white bar) cells were treated with gefitinib, cisplatin and etoposide for 48 hours. The mrna levels were corrected for differences in 18S rrna. The values are represented as the ratio of untreated controls (no drug). Results are presented as means ± SD (n=3, *p<5). C, EGFR sirna (30 nmol/l) was transfected into and cells. EGFR expression was detected by western blot using anti-egfr antibody. D, cells were knocked down by sirna against EGFR. The s were measured by qpcr. Results are presented as means ± S.D. (n=3, *p<5). lack and white bars represent or cells, respectively. Figure S4. Mutations of p16 INK4a, p14 ARF and p53 in the NSCLC cells. A, p16 INK4a and p14 ARF exon sequences were analyzed using,, HCC827 and H1975 genomes. Alterations in the nucleotides (top panel) are indicated by the arrow. Mutation sites are summarized in the lower panel. No mutations were observed in HCC827 cells., p53 mutations were analyzed by genomic sequence. Changes in nucleotides and protein are indicated by the arrow. C, cells with gefitinib treatment were stained with anti-p53 and anti-hsp60 antibodies. Localization of proteins was examined by fluorescent microscopy. Nuclei were stained with Hoechst Scale bar = 10 m. Figure S5. Subcellular localization of the truncated GFP-p14 ARF fusion proteins. and cells expressing GFP-p14 ARF (A) or MTS-GFP-p14 ARF () mutants were cultured for 24 h. Subcellular localization of each construct was visualized by fluorescent microscopy. Nuclei were stained with Hoechst Empty indicates empty vector containing GFP or MTS-GFP, respectively. Scale bar = 10 m. Figure S6. Growth suppression of the tumor cells by various anti-tumor peptides. A, sequences of peptides were used in this study. Nine residues of D-arginine (r) were 5

6 fused to all peptides to produce cell penetrating peptides. GPG sequence was used as a spacer., fluorescent microscopy analysis of living cells incubated with the FITC-labeled peptides at 37 C for 14 h. Scale bar = 10 m. C, growth inhibition is indicated by morphology of cells after 24 h of peptide treatment. Scale bar = 20 m. D, and cells were treated with peptide (20 mol/l) for 48 h at 37 C. Cell growth was measured by WST-8 reagent. The ratio of viable cells is shown relative to non-treated cells (pep ). Results are presented as means ± SD (n=3, *p<5). Figure S7. Immunohistochemistry of p14 ARF and p53 in the NSCLC tissues. p14 ARF and p53 expression were detected in NSCLC tissues using anti-p14 ARF and anti-p53 (DO-1) antibodies. NSCLC have gefitinib-sensitive EGFR mutations (A) and gefitinib-resistant EGFR mutations (). Three cases of the NSCLC were used. Scale bar = 50 m. 6

7 Relative cell growth (μmol/l) Relative cell growth HCC827 H (μmol/l) (-) Gefitnib (+) G1 orange SG2M green Phase C, (-), (+) Sub-G1 : 4 % G1/G0 : 44 S : G2/M : Sub-G1 : 7.28 % G1/G0 : S : 16 G2/M : (-) Gefitnib (+), (-), (+) Sub-G1 : 9 % G1/G0 : S : 21 G2/M : 35 Sub-G1 : 4.72 % G1/G0 : 51 S : G2/M : G1 G1/S S, G2, M D CDKN1A mrna level (h) CDKN1A mrna level (h) CDKN2A mrna level 2.5 * (h) CDKN2A mrna level (h) Supplementary Figure S1

8 M (bp) p16 p12 p14 variant2 ARF : p16 INK4a M (bp) p16 p12 p14 variant2 ARF : p16 INK4a HCC827 * * H1975 C p16 INK4a mrna level p16 INK4a mrna level p16 INK4a mrna level HCC827 p16 INK4a mrna level H1975 D E HCC827 H1975 M (kda) (h) p14 ARF 15 p16 INK4a HCC827 H1975 M (kda) (h) p14 ARF p16 INK4a HSP60 p14 ARF p21 Cip1 -Actin p21 Cip1 -Actin Merge (+ Hoechst) Supplementary Figure S2

9 Relative cell growth * Relative cell growth 1.2 * * No drug Cisplatin Etoposide No drug Cisplatin Etoposide No drug Cisplatin Etoposide No drug Cisplatin Etoposide C D M (kda) 210 si Control si EGFR si Control si EGFR EGFR 3.5 * * Actin si Control si EGFR si Control si EGFR Supplementary Figure S3

10 (Exon2) G CDKN2A (Exon2) G H1975 CDKN2A (Exon2) G TGCTCCACGTCGCGGAGCC TGCTCCACGTCGCGGAGCC TGCTCCACGGCGCGTAGCC Cell line Gene Mutation Amino acid p16 INK4a GGC GTC (Exon2) Gly 67 Val p14 ARF CGG CGT (Exon2) Arg 81 Arg (silent) p16 INK4a GGC GTC (Exon2) Gly 67 Val p14 ARF CGG CGT (Exon2) Arg 81 Arg (silent) HCC827 p16 INK4a No mutation wild type p14 ARF No mutation wild type H1975 p16 INK4a GAG TAG (Exon2) Glu 69 Amber (stop) p14 ARF GGA GTA (Exon2) Gly 83 Val G GGCATGAACCAGAGGCCCA C (+) (+) p53 p53 : CGG (Arg 248 ) CAG (Gln) G HSP60 GGCATGAACCAGAGGCCCA Hoechst p53 : CGG (Arg 248 ) CAG (Gln) Supplementary Figure S4

11 GFP GFP + Hoechst GFP GFP + Hoechst empty empty WT WT GFP-p14 ARF ( cells) MTS-GFP-p14 ARF ( cells) empty empty WT WT GFP-p14 ARF ( cells) MTS-GFP-p14 ARF ( cells) Supplementary Figure S5

12 Peptide r9 rrrrrrrrr Sequence FITC-r9-p FITC-r9-p phase p14-1c p p p16-mis r9-gpg-rrflvtlrirracgpprvrvfvvhipr r9-gpg-apaavalvlmll r9-gpg-apaavalvlmllrsqrlgqqplprrpg r9-gpg-fldtlvvlhr p21-s153a r9-gpg-hakrrlif Smac r9-gpg-avpiaqk ( r ; D-Arg ) C D pep (-) r9 p21-s153a 1.2 p14-1c p p16-mis Relative cell growth pep (-) r9 p14-1c p p p16-mis p21-s153a Smac pep (-) r9 p21-s153a 1.2 Relative cell growth pep (-) r9 p14-1c p p p16-mis p21-s153a Smac p14-1c p p16-mis Supplementary Figure S6

13 Ex19 EGFR L858R EGFR L858R EGFR p53 p14 ARF T790M EGFR T790M EGFR D761Y EGFR p53 p14 ARF Supplementary Figure S7