Supplementary Information

Transcription

1 Supplementary Information Supplementary Fig. 1 Morphology of NP69 and CNE1 cells. Phase contrast images of normal nasopharyngeal epithelial NP69 cells (left) and the well differentiated NPC CNE1 cells (right). Scale bars: 50 m. 1

2 Supplementary Fig. 2 Over-expression of IKK induces differentiation and G1 phase arrest in NPC cells. (a) Expression of indicated proteins was analyzed by western-blot in HONE1 and SUNE1 cells. (b and c) Vector (pbabe) or pbabe-ikk over-expressed CNE2 cells were subjected to flow cytometry (b) and western-blot (c) analysis. 2

3 Supplementary Fig. 3 Immunohistochemical analysis of cell differentiation markers in mouse tumors. Immunohistochemical analysis of tumors from mice injected with CNE2-IKK cells or CNE2-vector control cells with indicated antibodies. Consecutive sections from the representative cases are shown. Scale bars: 100 m. 3

4 Supplementary Fig. 4 IKK is found to be frequently silenced by methylation in poorly differentiated NPC cell lines and tissues. (a and b) MSP analysis of NPC cell lines and primary NPC samples. NP69, immortalized normal human nasopharyngeal epithelial cell line; HK-1 and CNE1, well differentiated NPC cell lines; CNE2, HONE1 and SUNE1, poorly differentiated NPC cell lines; Non-Ca: non-cancer nasopharyngeal mucositis tissues. M: methylated, U: unmethylated. (c) MSP of siezh2 treated-cne2 or control cells. M: methylated, U: unmethylated. 4

5 Supplementary Fig. 5 Suppression of EZH2 induces IKK expression and differentiation in HONE1 and SUNE1 cells. HONE1 and SUNE1 cells were transfected with siezh2-1, siezh2-2 or si-control (scramble) and subjected to western-blot analysis. 5

6 Supplementary Fig. 6 RA induces differentiation and inhibits proliferation in NPC cells. (a) HONE1 and SUNE1 cells were treated with RA (0, 10, 20 and 30 M) for 48 h and subjected to western-blot analysis with indicated proteins. (b and c) Proliferation of CNE2 cells treated with RA or control (DMSO) was analyzed by MTT (n=6), ***P<0.001, 2-tailed Student s t tests (b), and colony formation assay (n=3), ***P<0.001, 2-tailed Student s t tests (c). Error bars represent mean ± SD. 6

7 Supplementary Fig. 7 RA decreases tumorigenicity, increases cell differentiation markers in vivo. (a and b) Mice were injected with HONE1 and SUNE1 cells subcutaneously ( cells/mouse, n=5-8), *P<0.05, 2-tailed Student s t tests. Ten days after injection, the mice were divided into RA and control groups which were treated with RA (20 mg kg -1 (2d) -1 ) or water respectively. Tumor volumes were measured as described in Methods. (c) SUNE1 cells were cultured with RA (30 M) or control (DMSO) for 96 h and injected subcutaneously ( cells/mouse, n=5). Tumor volumes were measured as described in Methods. (d) Immunohistochemisty of tumors from mice injected with RA-treated CNE2 cells or control cells with indicated antibodies. Consecutive sections from the representative cases are shown. Error bars represent mean ± SD. Scale bars: 100 m. 7

8 Supplementary Fig. 8 IKK is required for RA-induced differentiation in CNE2 cells. (a-c) CNE2 cells transfected with si-control (scramble) or siikk were cultured with or without RA (30 M) for 48 h, and subjected to western-blot assay (a), phase contrast imaging (b) and colony formation assay (n=3), **P<0.01, 2-tailed Student s t tests (c). Scale bars: 50 m. Error bars represent mean ± SD. 8

9 Supplementary Fig. 9 Expression of EZH2 in human primary NPC samples. EZH2 level was analyzed by western-blot in human primary NPC and paired normal tissues. N: normal tissues; T: tumors. 9

10 Supplementary Fig. 10 Uncropped scans of the most important blots used in the manuscript. 10

11 Supplementary Table 1 Tumorigenicity of CNE2-IKK, CNE2 and CNE1 cells. a Days after injection (d) Vector 0/9 0/9 1/9 1/9 1/9 4/9 8/9 9/9 9/9 IKK 0/9 0/9 0/9 0/9 0/9 0/9 0/9 0/9 2/9 b Cell injection CNE2 6/6 6/6 6/6 6/6 CNE1 0/6 0/6 0/6 2/6 (a) Mice were injected with vector (pbabe) or pbabe-ikk over-expressed CNE2 cells subcutaneously ( cells/mouse). (b) Mice were injected with CNE2 or CNE1 cells subcutaneously at indicated cell number, and observed for ~90 days. 11

12 Supplementary Table 2 Patient characteristics. Characteristic No. of patients (%) Age (ys) Median 46 Range Gender Male 75 (78.1) Female 21 (21.9) Differentiation Non-Ca 9 (9.4) Differentiated 13 (13.5) Undifferentiated 74 (77.1) Non-Ca: nasopharyngeal mucositis tissues. 12

13 Supplementary Table 3 Primers used in qpcr assay. Name Forward 5 3 Reverse 5 3 ATF2 AATTGAGGAGCCTTCTGTTGTAG CATCACTGGTAGTAGACTCTGGG CHUK ATGAAGAAGTTGAACCATGCCA CCTCCAGAACAGTATTCCATTGC ETS2 CCCCTGTGGCTAACAGTTACA AGGTAGCTTTTAAGGCTTGACTC HES1 TCAACACGACACCGGATAAAC GCCGCGAGCTATCTTTCTTCA KLF4 CCCACATGAAGCGACTTCCC CAGGTCCAGGAGATCGTTGAA MAPK1 TACACCAACCTCTCGTACATCG CATGTCTGAAGCGCAGTAAGATT MAPK13 TGAGCCGACCCTTTCAGTC AGCCCAATGACGTTCTCATGC MAPK6 TGTCAAACATGCTCTACGTGAAA TCGTCTGTTAATTGGCTTCCAC NCOA6 AAAACGTGCCCAATTTGTTACAC GAGAATCCCTAAATCCCGAAGC NFATC4 CTTCTCCGATGCCTCTGACG CGGGGCTTGGACCATACAG NOTCH2 CAACCGCAATGGAGGCTATG GCGAAGGCACAATCATCAATGTT RXRA ATGGACACCAAACATTTCCTGC GGGAGCTGATGACCGAGAAAG SMAD4 CTCATGTGATCTATGCCCGTC AGGTGATACAACTCGTTCGTAGT GAPDH GGAGCGAGATCCCTCCAAAAT GGCTGTTGTCATACTTCTCATGG ACTB GTCTGCCTTGGTAGTGGATAATG TCGAGGACGCCCTATCATGG 13