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Oligo sequence for shRNA cloning TurboGFP shRNA upper strand CCGGCGTGATCTTCACCGACAAGATCTCGAGATCTTGTC GGTGAAGATCACGTTTTT TurboGFP shRNA bottom strand AATTAAAAACGTGATCTTCACCGACAAGATCTCGAGATC.

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Presentation on theme: "Oligo sequence for shRNA cloning TurboGFP shRNA upper strand CCGGCGTGATCTTCACCGACAAGATCTCGAGATCTTGTC GGTGAAGATCACGTTTTT TurboGFP shRNA bottom strand AATTAAAAACGTGATCTTCACCGACAAGATCTCGAGATC."— Presentation transcript:

1 Oligo sequence for shRNA cloning TurboGFP shRNA upper strand CCGGCGTGATCTTCACCGACAAGATCTCGAGATCTTGTC GGTGAAGATCACGTTTTT TurboGFP shRNA bottom strand AATTAAAAACGTGATCTTCACCGACAAGATCTCGAGATC TTGTCGGTGAAGATCACG Human PHF2 shRNA-I upper strand GGCCGGGAACGGGAAACTACTCCTTTACTCGAGTAAAG GAGTAGTTTCCCGTTCTTTTTG Human PHF2 shRNA-I bottom strand CTAGCAAAAAGAACGGGAAACTACTCCTTTACTCGAGTA AAGGAGTAGTTTCCCGTTCCC Human PHF2 shRNA-II upper strand CCGGGGCAAGCGCCTGACGTCAAGCCTCGAGGCTTGAC GTCAGGCGCTTGCCTTTTT Human PHF2 shRNA-II bottom strand AATTAAAAAGGCAAGCGCCTGACGTCAAGCCTCGAGGC TTGACGTCAGGCGCTTGCC Primer sequence for Sub-cloning A PHF2(h)_1-186_FORCCGGATCCATGGCGACGGTGCCCGTGTA PHF2(h)_1-186_REVGGGAATTCTTATTGCGGTTGGTGCTGTAGTA PHF2(h)_174-607_FORCCGGATCCCTGAAGGAGTTTGTGGACTAT PHF2(h)_174-607_REVGGGAATTCTTATTCTTGTACTTCCACTTGGC PHF2(h)_594-820_FORCCGGATCCCAAACCAAGAGCAAGTCAGAG PHF2(h)_594-820_REVGGGAATTCTTAGGCCTGGCCAGCTCCCCACG PHF2(h)_808-1097_FORCCGGATCCGACTCCTGCCTGCAGACCAC PHF2(h)_808-1097_REVGGGAATTCTTAAAGGAGTAGTTTCCCGTT p53(h)_D1-90_FORTGGCCCCTGTCATCTTCTGT p53(h)_D1-90_REVGAATTCTGCGTAATCCGGTA p53(h)_D90-292_FORAAGAAAGGGGAGCCTCACCA p53(h)_D90-293_REVGGAGGGGGCTGGTGCAGGGG p53(h)_D1-292_FORAAGAAAGGGGAGCCTCACCA p53(h)_D1-292_REVGAATTCTGCGTAATCCGGTA Supplemental Table 1 A Restriction sites are underlined

2 primer sequence for RT-PCR p21(h)_RT01 -ForAGCGATGGAACTTCGACTTTG p21(h)_ RT01-RevCGAAGTCACCCTCCAGTGGT mdm2(h)_RT01-ForCCGGATCTTGATGCTGGTGT mdm2(h)_RT01-RevCTGATCCAACCAATCACCTGAAT Bax(h)_RT01_ForGTCTTTTTCCGAGTGGCAGC Bax(h)_RT01_RevCCAGTTGAAGTTGCCGTCAGA primer sequence for ChIP p21(h)_P1GGCTGGTGGCTATTTTGTCC p21(h)_P2TCCCCTTCCTCCCTGAAAAC mdm2(h)_P1’AAACCATGCATTTTCCCAGC mdm2(h)_P2’CAGGTCTACCCTCCAATCGC p21(h)_NS1 CAGTGTGGCCAAAGGATCTGACGA p21(h)_NS1 TTGGGGCTGCCTATGTAGTGAA mdm2(h)_NS1’ TCTTGACTTTCATGCAAGTCAAACGG mdm2(h)_NS2’ GAAAGGAAAATATAAGAGGACTG Gapdh(h)_Fwd ATCGGGCCAATCTCAGTCCCTTCCC Gapdh(h)_Rev GCCTGGTTCAACTGGGCACGCACCGA Supplemental Table 1 cont. Supplemental table 1. The oligonucleotide sequences used for cloning of shRNA viral vectors, primers for subcloning of plasmid vectors, and primers for RT-qPCR and Chip-PCR are listed.

3 p53 functionality and p21 vs PHF2 levels in each tumor Supplemental Table 2 Colon cancers with positive p53 function Patient No. PHF2 levelp21 level SexageTP53 mutationp53 activitynormaltumornormaltumor 4F66WT1001.29.51.23.9 6F64WT1000.5140.61.7 7M56WT100104.30.82.1 9M64WT1009.62.51.61.3 10M71A78S (active)74.945.63.61.62.6 14M57Q104L (active)524.680.74.4 15F41Q104L (active)522.16.20.75.4 16M62WT10017.934.3 17M60Q104L (active)521.26.80.31.4 19M79WT1004.6110.83.4 20M77WT1002.61613.1 23M66S106R (active)50.20.6200.62.2 26M74WT1002.73.70.51 27M64WT10055.51.41.5 28F67WT100119.81.52.3

4 p53 functionality and p21 vs PHF2 levels in each tumor Supplemental Table 2 cont. Colon cancers with negative p53 function Patient No. PHF2 levelp21 level SexageTP53 mutationp53 activitynormaltumornormaltumor 2F64R273C (inactive)0.91.3130.71.5 3M75R248Q (inactive)03.9161.52.2 5F55R175H (inactive)12.45.92.81.12.3 8F58Y234C (inactive)2.12.85.41.12 11M68insertion after S106n/a3.380.61.9 12M62R342STOPn/a1.58.20.30.5 13F61V143E (inactive)9.135.79.50.62.1 18M56insertion after S106N/A1.43.50.20.8 21F70P151H (inactive)10.70.83.90.92.7 22F62F54I, C141Y (inactive)71.7, 9.80.79.20.32.1 24F62R248Q (inactive)0136.21.92.4 25F57R337P (inactive)4.21.29.90.91.5 29M62Y205N (inactive)9.34.6121.63.5 30F65R337P (inactive)4.24.61.722.4 Supplemental table 2. The p53 cDNA was sequenced in 30 colorectal cancer tissues. According to functional status of p53, the cancer tissues are divided into two groups, active p53 and mutant p53. PHF2 and p21 levels shown in Figure 6a are listed together.

5 Case No DiagnosisAGESEXT_stageN_stageM_stageDIFF 1Adenocarcinoma62FpT3pN1.Moderately 2Adenocarcinoma56MpT3pN1.Moderately 3Adenocarcinoma64FpT4bpN2.Moderately 4Adenocarcinoma58FpT3pN0.Moderately 5Adenocarcinoma68MpT3pN1.Moderately 6Adenocarcinoma79MpT3pN0.Poorly 7Adenocarcinoma62MpT3pN1b.Moderately 8Adenocarcinoma61FpT3pN2.Moderately 9Adenocarcinoma64FpT3pN0.Moderately 10Adenocarcinoma57FpT3pN0.Moderately 11Adenocarcinoma60MpT4bpN1.Well 12Adenocarcinoma57MpT2pN0.Moderately 13Adenocarcinoma71MpT3pN1.Moderately 14Adenocarcinoma62MpT3pN1.Well 15Adenocarcinoma62MpT3pN0.Moderately 16Adenocarcinoma41FpT3pN0.Moderately 17Adenocarcinoma64MpT3pN0.Well 18Adenocarcinoma75MpT3pN0.Moderately 19Adenocarcinoma66FpT3pN2.Moderately 20Adenocarcinoma62FpT3pN0.Moderately 21Adenocarcinoma70FpT4bpN0.Moderately 22Adenocarcinoma77MpT3pN1.Moderately 23Adenocarcinoma55FpT3pN1.Moderately 24Adenocarcinoma64MpT3pN0.Moderately 25Adenocarcinoma74MpT3pN0.Well 26Adenocarcinoma67FpT3pN0.Moderately 27Adenocarcinoma62MpT3pN1.Moderately 28Adenocarcinoma66MpT3pN0.Moderately 29Adenocarcinoma65FpT4bpN1.Moderately 30Adenocarcinoma56MpT3pN2.Moderately Supplemental Table 3 Patient Demographics for colon cancer tissue array

6 Case No. DiagnosisAgeSexT stageN stageDiff 1adenocarcinoma40MpT4apN3a poorly differentiated 2adenocarcinoma43FpT4apN0 poorly differentiated 3adenocarcinoma61FpT3pN3a moderately differentiated 4signet ring cell carcinoma45FpT3pN3a 5adenocarcinoma67MpT3pN1 poorly differentiated 6adenocarcinoma53FpT3pN3a poorly differentiated 7adenocarcinoma67MpT4apN3b moderately differentiated 8adenocarcinoma72MpT3pN3a poorly differentiated 9mucinous adenocarcinoma61MpT1bpN2 10adenocarcinoma65MpT4apN0 poorly differentiated 11adenocarcinoma70MpT4apN2 moderately differentiated 12adenocarcinoma62MpT3pN2 well differentiated 13signet ring cell carcinoma62FpT3pN2 14papillary adenocarcinoma52MpT2pN0 15adenocarcinoma60MpT3pN3a poorly differentiated 16adenocarcinoma66MpT4apN3b poorly differentiated 17signet ring cell carcinoma41MpT3pN3a 18adenocarcinoma60FpT3pN3a poorly differentiated 19adenocarcinoma59MpT4apN3b poorly differentiated 20adenocarcinoma62MpT3pN3b poorly differentiated 21adenocarcinoma49MpT4bpN3b poorly differentiated 22mucinous adenocarcinoma64MpT3pN1 23undifferentiated carcinoma55MpT4apN3b 24adenocarcinoma56FpT3pN2 poorly differentiated 25adenocarcinoma48MpT3pN2 poorly differentiated 26mucinous adenocarcinoma65FpT4apN3a 27adenocarcinoma63MpT3pN2 well differentiated 28adenocarcinoma60MpT3pN0 well differentiated 29signet ring cell carcinoma42MpT3pN1 30mucinous adenocarcinoma55MpT30pN3a Supplemental Table 3 cont. Patient Demographics for stomach cancer tissue array

7 31adenocarcinoma58MpT3pN3b moderately differentiated 32adenocarcinoma72FpT1bpN2 moderately differentiated 33adenocarcinoma53MpT3pN0 poorly differentiated 34adenocarcinoma46MpT4bpN0 poorly differentiated 35adenocarcinoma64MpT2pN1 moderately differentiated 36adenocarcinoma65MpT2pN1 poorly differentiated 37adenocarcinoma45FpT2pN1 poorly differentiated 38adenocarcinoma58FpT3pN0 well differentiated 39adenocarcinoma68FpT3pN3a poorly differentiated 40adenocarcinoma66MpT4apN3a poorly differentiated 41adenocarcinoma63MpT3pN0 poorly differentiated 42adenocarcinoma52FpT3pN2 moderately differentiated 43adenocarcinoma35FpT1apN0 poorly differentiated 44adenocarcinoma44FpT1bpN0 well differentiated 45adenocarcinoma55MpT1apN0 well differentiated 46signet ring cell carcinoma40MpT1apN0 47adenocarcinoma66MpT1bpN0 moderately differentiated 48adenocarcinoma65FpT1bpN0 moderately differentiated 49adenocarcinoma62FpT1bpN0 poorly differentiated 50adenocarcinoma60FpT1bpN0 moderately differentiated 51adenocarcinoma70FpT1bpN1 moderately differentiated 52adenocarcinoma52MpT1bpN0 well differentiated 53signet ring cell carcinoma40MpT1bpN1 54signet ring cell carcinoma34FpT1apN1 55adenocarcinoma60MpT1bpN0 well differentiated 56adenocarcinoma68MpT1bpN1 well differentiated 57adenocarcinoma52MpT2pN0 well differentiated 58signet ring cell carcinoma62MpT1apN0 59adenocarcinoma60MpT2pN0 well differentiated Supplemental Table 3 cont. Patient Demographics for stomach cancer tissue array Supplemental table 3. Demographic information on patients, stages and differentiation degree in colon and stomach cancer arrays used for Figure 6e-h is listed.

8 C. PARP C. Casp 3 C. Casp 9 Tubulin C. PARP C. Casp 3 C. Casp 9 Tubulin HepG2 Hep3B DOX PBSOxa5-FU Sh-Con+-+-+-+- Sh-PHF2-I-+-+-+-+ DOX PBSOxa5-FU Sh-Con+-+-+-+- Sh-PHF2-I-+-+-+-+ C. PARP C. Casp 3 C. Casp 9 Tubulin DOX PBSOxa5-FU Sh-Con+-+-+-+- Sh-PHF2-I-+-+-+-+ C. PARP C. Casp 3 C. Casp 9 Tubulin DOX PBSOxa5-FU Sh-Con+-+-+-+- Sh-PHF2-I-+-+-+-+ KATO IIIMKN-74 C. PARP C. Casp 3 C. Casp 9 Tubulin DOX PBSOxa5-FU Sh-Con+-+-+-+- Sh-PHF2-I-+-+-+-+ C. PARP C. Casp 3 C. Casp 9 Tubulin DOX PBSOxa5-FU Sh-Con+-+-+-+- Sh-PHF2-I-+-+-+-+ MDA-MB231 MCF7 Supplemental Fig. 1 Figure S1. Indicated stable cells were treated with PBS, 12.5 mM oxaliplatin, 50 mM 5- FU or 0.5 mM doxorubicin for 16 hours and apoptotic markers were analyzed by Western blotting using indicated antibodies.

9 Total H3 H3K4-Me2 H3K9-Me2 H3K27-Me2 H3K36-Me2 H3K72-Me2 Sh-Con +-- Sh-PHF2-I -+- Sh-PHF2-II --+ Supplemental Fig. 2 Figure S2. Histone methylation status in PHF2 knock down cell lines was measured. Protein extract from HCT116 stable cells underwent western blotting with methyl-specific antibodies..

10 Sh-Con Sh-PHF2 OxaPBS Supplemental Fig. 3 Figure S3. Xenograft tumor sizes after PBS or Oxaliplatin (Oxa) treatment.

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