Supplementary figures

Transcription

1 Relative intensity Relative intensity Relative intensity Supplementary figures a None Caffeine None Caffeine c None Caffeine ISG UBE1L UBCH EFP 1 1 DOX (h) 1 1 CPT (h) 1 1 UV (h) Supplementary Figure 1. Quantification of DNA damage-induced changes in the levels of ISG-conjugating system. Experiments were performed as in Fig. 1 and the and intensities were determined y using a densitometer and normalized y those of -actin. The normalized densities seen at time points were expressed as 1. and the others were as its relative values. Error ar, ± s.d. (n=3).

2 a UOS MCF7 A59 UOS MCF7 A59 DOX: None Caffeine None Caffeine None Caffeine CPT: None Caffeine None Caffeine None Caffeine 1 1 ISG- conjugates ISG- conjugates ISG p-chk1 ISG p-chk1 p-p53 p-p53 p53 p53 13 UBE1L 13 UBE1L UBCH8 UBCH8 EFP EFP -Actin -Actin c UOS MCF7 A59 None Caffeine None Caffeine None Caffeine UV: 1 ISG- conjugates ISG p-chk1 p-p53 13 p53 UBE1L UBCH8 EFP -Actin Supplementary Figure. Effect of caffeine on the expression of ISG- conjugating system in various cancer cells. Doxoruicin (a), camptothecin (), and UV (c) were treated to UOS, MCF7, and A59 cells with and with caffeine, followed y immunolot analysis as in Fig. 1.

3 HeLa HeLa HeLa HCT116 (p53 / ) None Caffeine None Caffeine None Caffeine None Caffeine DOX: CPT: UV: (h) ISG- conjugates ISG UBE1L UBCH8 EFP p-chk1 p53 -Actin Supplementary Figure 3. Effect of caffeine on the expression of ISG- conjugating system in HeLa cells. Experiments were performed as in Supplementary Fig. 1, ut using HeLa cells.

4 a HCT116 p53 / p53 -/- A59 H199 p53 / p53 -/ (h): UV UBE1L (h): UV UBE1L UBCH8 UBCH8 EFP EFP 1 1 ISG- conjugates ISG- conjugates ISG ISG p53 p53 p1 p1 GAPDH GAPDH c p53 / HCT116 d A59 shp53 shp (h): UV UBE1L (h): UV UBE1L UBCH8 EFP UBCH8 EFP 1 ISG- conjugates 1 ISG- conjugates ISG ISG p53 p53 p1 p1 GAPDH GAPDH Supplementary Figure. p53 induces the expression of ISG-conjugating system. After exposure to UV, HCT116 (p53 / and p53 -/- ) cells (a) and A59 (p53 / ) and H199 (p53 -/- ) cells () were incuated for various periods. They were then sujected to immunolot analysis. HCT116 (p53 / ) cells (c) and A59 (p53 / ) cells (d) that had een transfected with or shp53 were exposed to UV. After incuation, they were sujected to immunolot analysis.

5 a c d ISG RE1 RE RE3 RE3* ISRE ISRE* UBE1L RE1 RE RE3 RE1/* ISRE ISRE* UBCH8 RE1 RE RE3 RE RE1* ISRE ISRE* EFP RE1 RE RE3 RE1/* GGACATGTGCTCCGTTCCTGCTACTTGTTT TTCCAAGTTTACTAATTTTGCAAGTTG GGGCATGCCT GGGACGTCCT GAAAGGGAAACCGAAACTGAAG GAAAGGTCCACCTCCACTGAAG GGGCAAGGTGGGAGGATCACTTGAGGCCAGGCAT GGCCAGGCATTCGAGACCAGCCTGGGCAAGATA GAGCAAGCCT GGGACCTGTGGGAGGATCACTTGAGGCACTTCATTCGAGACCAGCCTGGGACCTATA CTTTCACTTTTCTTTTC CTGGAACTTGGATTGGA GAACTAGAACCCAGGTCTCCCTGGGGCTTGTTC CATCATGTTTCACAATACACTTGGAT CAACAAGTCTCATCTTCCCCAGATGCTGCTTGGTG CTGCAAGTACTCTGTCCTTGCAC GAAAGCTAACCCAGGTCTCCCTGGGGAGGTTTC AAAAGAGAAA AACCTATCCA AAACAAGATGTGGGACAGGCCC GGACAGGCCCATGGTGGCTCATGCCT GGACTTGCTCCGAGCGCAAGTTT AAAACCTATGTGGGAACTTCCCATGGTGGCTACGTCCT ISRE ISRE* TTTCGTTT TGGAGTGG Supplementary Figure 5. Sequences of ISREs and putative p53res in the promoter regions of the genes for ISG-conjugating system. Sequences of ISREs and putative p53res in the promoter regions of the ISG (a), UBE1L (), UBCH8 (c), and EFP genes (d) were shown. The asterisks indicate the mutant forms of p53res and ISREs. The mutated nucleotides were indicated with the red letters.

6 a Input a-p53 a-igg p53 RE3 (ISG) p53re (p1) Input a-p53 a-igg p53 RE1/ (UBE1L) p53re (p1) c Input a-p53 a-igg p53 RE1 (UBCH8) p53re (p1) d Input a-p53 a-igg p53 RE1/ (EFP) p53re (p1) Supplementary Figure 6. Binding of p53 to p53res in the promoter regions of the ISG, UBE1L, UBCH8, and EFP genes. HA-p53 was expressed in p53 -/- HCT116 cells. Cell lysates were sujected to ChIP assay using anti-p53 antiody or anti-mouse IgG. Bound DNAs were sujected to PCR using the following proes for: (a) ISG, 5 -TGCGCGATATTTAGGTGTTTCCAGGGTGTTGGGT-3 (forward) and 5 -CTGGTGGCCAAATTTGGCTTCAGTTTCGGTTTCC-3 (reverse); () UBE1L, 5 -CATGGTGGCTCACACCTGTAATCCCAGCAC-3 (forward) and 5 - ATGTTGCCTAGGCTGGTCTCGAATCTCTGG-3 (reverse); (c) UBCH8, 5 - AATGGTCCTGGCAAGCAGATAGGAGTTTCC-3 (forward) and 5 - CCACTGGTGTGCCTTTCATGTCCTTTCACA-3 (reverse); (d) EFP, 5 - TGGGCTGTGTGCCAATGTCCATTCTTAGAC-3 (forward) and 5 - ATACAAGCACGTGCCACCACACCCAGCTAA-3 (reverse); (A-D) p1, 5 - CAGGCTGTGGCTCTGATTGG-3 (forward) and 5 - TTCAGAGTAACAGGCTAAGG-3 (reverse).

7 UV IFNa (h) HA-p53 ISG- conjugates ISG p53 p1 p-stat1 (Tyr1) UBE1L UBCH8 EFP -Actin Supplementary Figure 7. UV and IFNa independently induce ISGylation of cellular proteins. p53 -/- HCT116 cells that had een transfected with an empty vector () or pcdna-ha-p53 () were exposed to UV and then incuated for increasing periods. The cells were also treated with IFNa for increasing periods. Cell lysates were sujected to immunolot analysis.

8 Relative mrna level a 1 HisMax-p (h): UV ISG- conjugates 13 ISG p53 UBE1L UBCH8 EFP GAPDH ISG UBE1L UBCH8 EFP HisMax-p53 UV (h) Supplementary Figure 8. p53 is required for UV-mediated increase in the expression of ISG-conjugating system. (a) H199 cells transfected with an empty vector () or pcdna-hismax-p53 () were exposed to UV followed y incuation for increasing periods. They were then sujected to immunolot analysis. () The cells prepared as in a were sujected to qrt-pcr. The level of mrna for each component of ISG-conjugating system seen without ectopic expression of p53 at time point was expressed as 1. and the others were as its relative values. Error ar, ± s.d. (n=3).

9 Relative intensity Relative intensity a UV UV CHX (h) p53 GAPDH KR GAPDH UV UV Wt KR CHX (h) Supplementary Figure 9. Effect of ISGylation-deficient KR mutation on p53 staility. (a) p53 and KR were expressed in p53 -/- HCT116 cells. After exposure to UV, cells were sujected to incuation with. mg/ml cycloheximide (CHX) for increasing periods followed y immunolot analysis. () Experiments in a were repeated and the and intensities were scanned y using a densitometer and normalized y those of GAPDH. The normalized densities seen at time points were expressed as 1. and the others were as its relative values. Error ar, ± s.d. (n=3).

10 Lysate Lysate IP : a-xpress IP: a-ha a p53 p 1 p p 3 p EFP-inding region Binding EFP E 1 E E 3 E p53-inding region Binding HisMax-p53 HisMax-p 1 HisMax-p HisMax-p 3 HisMax-p HA-EFP EFP p53 p 1 * p p 3 ** p 3 6 HA-EFP HA-E 1 HA-E HA-E 3 HA-E HisMax-p53 p53 EFP E 1 E 3 * E E 3 6 EFP p53 p p 3 p 3 6 p53 EFP E 1 E 3 ** E E Supplementary Figure 1. Identification of the regions for interaction etween p53 and EFP. (a) Deletions of p53 (p 1- p ) were expressed in HEK93T cells with and without HA-EFP. Cell lysates were sujected to immunoprecipitation with anti-xpress antiody followed y immunolot with anti- HA or anti-xpress antiody. They were also directly proed with anti-ha and anti- Xpress antiodies. The asterisk indicates the IgG heavy chain and the doule asterisk shows the IgG light chain. () Deletions of EFP (E 1-E ) were expressed in HEK93T cells with and without HisMax-p53. Cell lysates were sujected to immunoprecipitation with anti-ha antiody followed y immunolot with anti-xpress or anti-ha antiody. They were also directly proed with anti- Xpress and anti-ha antiodies. The asterisk indicates the IgG heavy chain and the doule asterisk shows a nonspecific and.

11 Relative activity of Luc PG13-Luc p1-luc BAX-Luc DOX(h) Mock Wt KR Mock Wt KR Mock Wt KR Supplementary Figure 11. ISGylation promotes p53 transactivity. Experiments were performed as in Fig. 6a, ut in the presence of doxoruicin in place of UV exposure. The luciferase activity seen without any treatment (i.e., time in Mock) was expressed as 1. andthe others were as its relative values.

12 a HisMax-p53 HisMax-KR UV (h) p53 1 MDM 5 5 BAX p1 5 5 shisg shefp Flag-ISG Myc-EFP UV p53 Flag-ISG Flag-ISG GAPDH ISG Myc-EFP EFP MDM BAX p1 GAPDH Supplementary Figure 1. Immunolot analysis of cells used for determining p53 transactivity. Cells prepared as in Fig. 6a (a) and 6 () were sujected to immunolot analysis.

13 Relative intensity a shisg shefp UV p53 p-chk p53 p-chk1 ISG 1 EFP GAPDH shisg shefp UV Supplementary Figure 13. Effect of knockdown of ISG or EFP on UVinduced Chk1 phosphorylation. (a) HCT116 (p53 / ) cells transfected with, shisg, or shefp were exposed UV. They were then sujected to immunolot analysis. () Experiments were performed as in a and the and intensities were determined y using a densitometer and normalized y those of GAPDH. The normalized densities for p53 and p-chk1 seen in the presence of and UV were expressed as 1. and the others were as its relative values. Error ar, ± s.d. (n=3).

14 % Input % Input % Input a p1 MDM BAX ISG HisMax-p53 Es/Flag-ISG Flag-UBP a-igg a-p53 a-igg a-p53 a-igg a-p53 a-igg a-p53 p1 MDM BAX ISG HisMax-p53 HisMax-KR Es/Flag-ISG a-igg a-p53 a-igg a-p53 a-igg a-p53 a-igg a-p53 c p1 MDM BAX ISG UV Flag-ISG shisg shisg shisg shisg a-igg a-p53 a-igg a-p53 a-igg a-p53 a-igg a-p53 Supplementary Figure 1. Recruitment of p53 to p53res of the p1, MDM, BAX, and ISG genes. (a-c) Experiments were performed as in Fig. 6c-e, respectively, ut the ound DNAs were sujected to qpcr using the proes for p53res of CDKN1, MDM, BAX, and ISG. Error ar, ± s.d. (n=).

15 Relative intensity Lysate a HisMax-p53 HisMax-KR UV Ac-p53 p-p53 p53 GAPDH Ac-p53 p-p53 HisMax-p53 HisMax-KR UV c IP: a-p UV (h) p53-isg p53 p-p53 Ac-p53 GAPDH Supplementary Figure. ISGylation of p53 promotes its acetylation and phosphorylation. (a) HisMax-tagged p53 or KR was expressed H199 cells. After exposure to UV, cells were sujected to incuation for h followed y immunolot analysis. () Experiments were performed as in a and the and intensities were determined y using a densitometer and normalized y those of GAPDH. The normalized densities for Ac-p53 and p-p53 seen with HisMax-p53 and without UV treatment were expressed as 1. and the others were as its relative values. Error ar, ± s.d. (n=3). (c) After UV treatment, p53 / HCT116 cells were incuated for increasing periods. Cell lysates were sujected to immunoprecipitation with anti-p53 antiody followed y immunolot with anti- ISG antiody. They were also directly sujected to immunolot analysis.

16 a shisg HisMax-p53 HisMax-KR DOX(h) p53 ISG GAPDH shisg Mock HisMax-p53 HisMax-KR DOX p53 ISG GAPDH c p53 / p53 / shisg Flag-ISG DOX p53 5 Flag-ISG ISG GAPDH Supplementary Figure 16. Immunolot analysis of cells used for determining cell growth and colony formation. Cells prepared as in Fig. 7a (a), 7 (), and 7d (c) were sujected to immunolot analysis.

17 % Apoptotic cells a.3 mm.6 mm (DOX) Mock p53 KR 5 3 DOX (mm) Mock p53 KR Supplementary Figure. Promotion of doxoruicin-induced apoptosis y p53 ISGylation. (a) p53 -/- HCT116 cells expressing HisMax-tagged p53, its KR mutant, or an empty vector (Mock) were treated with different concentrations of doxoruicin. They were then sujected to TUNEL assay. () Experiments were performed as in a and the numer of TUNEL-positive cells were counted and expressed as percentage of total numer of cells. Error ar, ± s.d. (n=3).

18 a shisg DOX shisg DOX p53 p-p53 1 MDM BAX p1 ISG 13 UBE1L UBCH8 EFP GAPDH Mock p53 KR DOX HisMax-p53 HisMax-KR DOX p53 p-p53 1 MDM BAX p1 ISG 13 UBE1L UBCH8 EFP GAPDH Supplementary Figure 18. ISGylation of p53 suppresses tumor growth. p53 / HCT116 cells expressing or shisg (a) and p53 -/- HCT116 cells expressing wild-type p53 or its ISGylation-deficient KR mutant () were injected to BALB/c nude mice. Mock in indicates mice injected with cells that had een transfected with an empty vector. After treatment with PBS or doxoruicin, tumors were dissected out and photographed (left panels). Lysates were prepared from the tumor tissues, and sujected to immunolot analysis (right panels). Bar, mm.

19 Relative intensity a shisg shisg DOX (h) 1. p53 p53 1. p p-p53 1. p-p53 p-p MDM.5.5 BAX p1 ISG MDM BAX MDM BAX UBE1L UBCH8.5 1 p1 p1.5 1 EFP UBP GAPDH ISG UBE1L ISG UBE1L UBCH8 UBCH EFP EFP UBP UBP DOX (h).5 Supplementary Figure 19. UBP represses p53-induced expression of ISG-conjugating system. (a) p53 / HCT116 cells that staly express or shisg were treated with doxoruicin for increasing periods. They were then sujected to immunolot analysis. () Experiments were performed as in a and the and intensities were determined y using a densitometer and normalized y those of GAPDH. The normalized peak densities of the indicated proteins seen with were expressed as 1. and the others were as its relative values. Error ar, ± s.d. (n=3).

20 Fig. 1 Fig. 1a Fig. 1c Fig. 1 1 Supplementary Figure. Full lots.

21 Fig. d Fig. e Fig Fig. 5c Fig. 5d

22 Fig. 5e Fig. 5f Fig. 6e Fig. 6c Fig. 6d

23 S. Fig. a S. Fig. S. Fig. c

24 S. Fig. 3 S. Fig. a S. Fig. c S. Fig. 8a

25 S. Fig. 9a S. Fig. 13a S. Fig. a S. Fig. c 1 1

26 S. Fig. 16a S. Fig. 16 S. Fig. 18a S. Fig. 19a