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Leo Sanders
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Supplementary figures Supplementary Figure 1 A WT -/- Percentage(%).6.4.

1 Supplementary figures Supplementary Figure 1 A WT -/- Percentage(%) Peripheral blood Cell number(1 6 ) MLN Cell number(1 7 ) Spleen Supplementary Figure 1 No baseline value differences in immune cells from Naïve mice Peripheral blood, MLN and spleen were collected from Naïve mice. Peripheral blood cells, MLN cells, and splenocytes were analyzed by flow cytometry after staining with antibodies for CD4,B22, CD11b, CD11c, and Gr-1. n=6 per group. Data represent means ± SEM(t-test).

2 Supplementary Figure 2 A B C IL-8 ** h h 1h 1h IL-1β * h h 1h 1h IL-6 * h h 1h 1h CXCL5 * h h 1h 1h TNFα 3 * 2 1 h h 1h 1h HCT116 shn HCT116 shr p-p t-p p-jnk t-jnk p-erk1/2 t-erk1/2 p-p38 t-p38 HCT116 shn shr TNFα(hrs) E kda HCT116 shn shr 54 TNFα /44 IP: p IP: IgG 42/44 38 Input IL-8 promoter region D ** TNFα + + SN IL-6 ** TNFα + + SN /+1 from the transcription start site of (mus) gene Luc1 Luc2 Luc3-186/ / /-252 F

3 Supplementary Figure 2 Mutual regulation between and NFκB. (A) Total RNA was extracted from colon cancer cell line HCT116 stably knocked down (shr) and control (shn) with or without TNFα stimulation. Expression of NFκB downstream genes were analyzed by RT-PCR. Data represent means ± SEM from three independent experiments; *p <.5; **p <.1(t-test). (B) Alteration of NFκB and MAPK pathways was estimated in HCT116 shn and shr cells. (C) ChIP assays of p recruitment to IL-8 promoter were performed in HCT116 shn and HCT116 shr cells treated with TNFα for one hour. Chromatin was immunoprecipitated with anti-p Ab. Ten percent of the precipitated chromatin (input) was assayed to ensure equal loading. (D) REG γ transcripts were measured before and after 6h TNF treatment with or without pretreatment of NFκB inhibitor in colon ex vivo culture. n=4, each group, Data represent means ± SEM from three independent experiments; *p <.5; **p <.1(t-test). (E) Potential NFĸB binding sequences on promoter was analyzed using TFSEARCH software ( /TFSEARCH.html). (F) ChIP-seq analysis for NFκB/p in primary MEFs treated with LPS for 1 hr or TNF for 3 min. We note a strong peak over the promoter-proximal region of the PSME3/ gene but not neighboring genes.

Supplementary Figure 3 A +/+ vs -/- Name T-test change PKD1/PKCmu(phosphorSer25).25.33217498 c-jun(phospho-ser63).935.319666351 Rac1/cdc42(phospho-Ser71).435.32642388 IκB-ε.829.

858242 B IκBβ -/- WT -/- kda 39 48 45 28 43 C 25 2 15 1 HCT116 shn HCT116 shr *** E GFP HCT116 shn HCT116 shr pegfp- pegfp pegfp - TNFα + TNFα - TNFα + TNFα - TNFα + TNFα 5 chx/hrs 2 4 6 2 4 6 p DAPI

4 Supplementary Figure 3 A +/+ vs -/- Name T-test change PKD1/PKCmu(phosphorSer25) c-jun(phospho-ser63) Rac1/cdc42(phospho-Ser71) IκB-ε Androgen Receptor(Ab -) Trk A(Ab-496) B IκBβ -/- WT -/- kda C HCT116 shn HCT116 shr *** E GFP HCT116 shn HCT116 shr pegfp- pegfp pegfp - TNFα + TNFα - TNFα + TNFα - TNFα + TNFα 5 chx/hrs p DAPI D Input IP: TNFα crel p p5 kda F Nucleus/cytoplasm (fold change) p location ** ** 1 TNFα HCT116 shn pegfp HCT116 shr pegfp HCT116 shn pegfp- 28 Supplementary Figure 3 modulating NFκB signaling via regulation of (A) High-throughput proteomic screen of antibody arrays (FullMoon Biosystems) revealed differential expression of in +/+ and -/- MEF cell. (B) Expression of IκBs in colon epithelial cells isolated from WT and -/- mice following 7 days of DSS treatment. Representative of three successful times.n=7,each group. (C) mrna levels were measured in HCT116 shn and shr cell following CHX treatment. Data represent means ± SEM from three independent experiments; ***p <.1(t-test). (D) protein complexes. HCT116 shn and shr cell were stimulated with/without TNFα and immunoprecipitated (IP) with antibody followed by Western blot as indicated. P and c-rel, but not p5, could be immunoprecipitated by. Moreover, elevated in shr cell contributed to more stable /p/c-rel complex that are resistant to TNFα stimulated degradation. Representative of three experiments. (E and F) HCT116 shn/shr cells transfected with a control vector (pegfp) or (pegfp-) were stimulated with or without TNFα for 1 hour. Cell were then fixed and labeled for GFP and p. Quantitative assessment of relative p distribution (nucleus/cytoplasm) reflects relative NFκB activities in 3 independent experiments. Data represent means ± SEM, **p <.1(t-test).

Supplementary Figure 4 A pg/mg 8, 4, KC ** ** ** pg/mg 2 1 MIP2α ***** ** 1

** ** 2 pg/mg 8 4 TNFα * ** * B C TNFα (1ng/ml) P-p h 1h shn shr dkd shn shr

-dependent regulation of NFκB responsive genes (A) Colon levels of cytokines

Data represent means±sem. *p <.5; **p <.1, ***p <.1(t-test).

stable knock-down) cells. Representative of two experiments.

experimental day 7, total RNA was extracted for analysis of NFkB regulated

5 Supplementary Figure 4 A pg/mg 8, 4, KC ** ** ** pg/mg 2 1 MIP2α ***** ** 1 pg/mg2 CXCL5 * *** *** -/- / -/- pg/mg 2 1 IL-1β *** * ** pg/mg 1 IL-6 *** 3 ** ** 2 pg/mg 8 4 TNFα * ** * B C TNFα (1ng/ml) P-p h 1h shn shr dkd shn shr dkd kda HCT16 Supplementary Figure 4 significantly contributes to -dependent regulation of NFκB responsive genes (A) Colon levels of cytokines were assessed from supernatants of organ culture by ELISA. n=5,each group. Data represent means±sem. *p <.5; **p <.1, ***p <.1(t-test). (B) Compound depletion of and restores NFκB activity in HCT116 dkd ( and stable knock-down) cells. Representative of two experiments. (C) Colon epithelial cells from WT, -/- and -/- / -/- mice were isolated at experimental day 7, total RNA was extracted for analysis of NFkB regulated down-stream genes by real- time RT-PCR; n= 5,each group. Data represent means±sem. *p <.5; **p <.1; n.s.=no significance (t-test).

6 Supplementary Figure 5 A B 2%DSS /days 7 kda WT -/- WT -/- days 7days C * IL-6 * D AOM 2%DSS Weeks Tumor evaluation E F %Tumors WT -/- WT -/- -/- / -/- G H Tumor number/mice %Tumors WT -/- -/- / -/-

7 Supplementary Figure 5 Elevated expression in colitis is associated with colon tumorigenesis in an dependent manner (A) protein level was measured in colon epithelial cells from day or 7 days DSS treated mice by Western blot. (B) Analysis of protein level in paraffin-embedded colon sections of control (day ) or 7 days DSS treated mice by immunohistochemistry. Representative images were shown, and the experiment was successfully repeated four times. Scale bars represent 1 μm. (C) Expression of and inflammatory cytokine IL6 were elevated in colon epithelial cells collected from DSS treated mice, n=6,each group. Data shown are representative of three independent experiments and denotes means ± SEM. *p <.5. (D) Schematic diagram of the experimental design for colitis-associated tumorigenesis. Mice were injected with AOM followed by one round of 2% DSS treatment for 7 days. (E) Diameter of the tumors in WT and -/- mice was measured. n =8,each group. (F) Representative appearances of colon tumors developed in WT, -/-, and / double knockout mice after AOM and DSS induction. n =8,each group. (G) The number of colon tumors in WT, -/-, and / double knockout mice was counted after AOM and DSS induction. n= 11,WT group; n=1, -/- group; n=9,/ double knockout group. **p <.1. (H) Summary of the tumor size in mice with different genotypes.

8 Supplementary Figure 6 A P-p WT -/- kda B sin si TNFα (1ng/ml,h) P-p kda 45 T-p IκBβ Supplementary Figure 6 Compensational upregulation of other IκBs in -/- colon epithelial cells. (A) Expression of /β and p-p in colon epithelial cells isolated from WT and -/- mice following 7 days of DSS treatment. Each lane represents a sample from an individual mouse. Representative data were from three experiments. (B) Silenced with transient sirna in HCT116 cell to detect p-p and.

Supplementary Figure 7 A BMDM REG γ +/+ REG γ -/- B BMDM LPS

REG γ kda 45 39 28 43 Relative mrna level IL-1β TNFα IL-6 IL12p4

REG γ +/+ REG γ -/- kda TNF 2 1h 2h 4h 8h 2 1h 2h 4h 8h p-p IκB ε

vitro and apoptotic responses in DSS-colon between WT and -/-

time and analyzed for NFκB activity by Western blot.

(B) BMDM were treated with LPS(1ng per ml) for h, 6h, and

BMDN were treated with TNFa (2ng per ml) or LPS (1ng per ml) for

(E) No significant differences in apoptotoc cell numbers in DSS

cell were collected from WT and -/- mice after DSS treatment for

9 Supplementary Figure 7 A BMDM REG γ +/+ REG γ -/- B BMDM LPS (1ng/ml,h,6h) LPS 15 1h 2h 4h 15 1h 2h 4h p-p T-P IκB ε p-iκb α REG γ kda Relative mrna level IL-1β TNFα IL-6 IL12p4 IL12p7 MCP1 CXCL1 MIP2α COX2 inos C BMDN D BMDN CXCL1 MIP2α CXCL5 REG γ +/+ REG γ -/- kda TNF 2 1h 2h 4h 8h 2 1h 2h 4h 8h p-p IκB ε 45 p-iκb α 39 Relative mrna level IL-1β TNFα IL-6 43 E Annexin V + PI - cells(%) Annexin V + PI + cells(%) n.s. n.s. Supplementary Figure 7 Comparable immune cell responses in vitro and apoptotic responses in DSS-colon between WT and -/- mice (A) BMDM were treated with LPS (1ng per ml) for indicated time and analyzed for NFκB activity by Western blot. Representative images were from three successful repeats. (B) BMDM were treated with LPS(1ng per ml) for h, 6h, and analyzed for expression of inflammatory genes by Q-PCR. n=6,each group. Data represent means ± SEM from three independent experiments. *p <.5, Student s t-test. (C) BMDN were treated with TNFa (2ng per ml) for indicated time and analyzed for NFκB activity by Western blot. Representative images were from three successful repeats. (D) BMDN were treated with TNFa (2ng per ml) or LPS (1ng per ml) for 6h, and analyzed for expression of inflammatory genes by Q-PCR. n=6,each group. Data represent means ± SEM from three independent experiments. Student s t-test. (E) No significant differences in apoptotoc cell numbers in DSS treated colons between WT and REG γ -/- mice Colon epithelial cell were collected from WT and -/- mice after DSS treatment for 7 days and analyzed by flow cytometry for annexin V and propidium iodide. n=6/each group. Data represent means±sem. n.s.=none significance.

10 Supplementary Figure 8 Uncropped gel images for Figure 4 Figure 4 A Figure 4 B Figure 4 C p-p p-p38 Longer exposure p-erk1/2 t-p t-p38 Shorter exposure t-erk1/2 Figure 4 E p-jnk Figure 4 F t-jnk Figure 4 I P-p

11 Supplementary Figure 9 Uncropped gel images for Figure 5A-G Figure5 A Figure5 B Figure5 C Shorter exposure 25kDa 15kDa Longer exposure Figure5 D Figure5 E Figure5 F LaminA 25kDa 1kDa 7kDa Flag-IκBs (Input) 7kDa 7kDa Flag-IκBs (Pull down) IκBβ Hsp9 1kDa 7kDa GST- (Pull down) Figure5 G p21 25kDa 15kDa GST-IκBs Shorter exposure 1kDa 7kDa Figure5 H Longer exposure 35KD 25KD

12 Supplementary Figure 1 Uncropped gel images from Figure 5H and Figure 6 Figure5 I Figure 6 G 1kDa C-Rel 7kDa p5 p P-p Supplementary Figure 2 B Supplementary Figure 3 B 7kDa P-p P-Jnk 7kDa t-p t-jnk IκBβ P-Erk P-p38 t-erk t-p38

13 Supplementary Figure 11 Supplementary Figure 3 D Supplementary Figure 4 B P-p Supplementary Figure 5 A C-Rel 7kDa P-p p5 Supplementary Figure 6 A Supplementary Figure 6 B 7kDa P-p 7kDa P-p 7kDa t-p IκBβ

14 Supplementary Figure 12 Supplementary Figure 7 A P-P 7 55 Supplementary Figure 7 C 4 35 P- IκB ε P P-P t-p

15 Supplementary Tables Supplementary Table 1 Proteins differentially regulated in +/+ and -/- MEF cells. The Full Moon arrays contain antibodies against nearly 1,3 phospho and total proteins, which involves in more than 3 different regulatory pathways. +/+ vs -/- +/+ vs -/- Name change Name change BCL-2 (Ab-7) PAK1/2/3(PhosphoSer141) Rb (Ab-68) GluR1 (Phospho-Ser863) CREB (Phospho-Ser142) MYPT1(PhosphoThr853) Vinculin (Ab-821) MER/SKY(PhosphoTyr749/Tyr681) PKC pan activation site ASK1 (Phospho-Ser966) ACC1 (Phospho-Ser8) Pyk2 (Ab-881) c-pla2 (Phospho-Ser55) ALK (Phospho-Tyr157) Smad3 (Ab-24) CDK7 (Phospho-Thr17) ATF-1 (Ab-63) CASP9 (Ab-125) Rb (Phospho-Ser795) c-jun (Ab-93) PKCdelta(PhosphoSer645) MKP-1/2 (Phospho-Ser296) c-jun (Phospho-Thr91) FER (Phospho-Tyr42) HDAC4 (Phospho-Ser632) CD227/mucin1 (Phospho-Tyr1243) Caspase3(PhosphoSer15) CDC25C (Phospho-Ser216) IL3R (Phospho-Tyr593) ACC1 (Phospho-Ser79) FER (Phospho-Tyr42) CD32 (FcgammaRIIb) (Ab-292) GSK3 alpha (Ab-21) NFkB-p (Phospho-Ser536) p53 (Phospho-Ser46) SHP-2 (Phospho-Tyr542) CASP2 (Ab-157) merlin (Ab-1) Chk1 (Phospho-Ser317) RelB (Phospho-Ser552) P7S6K (Phospho-Ser411) HDAC8 (Ab-39) DARPP-32(PhosphoThr34) PPAR-b (Phospho-Thr1457) ATPase (Phospho-Ser16) P38 MAPK (Phospho-Tyr182) XIAP (Phospho-Ser87) ITGB4 (Phospho-Tyr151) Cateninbeta(PhosphoSer3) VEGFR2 (Phospho-Tyr1214) AKT1 (Ab-38) PAK1 (Phospho-Ser24) MAP3K7/TAK1 (Ab-439) Caspase 9 (Ab-196) HSP 9-beta (Ab-226) Connexin 43 (Ab-367) HSP27 (Phospho-Ser78) NFkB-p (Phospho-Ser311) WASP (Ab-29) Cyclin E1 (Ab-77) Dok-1 (Phospho-Tyr362) p53 (Phospho-Ser392) MKP-1/2(Phospho-Ser296) NFkB-p (Phospho-Ser276)

16 +/+ vs +/+ vs -/- -/- Name change Name change CoagulationFactorIII(PhosphoSer2 9) Stathmin 1(Phospho-Ser37) Rb (Ab-68) Mst1/Mst2 (Phospho-Thr183) CD4 (Ab-433) EGFR (Phospho-Thr678) FADD (Phospho-Ser194) c-jun (Ab-239) NFkB-p1 (Phospho-Ser872) BCL-2 (Phospho-Thr56) PAK1/2/3 (Phospho-Ser141) PAK2 (Ab-192) Cyclin D3 (Phospho-Thr283) Hsp9cochaperoneCdc37(PhosphoSer13) AKT1 (Phospho-Thr72) MSK1 (Phospho-Thr581) COT (Ab-29) AKT (Phospho-Thr38) Tuberin/TSC2 (Ab-939) CDC25A (Ab-75) FADD (Phospho-Ser194) RSK1/2/3/4 (Ab-221/227/218/232) MEF2A (Ab-312) CDK2 (Phospho-Thr16) claudin 3 (Phospho-Tyr219) Synuclein alpha (Ab-133) P7S6K (Ab-229) KIT (Phospho-Tyr73) JunB (Phospho-Ser79) Shc (Phospho-Tyr349) RelB (Ab-552) ATPase (Ab-16) p21cip1 (Phospho-Thr145) KIT (Phospho-Tyr73) G3BP-1 (Ab-232) MAPKAPK2 (Phospho-Thr334) MEF2D (Phospho-Ser444) Abl1 (Ab-754/735) ATP1A1/Na+K+ ATPase1 (Ab-23) EGFR (Phospho-Thr693) Pim-1 (Ab-39).824 AMPK1 (Ab-174) MARCKS (Phospho-Ser158) Pyk2 (Phospho-Tyr579) Raf1 (Phospho-Ser338) Chk2 (Phospho-Thr68) eef2k (Ab-366) Tau (Ab-25) NFkB-p15/p5 (Ab-932) Trk A (Phospho-Tyr71) ATP-Citrate Lyase (Ab-454) Abl1 (Ab-24) VASP (Ab-157) p53 (Ab-376) ERK3 (Phospho-Ser189) CASP8 (Ab-347) BAD (Ab-112) Tau (Ab-25) Catenin beta (Phospho-Tyr4) PKD1/PKC mu (Phospho-Ser25) E-BP1 (Phospho-Thr45) c-jun (Phospho-Ser63) CDK1/CDC2 (Ab-14) Rac1/cdc42 (Phospho-Ser71) MEF2D (Phospho-Ser444) E-BP1 (Ab-) Androgen Receptor (Ab-) NFkB-p (Ab-281) Trk A (Ab-496) PLC-beta (Phospho-Ser115)

17 Supplementary table2 IκBs N-terminal sequence alignment

18 Supplementary Table 3 Sequences of primers used for Q-PCR Gene Forward (5 3 ) Reverse (5 3 ) IL-8(homo) GCATAAAGACATACTCCAAACC AAAACTTCTCCACAACCCTC IL-6(homo) AATTCGGTACATCCTCGACGG TTGGAAGGTTCAGGTTGTTTTCT IL-1β(homo) AGCTACGAATCTCCGACCAC CGTTATCCCATGTGTCGAAGAA CXCL5(homo) AGCTGCGTTGCGTTTGTTTAC TGGCGAACACTTGCAGATTAC TNFα(homo) CCTCTCTCTAATCAGCCCTCTG GAGGACCTGGGAGTAGATGAG KC(mus) ACTGCACCCAAACCGAAGTC TGGGGACACCTTTTAGCATCTT MIP2α(mus) ATCCAGAGCTTGAGTGTGACGC AAGGCAAACTTTTTGACCGCC MIP2β(mus) CATAGCCACT CTCAAGGATG AGAATGCAGGTCCTTCATCATG CXCL5(mus) GTTCCATCTCGCCATTCATGC GCGGCTATGACTGAGGAAGG IL-1β(mus) GAAATGCCACCTTTTGACAGTG TGGATGCTCTCATCAGGACAG IL-6(mus ) CTGCAAGAGACTTCCATCCAG AGTGGTATAGACAGGTCTGTTGG TNFα(mus ) CCTGTAGCCCACGTCGTAG GGGAGTAGACAAGGTACAACCC CyclinD1(mus) GCGTACCCTGACACCAATCTC CTCCTCTTCGCACTTCTGCTC Cox2(mus) CACCCTGACATAGACAGTGAAAG CTGGGTCACGTTGGATGAGG Survivin(mus) TGGCAGCTGTACCTCAAGAA AGCTGCTCAATTGACTGACG

Supplementary Information

Supplementary Information mediates STAT3 activation at retromer-positive structures to promote colitis and colitis-associated carcinogenesis Zhang et al. a b d e g h Rel. Luc. Act. Rel. mrna Rel. mrna