Figure 1 pβcat552 β-cat pgsk3β GSK3β 110 110 pgsk3β/du -cad/ TL 20 µm 20 µm pkt473 pankt pkt473 pankt (days) 0 1 2 3 4 (h) 0 1 3 6 9 12 pβ-cat552/nuclei ensitometricanalysis (Pixel intensity) F ensitometricanalysis (Pixel intensity) 2.0 1.5 1.0 0.5 2.0 1.5 1.0 0.5 20 µm kt308 kt473 1d 2d 3d 4d kt308 kt473 1h 3h 6h 9h 12h
Figure 2 pkt 4 pkt 246 (days) 0 1 2 3 4 pkt 4 pkt 246 (h) 0 1 3 6 9 12
Figure 3 ppk1 PK1 ensitometricanalysis (Pixel intensity) 2 1.5 1 0.5 0 ppk1 14-3-3 η 14-3-3 θ 14-3-3 β 14-3-3 ε 14-3-3 ζ ppk1 ppk-1 PK-1 ontrol θ 14-3-3 eta 14-3-3 zeta η 14-3-3 β ε ζ ensitom: 1 1.1 0. 1.2 1.0 1.1 OT- Flag 20 µm
Figure 4 ensitometricanalysis (Pixel intensity/) ppk1 PK1 J TG5 Raptor Oct- 1. 1 0. 0.5 0. 0 yt In vivo / TNFα Rapt SW480 In vitro +Q yt Q S58/Rapt S58 yt MG132 yt LLN L3 I II TG5 G FLG L3/Nuc K F WT ps58 pan14-3-3 (days) 0 4 IP IgG IP: Flag Input IFN/TNF Raptor ensi: 1 1.12.5 2 L3 S58 1 (days) 0 1 2 3 4 H S58 14-3-3 η ppk1 PK1 14-3-3 ζ Flag Pon Red S58 WT+ 10 µm I pkt473 10 µm Vector 10 µm S58 p62 S58
Figure 5 ensitometricanalysis (Pixel intensity) MSO V02 PRP asp-3 L asp-3 H p-hist3 PN 130 /V02 1cm V02 V02/ SW480 PRP asp-3 p-hist3 130 Hist3 15 SW480 V02 V02/ pkt473 pkt473 MSO V02
Figure 6 I (isease ctivity Index) asp-3/nuclei 12.0 9.0 6.0 3.0 TL kt VIII 0.0 (ays): 0 1 2 3 4 5 6 TL /MSO /kti VIII ktinviii 1cm Length (cm) pkt 308 Red Ponc 9 6 3 0 20µm /kti VIII asp-3 positive cells/100 crypts 4 360 270 180 90 0 pkt473 PRP asp-3 S/kti VIII /kti VIII 130 pβ-cat552 PRP H PRPc L S 110 130 110 F In vivo Nucleus ytosol pkt473 /V02 /V02 Hist3 GPH 17
Figure 7 SW480 +TNFα Vector 14-3-3 η +TNFα +TNFα /GSK23334470 asp-3 PRP ppk1 PK1 14-3-3 η /TNFα Receptor? PI3 K 130 %Piknotic Nuclei 70 60 40 30 20 10 0 SW480 +TNFα PIP3 NS GSK23334470 Oct- leaved asp-3 Membrane SW480 +TNFα GSK23334470 10 µm KT1 pkt1308 ps58 PK1 TOR1 Nucleus utophagosome
Supplementary figure 1 8.0 ay 1 ay 2 ay 3 ay 4 ensitometricanalysis (Pixel intensity) (ays): 0 1 2 3 4 6.0 4.0 2.0 0.0 I (isease ctivity Index) 1 cm induced inflammation decreases cell proliferation and colon length. () ffects of -induced inflammation on cell proliferation markers (PN, phistone3) and disease activity (isease ctivity Index (I)). 57L/6J were treated with 3% dissolved in drinking water for 1-4 days. Relative values for PN and phistone3 obtained from densitometric analysis were normalized to actin. I was analyzed as previously reported (Laukoetteret al, 2007). n=6. () Representative image of colon tissue obtained from control and colitic mice. 57L/6J mice were treated with 3% dissolved in drinking water for 1-4 days. n=6. ar= 1cm.
Supplementary figure 2 L3 I II /TNFα 3h 6h pβ-cat552 β-catenin /TNFα 0 3h 6h /TNFα WT S58 S58 5µm 110 110 pkt473 ps58 F : - + + + ps6 G In vivo Rapam ktinhviii W: 14-3-3 IP, W: Flag H (4 ) -GFP Input IgG WT-Flag S58-Flag S58-Flag 14-3-3 η ppk1 PK1 14-3-3 ζ Flag Ponceau ps58 10µm S58 and ps58 increased in the mucosa of colitic mice and regulate kt activation. (, upper panel) L3I and L3II were analyzed in SW480 cells treated with cytokines. was used as loading control. (, lower panel) cridine orange (green) was used to analyze the presence of autophagosomes. Nuclei blue. ar= 5µm. () ps58 and p were analyzed in colonic cell lysates of 57L/6J mice injected intraperitoneally with /TNFα. was used as loading control. () WT-Flag, 14-3- 3ζWT-Flag and WT-Flag were precipitated from cells cotransfected with a plasmid expressing 14-3- 3ζ-GFP plus WT-Flag, WT-Flag or WT-Flag. Westernblot against pan-14-3-3 and Flag were performed. (), ppk1, PK1, and Flagwere analyzed in SW480 cells expressing increasing concentrations of 14-3-3 ζ S58. Ponceau read was used as loading control. () pβ-cat552, β- catenin,, pkt473, and were analyzedin SW480 cells transfected with 14-3-3 ζwt, 14-3-3 ζs58 and 14-3-3 ζ S58 and treated with (12h). was used as loading control. (F) and ps6 were analyzed in SW480 cellstreated with, plus rapamycin (200 nm) and plus kt InhibVIII (2.12µM). was carried out for 12h. was used as loading control. (G) 14-3-3 ζ, p14-3-3 ζ S58 and 14-3-3σ were analyzed in the mucosa of mice treated with 1-4 days. ensitometric analysis is shown. n=6. (H) The distribution of and ps58 was analyzed in the mucosa of coliticmice by immunofluorescence. and ps58, green. Highly enriched cells are marked by arrow () and arrowhead (ps58). iscontinuous line denotes crypt shape. Nuclei, blue. du= red ar= 10µm.
Supplementary figure 3 asp-3 L asp-3 H PN TUNL/Nuclei 30µm ensitometricanalysis (Pixel intensity/) 3.0 2.0 1.0 0.0 PRP cleav asp-3 cleav phist3 (ays): 0 1 2 3 4 5 Ponceau M30/Nuclei 24h 48h ps58 3% MSO V02 Inflammation enhances apoptosis in Is. () aspase-3, PN and phist3 were analyzed in control and treated mice. 57L/6J were treated with 3% dissolved in drinking water for 4 days. Low xposure (L) and High xposure (H) for caspase-3 are shown. Ponceau red was used as loading control. () poptosis was analyzed in the mucosa of control and treated mice by Tunelstaining (green). 57L/6J were treated with 3% dissolved in drinking water for 1-4 days. Nuclei, blue. ar=30µm. () Graph shows relative densitometric values for PRP and asp3 cleavage in the mucosa of treated animals. 57L6/J mice received 3% in drinking water for 1-5 days. () poptosis was analyzed in SW480 cells treated with 100U/ml of by M30 staining (green). Nuclei, blue. ar=20µm.() and ps58 levels were analyzed in the mucosa of mice treated with /MSO or /V02.57L6/J mice received 3% in drinking water for 4 days. Mice were injected daily with 10 mg/kg of weight of V02 via peritoneum. was used as loading control.
Supplementary figure 4 pkt (Thr308) Surface ottom In vivo Nucleus pkt473/du pkt473 Hist3 pkt473/du PN/Nuclei ktinviii 20µm PRP PN positive cells per crypt 40 30 20 10 0 Vector Vector myr 130 ktaccumulates in the nucleus of Is and regulates cell proliferation during inflammation. () pkt 473( White) was analyzed by immunofluorescence in samples of mice exposed to 4 days. pkt473 is observed in cytosol in proliferating cells green star and nuclear staining is observed in non-proliferating cells green arrow. du, red. Nuclei, lue. ar= 10µm. () pkt 308 (Green) was analyzed by immunofluorescence in samples of mice exposed to 2 and 4 days. is observed at membrane (White arrow) and nucleus. Nuclei, lue. ar=10µm. () pkt 473, and presence were analyzed in nuclear fractions of Is obtained from mice exposed to 4 days. pkt473, and were enriched in nuclear fractions of colitic mice. Histone 3 was used as marker for nuclear fraction. ()ell proliferation was analyzed in the mucosa of control and mice treated with /MSO or /kt inhibitor VIII by PN staining (red). rypts are marked by dotted line. 57L6/J mice received 3% in drinking water for 4 days. Mice were injected daily with 10 mg/kg of weight of kt inhibitor VII via peritoneum. Nuclei, blue. ar=20µm. Quantification is showed in the graph. p<0.05; p<0.001 (), and PRP cleavage were analyzed in SW480 cells expressing myristoylated(.myr) that were exposed to for 36h. was used as loading control.