Figure 3A Self - ribosylation of SIRT6 using biotin - labeled NAD + . Recombinant SIRT6 was incubated with NAD + conjugated with a biotin residue and then run on an SDS - PAGE . Each allele was assessed relative to its 0 hr time point and normalized to SIRT6 total protein loading controls . n = 3 TR ; error bars = SD . Students t - test , two tailed . Asterisk indicate p < 0 . 05 . Black asterisks indicate significance over 0 hr control . Red asterisks indicated significance over time - matched WT SIRT6 . B Self - ribosylation of SIRT6 with titration of NAD + . mADPr - specific antibody was used to detect ribosylated wild type SIRT6 and centSIRT6 proteins . Plot was fit the Michaelis - Menten best - fit with GraphPad software . Km NAD was 142 + 29 for centSIRT6 and 106 + 45 for wild type SIRT6 and maximal signal was ~ 2x greater for centSIRT6 compared to the wild type SIRT6 . Two separate batches of recombinant protein of each SIRT6 allele were prepared and assayed . n = 3 TR ; error bars = SD . C Activation of PARP1 by SIRT6 variants . SIRT6 protein was incubated with human PARP1 protein and then analyzed by immunoblotting with poly - ADPr antibody . Poly - ADPr activity of PARP1 results in a wide range of product size . Activity was assessed by quantifying poly - ADPr signal in whole lanes for each sample . n = 3 TR ; error bars = SD . Two - way ANOVA . Asterisk indicate p < 0 . 05 . Black asterisks indicate significance over HPRT control . Red asterisks indicated significance over wild type SIRT6 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3A Self-ribosylation of SIRT6 using biotin-labeled NAD+. Recombinant SIRT6 was incubated with NAD+ conjugated with a biotin residue and then run on an SDS-PAGE. Each allele was assessed relative to its 0 hr time point and normalized to SIRT6 total protein loading controls. n=3 TR; error bars=SD. Students t-test, two tailed. Asterisk indicate p<0.05. Black asterisks indicate significance over 0 hr control. Red asterisks indicated significance over time-matched WT SIRT6.B Self-ribosylation of SIRT6 with titration of NAD+. mADPr-specific antibody was used to detect ribosylated wild type SIRT6 and centSIRT6 proteins. Plot was fit the Michaelis-Menten best-fit with GraphPad software. Km NAD was 142+29 for centSIRT6 and 106+45 for wild type SIRT6 and maximal signal was ~2x greater for centSIRT6 compared to the wild type SIRT6. Two separate batches of recombinant protein of each SIRT6 allele were prepared and assayed. n=3 TR; error bars=SD.C Activation of PARP1 by SIRT6 variants. SIRT6 protein was incubated with human PARP1 protein and then analyzed by immunoblotting with poly-ADPr antibody. Poly-ADPr activity of PARP1 results in a wide range of product size. Activity was assessed by quantifying poly-ADPr signal in whole lanes for each sample. n=3 TR; error bars=SD. Two-way ANOVA. Asterisk indicate p<0.05. Black asterisks indicate significance over HPRT control. Red asterisks indicated significance over wild type SIRT6.


Figure 4A qRT - PCR analysis of LINE1 expression in cumate - inducible SIRT6 fibroblasts . Primers assessed both 5 ' ( ORF1 ) and 3 ' ( ORF2 ) LINE1 sequences from the L1HS family of evolutionarily active LINE1 retrotransposons . Assessment of both regions was conducted to mitigate contributions from partial insertion sequences in coding genes . Asterisk indicate p < 0 . 05 . n = 3 TR ; error bars = SD . Students t - test , two tailed . B LINE1 EGFP retrotransposition assay . Three independent transfections with LINE1 EGFP reporter plasmid were conducted and quantified by flow cytometry . Asterisk indicate p < 0 . 05 . Red asterisk indicated significance over SIRT6 KO control . Black asterisk indicate significance over WT SIRT6 . n = 3 BR ; error bars = SD . Two - way ANOVA . C In vitro KAP1 ribosylation by SIRT6 . Asterisk indicate p < 0 . 05 . Red asterisk indicate significance over 0 hr , black asterisk indicate significance over compared to WT SIRT6 . n = 3 BR ; error bars = SD . Students t - test , two tailed . D , E Stimulation of NHEJ and HR by SIRT6 variants . Reporter cell lines were co - transfected with SIRT6 - expressing plasmid , I - Sce1 plasmid , and DsRed transfection control . After 72 hr recovery , reactivation of the GFP reporter was measured by flow cytometry . Stimulation of NHEJ or HR was calculated as radio of GFP + / DsRed + positive cells . Blots demonstrate equivalent SIRT6 abundance in transfected cells . Asterisk indicate p < 0 . 05 Black asterisk indicate significance over control , red asterisk indicate significance over WT SIRT6 . n = 3 BR ; error bars = SD . Two - way ANOVAF Basal γH2AX foci in cumate - inducible SIRT6 fibroblasts . Foci were scored in at least 80 cells per condition . Asterisk indicate p < 0 . 05 . n = 3 BR ; error bars = SD . Students t - test , two tailed . G DNA repair kinetics in cumate - inducible SIRT6 fibroblasts . Cells were grown on slides and irradiated with 2 Gy gamma radiation , followed by immunostaining for γH2AX . Irradiation was conducted when the cells were at 75 % confluency on slides . Cells were fixed and foci scored at t = 0 . 5 hr , 2 hr , 4 hr , 6 hr , and 24 hr post - irradiation . Foci were scored in at least 80 cells per genotype per time point . Asterisk indicate p < 0 . 05 . Asterisks indicate significance over WT SIRT6 . Color of the asterisk corresponds to the SIRT6 allele . n = 3 BR ; error bars = SD . Students t - test , two tailed . H , I Oxidative stress resistance . Cumate - inducible SIRT6 fibroblasts were induced for SIRT6 expression and exposed to paraquat for 24 hours . Resistance was determined by apoptosis staining 48 hours after exposure . Asterisk indicate p < 0 . 05 . Asterisks indicate significance over WT SIRT6 . Color of the asterisk corresponds to the SIRT6 allele . n = 3 BR ; error bars = SD . Students t - test , two tailed .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4A qRT-PCR analysis of LINE1 expression in cumate-inducible SIRT6 fibroblasts. Primers assessed both 5' (ORF1) and 3' (ORF2) LINE1 sequences from the L1HS family of evolutionarily active LINE1 retrotransposons. Assessment of both regions was conducted to mitigate contributions from partial insertion sequences in coding genes. Asterisk indicate p<0.05. n=3 TR; error bars=SD. Students t-test, two tailed.B LINE1 EGFP retrotransposition assay. Three independent transfections with LINE1 EGFP reporter plasmid were conducted and quantified by flow cytometry. Asterisk indicate p<0.05. Red asterisk indicated significance over SIRT6 KO control. Black asterisk indicate significance over WT SIRT6. n=3 BR; error bars=SD. Two-way ANOVA.C In vitro KAP1 ribosylation by SIRT6. Asterisk indicate p<0.05. Red asterisk indicate significance over 0 hr, black asterisk indicate significance over compared to WT SIRT6. n=3 BR; error bars=SD. Students t-test, two tailed.D, E Stimulation of NHEJ and HR by SIRT6 variants. Reporter cell lines were co-transfected with SIRT6-expressing plasmid, I-Sce1 plasmid, and DsRed transfection control. After 72 hr recovery, reactivation of the GFP reporter was measured by flow cytometry. Stimulation of NHEJ or HR was calculated as radio of GFP+/DsRed+ positive cells. Blots demonstrate equivalent SIRT6 abundance in transfected cells. Asterisk indicate p<0.05 Black asterisk indicate significance over control, red asterisk indicate significance over WT SIRT6. n=3 BR; error bars=SD. Two-way ANOVAF Basal γH2AX foci in cumate-inducible SIRT6 fibroblasts. Foci were scored in at least 80 cells per condition. Asterisk indicate p<0.05. n=3 BR; error bars=SD. Students t-test, two tailed.G DNA repair kinetics in cumate-inducible SIRT6 fibroblasts. Cells were grown on slides and irradiated with 2 Gy gamma radiation, followed by immunostaining for γH2AX. Irradiation was conducted when the cells were at 75% confluency on slides. Cells were fixed and foci scored at t=0.5 hr, 2 hr, 4 hr, 6 hr, and 24 hr post-irradiation. Foci were scored in at least 80 cells per genotype per time point. Asterisk indicate p<0.05. Asterisks indicate significance over WT SIRT6. Color of the asterisk corresponds to the SIRT6 allele. n=3 BR; error bars=SD. Students t-test, two tailed.H, I Oxidative stress resistance. Cumate-inducible SIRT6 fibroblasts were induced for SIRT6 expression and exposed to paraquat for 24 hours. Resistance was determined by apoptosis staining 48 hours after exposure. Asterisk indicate p<0.05. Asterisks indicate significance over WT SIRT6. Color of the asterisk corresponds to the SIRT6 allele. n=3 BR; error bars=SD. Students t-test, two tailed.


Figure 5A Number of adherent cells after transfection with SIRT6 variants . Cells were transfected with SIRT6 plasmids encoding different SIRT6 alleles and cell numbers were counted after 72 hours . HCA2 are normal human foreskin fibroblasts . Asterisk indicate p < 0 . 05 . n = 3 BR ; error bars = SD . Students t - test , two tailed . B , C Apoptosis staining of cancer cell lines 48 hours after transfection . Cells were stained with Annexin V / PI and analyzed by flow cytometry . Asterisk indicate p < 0 . 05 . Red asterisk indicates significance over control , black asterisk indicates significance over WT SIRT6 . n = 3 BR ; error bars = SD . Two - way ANOVA .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5A Number of adherent cells after transfection with SIRT6 variants. Cells were transfected with SIRT6 plasmids encoding different SIRT6 alleles and cell numbers were counted after 72 hours. HCA2 are normal human foreskin fibroblasts. Asterisk indicate p<0.05. n=3 BR; error bars=SD. Students t-test, two tailed.B, C Apoptosis staining of cancer cell lines 48 hours after transfection. Cells were stained with Annexin V/PI and analyzed by flow cytometry. Asterisk indicate p<0.05. Red asterisk indicates significance over control, black asterisk indicates significance over WT SIRT6. n=3 BR; error bars=SD. Two-way ANOVA.


Figure 6B IP experiments on lysates from cumate - induced fibroblasts expressing wild type or centSIRT6 alleles with antibodies to SIRT6 , LMNA , and mADPr . SIRT6 expression was induced 48 hours prior to IP . n = 3 BR . One representative set of IPs is shown . C CentSIRT6 shows stronger interaction with LMNA compared to the wild type SIRT6 . Quantification of the IP experiment shown in ( B ) . SIRT6 IP followed by Western blot with antibodies to LMNA . D LMNA shows enhanced interaction with centSIRT6 compared to the wild type . Quantification of the IP experiment shown in ( B ) . LMNA IP from cumate - inducible SIRT6 fibroblasts followed by Western blot with antibodies to SIRT6 . Data information : n = 3 BR ; error bars = SD . Students t - test , two tailed . Asterisk indicate p < 0 . 05E centSIRT6 shows enhanced mADPr . Quantification of the IP experiment shown in ( B ) . SIRT6 IP from cumate - inducible SIRT6 fibroblasts followed by Western blot with antibody to mADPr residues F LMNA shows enhanced mADPr signal in cells expressing centSIRT6 . Quantification of the IP experiment shown in ( B ) . IP with mADPr antibody using extract from cumate - induced SIRT6 fibroblasts , followed by Western blot with antibodies to LMNA . Data information : n = 3 BR ; error bars = SD . Students t - test , two tailed . Asterisk indicate p < 0 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6B IP experiments on lysates from cumate-induced fibroblasts expressing wild type or centSIRT6 alleles with antibodies to SIRT6, LMNA, and mADPr. SIRT6 expression was induced 48 hours prior to IP. n=3 BR. One representative set of IPs is shown.C CentSIRT6 shows stronger interaction with LMNA compared to the wild type SIRT6. Quantification of the IP experiment shown in (B). SIRT6 IP followed by Western blot with antibodies to LMNA. D LMNA shows enhanced interaction with centSIRT6 compared to the wild type. Quantification of the IP experiment shown in (B). LMNA IP from cumate-inducible SIRT6 fibroblasts followed by Western blot with antibodies to SIRT6. Data information: n=3 BR; error bars=SD. Students t-test, two tailed. Asterisk indicate p<0.05E centSIRT6 shows enhanced mADPr. Quantification of the IP experiment shown in (B). SIRT6 IP from cumate-inducible SIRT6 fibroblasts followed by Western blot with antibody to mADPr residues F LMNA shows enhanced mADPr signal in cells expressing centSIRT6. Quantification of the IP experiment shown in (B). IP with mADPr antibody using extract from cumate-induced SIRT6 fibroblasts, followed by Western blot with antibodies to LMNA. Data information: n=3 BR; error bars=SD. Students t-test, two tailed. Asterisk indicate p<0.05


Figure 1 ( A ) 22 Tie1 - binding antibodies were screened for phospho - AKT levels in an in vitro Ang1 - stimulation experiment . Only one clone ( AB - Tie1 - 39 ) reduced AKT phosphorylation indicating suppressed Tie2 signaling ( n = 1 experiment ) . ( C ) Tumor growth curves show delayed primary tumor growth upon treatment with AB - Tie1 - 39 antibody as compared to IgG treatment ( mean ± SEM , n = 9 mice ) . * , P < 0 . 05 ; * * * , P < 0 . 001 ( 2 - way ANNOVA test ) . ( D ) Representative immunofluorescence images of tumor sections stained with CD31 ( EC - specific marker ) and Desmin ( mural cell - specific marker ) . Scale bars = 200 μm . ( E ) Dot plots show quantitation of intratumoral vessel density , mural cell coverage using Desmin and aSMA co - staining , and tumor necrosis ( mean ± SD , n = 9 mice ) . All comparisons were rendered non - significant according to two - tailed Mann - Whitney U test .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1(A) 22 Tie1-binding antibodies were screened for phospho-AKT levels in an in vitro Ang1-stimulation experiment. Only one clone (AB-Tie1-39) reduced AKT phosphorylation indicating suppressed Tie2 signaling (n = 1 experiment).(C) Tumor growth curves show delayed primary tumor growth upon treatment with AB-Tie1-39 antibody as compared to IgG treatment (mean ± SEM, n = 9 mice). *, P<0.05; ***, P<0.001 (2-way ANNOVA test).(D) Representative immunofluorescence images of tumor sections stained with CD31 (EC-specific marker) and Desmin (mural cell-specific marker). Scale bars = 200 μm.(E) Dot plots show quantitation of intratumoral vessel density, mural cell coverage using Desmin and aSMA co-staining, and tumor necrosis (mean ± SD, n = 9 mice). All comparisons were rendered non-significant according to two-tailed Mann-Whitney U test.


Figure 2 ( A ) Following a neoadjuvant treatment regimen with IgG or AB - Tie1 - 39 , mice were monitored by whole - body bioluminescence imaging . Shown are the images acquired two weeks after primary tumor resection . ( B ) Dot plots quantifying total photon flux during in vivo whole body as well as ex vivo lung and lymph node bioluminescence imaging ( mean ± SD , nIgG = 8 , nAB - Tie1 - 39 = 9 mice ) . * , P < 0 . 05 ; * * , P < 0 . 01 ( two - tailed Mann - Whitney U test ) . ( D ) Kaplan - Meier graph comparing percent survival between mice treated with either IgG or AB - Tie1 - 39 ( nIgG = 10 , nAB - Tie1 - 39 = 9 mice ) . * * , P < 0 . 01 ( Log - rank ( Mantel - Cox ) test ) .

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Figure 2(A) Following a neoadjuvant treatment regimen with IgG or AB-Tie1-39, mice were monitored by whole-body bioluminescence imaging. Shown are the images acquired two weeks after primary tumor resection.(B) Dot plots quantifying total photon flux during in vivo whole body as well as ex vivo lung and lymph node bioluminescence imaging (mean ± SD, nIgG = 8, nAB-Tie1-39 = 9 mice). *, P<0.05; **, P<0.01 (two-tailed Mann-Whitney U test).(D) Kaplan-Meier graph comparing percent survival between mice treated with either IgG or AB-Tie1-39 (nIgG = 10, nAB-Tie1-39 = 9 mice). **, P<0.01 (Log-rank (Mantel-Cox) test).


Figure 3 ( B ) Representative images of the whole lungs are shown on the left . Scale bars = 5 mm . Quantitation of the number of lung metastatic foci is plotted on the right ( mean ± SD , nIgG = 9 , nAB - Tie1 - 39 = 8 mice ) . * , P < 0 . 05 ( two - tailed Mann - Whitney U test ) . ( C ) EC were seeded on gelatin - coated Transwell inserts . RFP - labeled LLC cells were allowed to transmigrate for 8 h through the endothelial layer . Thereafter , transmigrated tumor cells were counted . The dot plot presents the normalized data ( mean ± SD , n = 9 inserts from three independent experiments ) . * * * , P < 0 . 001 ( two - tailed Mann - Whitney U test ) . ( D ) LLC tumor - bearing mice were pretreated with IgG or AB - Tie1 - 39 . On day 14 , lung tissue lysates were immunoblotted with anti - phospho - TIE2 , total - TIE2 and ACTB ( upper panel ; n = 6 mice ) . Densitometric quantitation of the blots presented in the upper panel is shown ( mean ± SD , n = 6 mice ) . * * , P < 0 . 01 ( two - tailed Mann - Whitney U test ) . ( E ) LLC tumor - bearing mice were pretreated with IgG or AB - Tie1 - 39 . On day 14 , lung EC were FACS - sorted and used for global gene expression ( n = 4 mice ) . On the left , a heatmap shows significantly - regulated genes ( red outline = AB - Tie1 - 39 - treated ; black outline = IgG - treated ) . On the right , top significantly regulated bio - functions in AB - Tie1 - 39 - treated as compared to IgG - treated mice in Ingenuity Pathway Analysis .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3(B) Representative images of the whole lungs are shown on the left. Scale bars = 5 mm. Quantitation of the number of lung metastatic foci is plotted on the right (mean ± SD, nIgG = 9, nAB-Tie1-39 = 8 mice). *, P<0.05 (two-tailed Mann-Whitney U test).(C) EC were seeded on gelatin-coated Transwell inserts. RFP-labeled LLC cells were allowed to transmigrate for 8 h through the endothelial layer. Thereafter, transmigrated tumor cells were counted. The dot plot presents the normalized data (mean ± SD, n = 9 inserts from three independent experiments). ***, P<0.001 (two-tailed Mann-Whitney U test).(D) LLC tumor-bearing mice were pretreated with IgG or AB-Tie1-39. On day 14, lung tissue lysates were immunoblotted with anti-phospho-TIE2, total-TIE2 and ACTB (upper panel; n = 6 mice). Densitometric quantitation of the blots presented in the upper panel is shown (mean ± SD, n = 6 mice). **, P<0.01 (two-tailed Mann-Whitney U test).(E) LLC tumor-bearing mice were pretreated with IgG or AB-Tie1-39. On day 14, lung EC were FACS-sorted and used for global gene expression (n = 4 mice). On the left, a heatmap shows significantly- regulated genes (red outline = AB-Tie1-39-treated; black outline = IgG-treated). On the right, top significantly regulated bio-functions in AB-Tie1-39-treated as compared to IgG-treated mice in Ingenuity Pathway Analysis.


Figure 1Formalin - fixed paraffin - embedded ( FFPE ) sections from control or Tgif1KO mice were immunostained with anti - pSmad2 antibody and revealed by immunofluorescence ( IF ) and DAPI . Representative pictures at 40x are shown ( n = 30 ) . Scale bars , 100 μM . FFPE pancreatic sections from control or Tgif1KO mice were stained with hematoxylin and eosin ( H & E ) or immunostained with antibodies to CK19 or Amylase and revealed by IHC . Representative pictures at 20x are shown ( n = 30 ) . Scale bars , 200 μM . Kaplan - Meyer survival analysis of control , Tgif1KO , KC and KTC mice . A regular mosaic two - colors line was used to discriminate between control and Tgif1KO mice . Pictures of whole pancreas and spleen tissues from control , Tgif1KO , KC or KTC mice ( left ) . Weight of pancreas was measured and presented as dot plot ( n = 30 ) . FFPE pancreatic sections from control , Tgif1KO , KC or KTC were stained with H & E or subjected to IHC analysis using antibodies to CK19 or α - SMA . Yellow arrows indicate PanINs in KC mice . Representative pictures at 20x are shown ( n = 30 ) . Scale bars , 200 μM .

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Figure 1Formalin-fixed paraffin-embedded (FFPE) sections from control or Tgif1KO mice were immunostained with anti-pSmad2 antibody and revealed by immunofluorescence (IF) and DAPI. Representative pictures at 40x are shown (n=30). Scale bars, 100 μM.FFPE pancreatic sections from control or Tgif1KO mice were stained with hematoxylin and eosin (H&E) or immunostained with antibodies to CK19 or Amylase and revealed by IHC. Representative pictures at 20x are shown (n=30). Scale bars, 200 μM.Kaplan-Meyer survival analysis of control, Tgif1KO, KC and KTC mice. A regular mosaic two-colors line was used to discriminate between control and Tgif1KO mice.Pictures of whole pancreas and spleen tissues from control, Tgif1KO, KC or KTC mice (left). Weight of pancreas was measured and presented as dot plot (n=30).FFPE pancreatic sections from control, Tgif1KO, KC or KTC were stained with H&E or subjected to IHC analysis using antibodies to CK19 or α-SMA. Yellow arrows indicate PanINs in KC mice. Representative pictures at 20x are shown (n=30). Scale bars, 200 μM.


Figure 2Representative pictures of dissected abdominal region of KC and KTC mice . Yellow arrows indicate metastatic nodules in liver . Yellow dotted lines show the pancreas . FFPE sections of pancreas , liver , lungs or lymph - nodes from control , KC or KTC mice were stained with H & E or immunostained with anti - Vimentin antibody and revealed by IF . Representative pictures at 4x ( Lymph node , H & E ) , 10x ( Liver , H & E ) and 20x ( Pancreas , H & E ; Lung , H & E ; Lymph node , IF - Vimentin ) are shown ( n = 18 to 30 ) . Scale bars , 1 , 000 μM ( Lymph node , H & E ) , 400 μM ( Liver , H & E ) and 200 μM ( Pancreas , H & E ; Lung , H & E ; Lymph node , IF - Vimentin ) . Tumor volume and metastasis in live KCLuc and KTCLuc mice were analyzed by IVIS bioluminescence . Following imaging live animals , pancreas , brain , liver and lung were dissected and analyzed again by IVIS bioluminescence ( n = 6 , 5 out of 6 mice developed metastasis to liver and lung ) . Total cell lysates from pancreas of control , KC or KTC mice were pooled and analyzed by immunoblotting using antibodies to pSmad2 , Smad2 or JunB ( n = 6 ) . Expression of JunB in pancreas from control , KC or KTC mice ( same as in D ) was analyzed by qRT - PCR ( n = 6 ) . Data are expressed as mean ± SEM . * * p < 0 . 01 ; based on a two - tailed Student ' s test .

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Figure 2Representative pictures of dissected abdominal region of KC and KTC mice. Yellow arrows indicate metastatic nodules in liver. Yellow dotted lines show the pancreas.FFPE sections of pancreas, liver, lungs or lymph-nodes from control, KC or KTC mice were stained with H&E or immunostained with anti-Vimentin antibody and revealed by IF. Representative pictures at 4x (Lymph node, H&E), 10x (Liver, H&E) and 20x (Pancreas, H&E; Lung, H&E; Lymph node, IF-Vimentin) are shown (n=18 to 30). Scale bars, 1,000 μM (Lymph node, H&E), 400 μM (Liver, H&E) and 200 μM (Pancreas, H&E; Lung, H&E; Lymph node, IF-Vimentin).Tumor volume and metastasis in live KCLuc and KTCLuc mice were analyzed by IVIS bioluminescence. Following imaging live animals, pancreas, brain, liver and lung were dissected and analyzed again by IVIS bioluminescence (n= 6, 5 out of 6 mice developed metastasis to liver and lung).Total cell lysates from pancreas of control, KC or KTC mice were pooled and analyzed by immunoblotting using antibodies to pSmad2, Smad2 or JunB (n=6).Expression of JunB in pancreas from control, KC or KTC mice (same as in D) was analyzed by qRT-PCR (n=6). Data are expressed as mean ± SEM. **p< 0.01; based on a two-tailed Student's test.


Figure 3Protein extracts from MIAPaCa - 2 cells ( left ) and control or Tgif1KO mice ( right ) were immunoprecipitated using normal IgG , anti - TGIF1 or anti - Twist1 antibody and analyzed by immunoblotting using antibodies to Twist1 or TGIF1 ( A - C ) . Protein extracts from control or Tgif1KO mice ( right ) were immunoprecipitated using normal IgG , anti - TGIF1 or anti - Twist1 antibody and analyzed by immunoblotting using antibodies to Twist1 or TGIF1 To control for loading , total cell lysates were analyzed by immunoblotting using antibodies to Twist1 , TGIF1 or β - Actin . Protein extracts from MIAPaCa - 2 cells ( left ) and control were immunoprecipitated using normal IgG , anti - TGIF1 or anti - Twist1 antibody and analyzed by immunoblotting using antibodies to Twist1 or TGIF1 In C , cell lysates were treated with DNase I before coimmunopreciptation . Panc - 1 cells were treated with TGF - β for 30 min , and protein extracts were immunoprecipitated with anti - TGIF1 antibody and analyzed by immunoblotting using anti - Twist1 antibody . To control for activation of TGF - β signaling , total cell lysates were analyzed by immunoblotting using anti - pSmad2 antibody . PL45 stably expressing Dox - inducible control or sh - RNA targeting TGIF were treated with vehicle or Dox for 48 hr and analyzed for Twist1 expression by immunoblotting or qRT - PCR ( n = 6 ) . In left panel , the intensity of the bands was assessed by densitometry , and the result was presented as a ratio of Twist1 / TGIF1 . PL45 stably expressing Dox - inducible TGIF1 were treated with vehicle or Dox for 48 hr and analyzed for binding of TGIF1 to the Twist1 promoter by ChIP and agarose gel . Pancreatic chromatin from control or Tgif1KO mice was analyzed for binding of TGIF1 to the Twist1 promoter by ChIP and agarose gel .

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Figure 3Protein extracts from MIAPaCa-2 cells (left) and control or Tgif1KO mice (right) were immunoprecipitated using normal IgG, anti-TGIF1 or anti-Twist1 antibody and analyzed by immunoblotting using antibodies to Twist1 or TGIF1 (A-C).Protein extracts from control or Tgif1KO mice (right) were immunoprecipitated using normal IgG, anti-TGIF1 or anti-Twist1 antibody and analyzed by immunoblotting using antibodies to Twist1 or TGIF1 To control for loading, total cell lysates were analyzed by immunoblotting using antibodies to Twist1, TGIF1 or β-Actin.Protein extracts from MIAPaCa-2 cells (left) and control were immunoprecipitated using normal IgG, anti-TGIF1 or anti-Twist1 antibody and analyzed by immunoblotting using antibodies to Twist1 or TGIF1 In C, cell lysates were treated with DNase I before coimmunopreciptation.Panc-1 cells were treated with TGF-β for 30 min, and protein extracts were immunoprecipitated with anti-TGIF1 antibody and analyzed by immunoblotting using anti-Twist1 antibody. To control for activation of TGF-β signaling, total cell lysates were analyzed by immunoblotting using anti-pSmad2 antibody.PL45 stably expressing Dox-inducible control or sh-RNA targeting TGIF were treated with vehicle or Dox for 48 hr and analyzed for Twist1 expression by immunoblotting or qRT-PCR (n=6). In left panel, the intensity of the bands was assessed by densitometry, and the result was presented as a ratio of Twist1/TGIF1.PL45 stably expressing Dox-inducible TGIF1 were treated with vehicle or Dox for 48 hr and analyzed for binding of TGIF1 to the Twist1 promoter by ChIP and agarose gel.Pancreatic chromatin from control or Tgif1KO mice was analyzed for binding of TGIF1 to the Twist1 promoter by ChIP and agarose gel.


Figure 4PL45 stably expressing both Dox - inducible TGIF1 and Tam - inducible Twist1 ( Twist1ER ) were treated with Tam and / or Dox for 48 hr and analyzed for Twist1 expression by immunoblotting or qRT - PCR ( n = 6 ) . MIAPaCa - 2 cells were transfected with wild - type or mutated ( Twist1 binding site ) Twist1 luciferase reporters ( TGTLuc ) together with increasing amounts of Twist1 expression vector in the presence of empty vector ( EV ) or TGIF1 expression vector . Luciferase activity was measured 48 hr following transfection and normalized on the basis of co - transfected Renilla luciferase ( n = 6 ) . Total lysates from pancreas of control , KC or KTC mice were pooled and analyzed by immunoblotting using antibodies to E - Cadherin , Vimentin , p16Ink4A and β - Actin ( n = 6 ) . Expression of Cdh1 , Vimentin , and p16Ink4A in pancreas from control , KC or KTC mice ( same as in C ) was analyzed by qRT - PCR ( n = 6 ) . FFPE pancreatic sections from control , KC or KTC mice were stained with H & E or immunostained with antibodies to E - Cadherin , Vimentin or p16Ink4a and revealed by IF or IHC . Representative pictures at 20x are shown ( n = 30 ) . Scale bars , 200 μM .

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Figure 4PL45 stably expressing both Dox-inducible TGIF1 and Tam-inducible Twist1 (Twist1ER) were treated with Tam and/or Dox for 48 hr and analyzed for Twist1 expression by immunoblotting or qRT-PCR (n=6).MIAPaCa-2 cells were transfected with wild-type or mutated (Twist1 binding site) Twist1 luciferase reporters (TGTLuc) together with increasing amounts of Twist1 expression vector in the presence of empty vector (EV) or TGIF1 expression vector. Luciferase activity was measured 48 hr following transfection and normalized on the basis of co-transfected Renilla luciferase (n=6).Total lysates from pancreas of control, KC or KTC mice were pooled and analyzed by immunoblotting using antibodies to E-Cadherin, Vimentin, p16Ink4A and β-Actin (n=6).Expression of Cdh1, Vimentin, and p16Ink4A in pancreas from control, KC or KTC mice (same as in C) was analyzed by qRT-PCR (n=6).FFPE pancreatic sections from control, KC or KTC mice were stained with H&E or immunostained with antibodies to E-Cadherin, Vimentin or p16Ink4a and revealed by IF or IHC. Representative pictures at 20x are shown (n=30). Scale bars, 200 μM.


Figure 5Kaplan - Meyer survival analysis of control , KTC and KTWC mice . FFPE sections of pancreas or liver from control , KTC or KTWC mice were stained with H & E or immunostained with anti - CK19 antibody and revealed by IHC . Representative pictures at 10x ( Liver , H & E ) or 20x ( Pancreas , H & E ; Pancreas , IHC - CK19 ) are shown ( n = 30 ) . Scale bars , 400 μM ( Liver , H & E ) and 200 μM ( Pancreas , H & E ; Pancreas , IHC - CK19 ) . Expression of Cdh1 , Vimentin and p16Ink4A in pancreas from control , KTC or KTWC mice was analyzed by qRT - PCR ( n = 6 ) .

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Figure 5Kaplan-Meyer survival analysis of control, KTC and KTWC mice.FFPE sections of pancreas or liver from control, KTC or KTWC mice were stained with H&E or immunostained with anti-CK19 antibody and revealed by IHC. Representative pictures at 10x (Liver, H&E) or 20x (Pancreas, H&E; Pancreas, IHC-CK19) are shown (n=30). Scale bars, 400 μM (Liver, H&E) and 200 μM (Pancreas, H&E; Pancreas, IHC-CK19).Expression of Cdh1, Vimentin and p16Ink4A in pancreas from control, KTC or KTWC mice was analyzed by qRT-PCR (n=6).


Figure 6Specificity of anti - phospho - TGIF1 ( pTGIF1 ) antibody . Cell extracts from Tgif1 - / - MEFs cells reconstituted with empty vector ( EV ) , wild - type ( TGIF1 ) or non - phosphorylated form ( TGIF1 . 2TA ) of Flag - TGIF1 were immunoblotted with anti - phospho - TGIF1 or β - Actin as a loading control . Expression of pTGIF1 and Twist1 in human tissue microarrays of human PDAC samples was analyzed by immunohistochemistry . Representative pictures at different stages ( 40x ) are shown . The percentages of samples with high versus low expression of pTGIF1 and Twist1 in normal tissues versus PDAC tissues are shown . Scale bars , 100 μM . MIAPaCa - 2 cells were transfected with TGIF1 or TGIF1 . 2TD expression vectors , selected with G418 and resistance colonies were pooled ( n = 6 ) . Expression of Twist1 was examined by qRT - PCR ( D ) . MIAPaCa - 2 cells were transfected with TGIF1 or TGIF1 . 2TD expression vectors , selected with G418 and resistance colonies were pooled ( n = 6 ) . Binding of TGIF1 to the Twist1 promoter was examined by ChIP and agarose gel . BxPC3 cells were transfected with TGTLuc together with TGIF1 or TGIF1 . 2TA in the presence or absence of KrasG12D . Luciferase activity was measured 48 hr following transfection and normalized on the basis of co - transfected Renilla luciferase ( n = 6 ) . BxPC3 cells were transfected with CDH1Luc or p16Luc reporter together with TGIF1 or TGIF1 . 2TA in the presence or absence of KrasG12D and luciferase activity was measured 48 hr following transfection as described in ( E ) ( n = 6 ) .

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Figure 6Specificity of anti-phospho-TGIF1 (pTGIF1) antibody. Cell extracts from Tgif1-/- MEFs cells reconstituted with empty vector (EV), wild-type (TGIF1) or non-phosphorylated form (TGIF1.2TA) of Flag-TGIF1 were immunoblotted with anti-phospho-TGIF1 or β-Actin as a loading control.Expression of pTGIF1 and Twist1 in human tissue microarrays of human PDAC samples was analyzed by immunohistochemistry. Representative pictures at different stages (40x) are shown.The percentages of samples with high versus low expression of pTGIF1 and Twist1 in normal tissues versus PDAC tissues are shown. Scale bars, 100 μM.MIAPaCa-2 cells were transfected with TGIF1 or TGIF1.2TD expression vectors, selected with G418 and resistance colonies were pooled (n=6). Expression of Twist1 was examined by qRT-PCR (D).MIAPaCa-2 cells were transfected with TGIF1 or TGIF1.2TD expression vectors, selected with G418 and resistance colonies were pooled (n=6). Binding of TGIF1 to the Twist1 promoter was examined by ChIP and agarose gel.BxPC3 cells were transfected with TGTLuc together with TGIF1 or TGIF1.2TA in the presence or absence of KrasG12D. Luciferase activity was measured 48 hr following transfection and normalized on the basis of co-transfected Renilla luciferase (n=6).BxPC3 cells were transfected with CDH1Luc or p16Luc reporter together with TGIF1 or TGIF1.2TA in the presence or absence of KrasG12D and luciferase activity was measured 48 hr following transfection as described in (E) (n=6).


Figure 7Representative microscope pictures ( 20x ) of MIAPaCa - 2 , Suit - 2 , Capan - 2 and Panc - 1 cells . Scale bars , 200 μM . Expression of TGIF1 and Twist1 in MIAPaCa - 2 , Suit - 2 , Capan - 2 and Panc - 1 cells was analyzed by immunoblotting . MIAPaCa - 2 , Suit - 2 , Capan - 2 and Panc - 1 cells were transfected with empty vector ( EV ) , wild - type ( TGIF1 ) or phosphorylation mimic mutant ( TGIF1 . 2TD ) of TGIF1 . Cell extracts were analyzed 48 hr following transfection by immunoblotting using anti - Flag antibody ( C ) . MIAPaCa - 2 , Suit - 2 , Capan - 2 and Panc - 1 cells were transfected with empty vector ( EV ) , wild - type ( TGIF1 ) or phosphorylation mimic mutant ( TGIF1 . 2TD ) of TGIF1 . Cells were selected with neomycin for 2 - 3 weeks , and counted using an automatic cell counter ( D ) ( n = 6 ) .

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Figure 7Representative microscope pictures (20x) of MIAPaCa-2, Suit-2, Capan-2 and Panc-1 cells. Scale bars, 200 μM.Expression of TGIF1 and Twist1 in MIAPaCa-2, Suit-2, Capan-2 and Panc-1 cells was analyzed by immunoblotting.MIAPaCa-2, Suit-2, Capan-2 and Panc-1 cells were transfected with empty vector (EV), wild-type (TGIF1) or phosphorylation mimic mutant (TGIF1.2TD) of TGIF1. Cell extracts were analyzed 48 hr following transfection by immunoblotting using anti-Flag antibody (C).MIAPaCa-2, Suit-2, Capan-2 and Panc-1 cells were transfected with empty vector (EV), wild-type (TGIF1) or phosphorylation mimic mutant (TGIF1.2TD) of TGIF1. Cells were selected with neomycin for 2-3 weeks, and counted using an automatic cell counter (D) (n=6).


Figure 1A . Structure of Brca1ex2 allele . * Note that exon 4 was annotated in error in original descriptions of the gene structure and is not drawn . RT - PCR shows a novel product from the Brca1ex2 allele , corresponding to splicing of exon 1 directly to exon 3 . B . Western blot to detect BRCA1 and BARD1 in MEFs expressing WT and mutant Brca1 . C . Immunofluorescent ( IF ) detection of BRCA1 and RAD51 at IR - induced nuclear foci in cells expressing WT and mutant forms of Brca1 . Scale bar : 10 ingD . Quantification of IF , showing proportion of cells with RAD51 foci that also had > 5 BRCA1 foci . N = 2 . E . Quantification of IF , showing proportion of RAD51 foci that colocalized with BRCA1 foci . N = 2 . F . Immunofluorescent detection of BARD1 at IR - induced nuclear foci in MEFs expressing WT and mutant forms of Brca1 . Scale bar : 10 μ

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Figure 1A. Structure of Brca1ex2 allele. * Note that exon 4 was annotated in error in original descriptions of the gene structure and is not drawn. RT-PCR shows a novel product from the Brca1ex2 allele, corresponding to splicing of exon 1 directly to exon 3.B. Western blot to detect BRCA1 and BARD1 in MEFs expressing WT and mutant Brca1.C. Immunofluorescent (IF) detection of BRCA1 and RAD51 at IR-induced nuclear foci in cells expressing WT and mutant forms of Brca1. Scale bar: 10 ingD. Quantification of IF, showing proportion of cells with RAD51 foci that also had >5 BRCA1 foci. N=2.E. Quantification of IF, showing proportion of RAD51 foci that colocalized with BRCA1 foci. N=2.F. Immunofluorescent detection of BARD1 at IR-induced nuclear foci in MEFs expressing WT and mutant forms of Brca1. Scale bar: 10 μ S


Figure 2A . Western blot showing abundance of BRCA1 and BARD1 protein in Brca1Δ2 / Δ2 ; Trp53bp1 - / - mouseB cells . B . Analysis of chromosome aberrations in mice with targeted deletion of Brca1 exon 2 and Trp53bp1 . Left , metaphase spreads were prepared from B cells treated with olaparib . Arrows show examples of chromosome aberrations . Right , quantification of chromosome breaks ( CSB ) , chromatid - type breaks ( CTD ) , radial chromosomes , and other abnormalities in mouseB cells . Error bars indicate SD , N = 3C . RAD51 foci in B cells after 10 Gy IR exposure . Chart shows mean percentage of cells with > 5 foci , N = 3 . Error bars indicate SD , statistical analysis by two - tailed Student t test . D . Percentage of cells showing > 5 RAD51 foci after 4hrs treatment with 10 μM olaparib ( OLA ) or 10 μM camptothecin ( CPT ) . N = 2 . E . Average number of γ - H2AX foci per cell in cells that were not treated ( NT ) or exposed to 2 Gy ionizing radiation . N = 2 . F . Sister chromatid exchanges in cells that were not treated ( NT ) or exposed to 2 μM olaparib ( OLA ) for 16 hrs . Black arrows in images show SCEs in olaparib - treated cells . Scale bar : 5 μm . N = 2 .

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Figure 2A. Western blot showing abundance of BRCA1 and BARD1 protein in Brca1Δ2/Δ2;Trp53bp1-/- mouseB cells.B. Analysis of chromosome aberrations in mice with targeted deletion of Brca1 exon 2 and Trp53bp1. Left, metaphase spreads were prepared from B cells treated with olaparib. Arrows show examples of chromosome aberrations. Right, quantification of chromosome breaks (CSB), chromatid-type breaks (CTD), radial chromosomes, and other abnormalities in mouseB cells. Error bars indicate SD, N=3C. RAD51 foci in B cells after 10 Gy IR exposure. Chart shows mean percentage of cells with >5 foci, N=3. Error bars indicate SD, statistical analysis by two-tailed Student t test.D. Percentage of cells showing >5 RAD51 foci after 4hrs treatment with 10 μM olaparib (OLA) or 10 μM camptothecin (CPT). N=2.E. Average number of γ-H2AX foci per cell in cells that were not treated (NT) or exposed to 2 Gy ionizing radiation. N=2.F. Sister chromatid exchanges in cells that were not treated (NT) or exposed to 2 μM olaparib (OLA) for 16 hrs. Black arrows in images show SCEs in olaparib-treated cells. Scale bar: 5 μm. N=2.


Figure 3A . Experimental scheme and representative images of DNA fibers . Scale bar : 5 μm . B . Analysis of fibers showing proportions of replication forks that showed restart after HU treatment or remained stalled . N = 2 , > 500 fibers scored per experiment . C . Proportion of total forks showing de novo initiation after HU treatment . N = 2 , > 500 fibers scored per experiment . D . Length of initial replication fork tracts ( CldU tracts ) . N = 2 , > 200 fibers measured per experiment .

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Figure 3A. Experimental scheme and representative images of DNA fibers. Scale bar: 5 μm.B. Analysis of fibers showing proportions of replication forks that showed restart after HU treatment or remained stalled. N=2, >500 fibers scored per experiment.C. Proportion of total forks showing de novo initiation after HU treatment. N=2, >500 fibers scored per experiment.D. Length of initial replication fork tracts (CldU tracts). N=2, >200 fibers measured per experiment.


Figure 4A . Kaplan - Meier survival curve for Brca1ex2 / + ; Trp53bp1 - / - and Brca1ex2 / ex2 ; Trp53bp1 - / - mice . N = 20 animals in each group . Statistical analysis by Mantel - Cox log - rank test . B . Kaplan - Meier survival curve for Brca1ex2 / + ; Trp53bp1 - / - ; Trp53 + / - and Brca1ex2 / ex2 ; Trp53bp1 - / - ; Trp53 + / - mice . N = 20 animals in each group . Statistical analysis by Mantel - Cox log - rank test . C . Percentage of Brca1ex2 / + ; Trp53bp1 - / - and Brca1ex2 / ex2 ; Trp53bp1 - / - mice showing signs of abnormal tissue morphology at death . N = 46 animals inspected for Brca1ex2 / + ; Trp53bp1 + / + , N = 54 animals inspected for Brca1ex2 / ex2 / Trp53bp1 - / - . D . Tissues affected by abnormal growth from N = 46 Brca1ex2 / + / Trp53bp1 - / - and N = 54 Brca1ex2 / ex2 / Trp53bp1 - / - mice .

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Figure 4A. Kaplan-Meier survival curve for Brca1ex2/+;Trp53bp1-/- and Brca1ex2/ex2;Trp53bp1-/- mice. N=20 animals in each group. Statistical analysis by Mantel-Cox log-rank test.B. Kaplan-Meier survival curve for Brca1ex2/+;Trp53bp1-/-;Trp53+/- and Brca1ex2/ex2;Trp53bp1-/-;Trp53+/- mice. N=20 animals in each group. Statistical analysis by Mantel-Cox log-rank test.C. Percentage of Brca1ex2/+;Trp53bp1-/- and Brca1ex2/ex2;Trp53bp1-/- mice showing signs of abnormal tissue morphology at death. N=46 animals inspected for Brca1ex2/+;Trp53bp1+/+, N=54 animals inspected for Brca1ex2/ex2/Trp53bp1-/-.D. Tissues affected by abnormal growth from N=46 Brca1ex2/+/Trp53bp1-/- and N=54 Brca1ex2/ex2/Trp53bp1-/- mice.


Figure 5A . H & amp ; amp ; E - stained sections of seminiferous tubules from testes of WT and Brca1ex2 / ex2 / Trp53bp1 - / - male mice . Scale bar : 100 μm . B . Immunofluorescence on meiotic chromosomes from WT and Brca1ex2 / ex2 ; Trp53bp1 - / - spermatocytes . Spreads were stained for SYCP3 to indicate chromosome axes and extent of synapsis and were stained for SYCP3 to indicate chromosome axex body . Scale bar : 5 apsiC . Quantification of the number of γ - H2AX foci observed at the pachytene stage in the indicated genotypes . P value is Mann - Whitney , two - tailed . D . G2M checkpoint analysis in mouse B cells after IR treatment . Left , mitotic cells were identified by flow cytometry as having 4c DNA content ( based on propidium iodide staining ) and pSer10 - H3 + . Right , quantification of flow cytometry data , showing mitotic cells after IR as percentage of that seen in untreated cells . N ≥ 2 .

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Figure 5A. H&amp;E-stained sections of seminiferous tubules from testes of WT and Brca1ex2/ex2/Trp53bp1-/- male mice. Scale bar: 100 μm.B. Immunofluorescence on meiotic chromosomes from WT and Brca1ex2/ex2;Trp53bp1-/-spermatocytes. Spreads were stained for SYCP3 to indicate chromosome axes and extent of synapsis and were stained for SYCP3 to indicate chromosome axex body. Scale bar: 5 apsiC. Quantification of the number of γ-H2AX foci observed at the pachytene stage in the indicated genotypes. P value is Mann-Whitney, two-tailed.D. G2M checkpoint analysis in mouse B cells after IR treatment. Left, mitotic cells were identified by flow cytometry as having 4c DNA content (based on propidium iodide staining) and pSer10-H3+. Right, quantification of flow cytometry data, showing mitotic cells after IR as percentage of that seen in untreated cells. N≥2.


Figure 1Normalised Wnt / STOP signalling activity by quantification of the protein expression of two Wnt / STOP target proteins , β - catenin and c - Myc , in mitotic ( M ) and asynchronised cells ( A ) after silencing 47 Wnt signalling regulators . ELISA validation of selected Wnt / STOP signalling regulators ( n = 3 ) . Immunoblotting and ( D ) quantification of the stability of four STOP target proteins in both G1S and natural mitotic cells treated with BCL9 siGenome pools and CHX ( n = 3 ) . Representative pictures ( left panel ) and the normalised biosensor intensity based on live cell imaging of the cells with a GSK3 - GFP biosensor following different treatments before and after metaphase ( n = 10 ) . Scale bars represent 10 μm . Myc and β - catenin target gene enrichment analysis based on the transcriptome targets regulated by BCL9 . The representative xenograft tumours ( top panel ) and tumour growth analysis ( bottom panel ) of scramble and BCL9 stable knockdown cells . LF : left flank of mouse , RF : right flank . n = 6 for shS1 vs . shB1 , n = 5 for shS1 vs . shB2 . Immunohistochemical staining of the xenograft ; the red arrows indicate the representative mitotic cells . Scale bars represent 200 μm .

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Figure 1Normalised Wnt/STOP signalling activity by quantification of the protein expression of two Wnt/STOP target proteins, β-catenin and c-Myc, in mitotic (M) and asynchronised cells (A) after silencing 47 Wnt signalling regulators.ELISA validation of selected Wnt/STOP signalling regulators (n=3).Immunoblotting and (D) quantification of the stability of four STOP target proteins in both G1S and natural mitotic cells treated with BCL9 siGenome pools and CHX (n=3).Representative pictures (left panel) and the normalised biosensor intensity based on live cell imaging of the cells with a GSK3-GFP biosensor following different treatments before and after metaphase (n=10). Scale bars represent 10 μm.Myc and β-catenin target gene enrichment analysis based on the transcriptome targets regulated by BCL9.The representative xenograft tumours (top panel) and tumour growth analysis (bottom panel) of scramble and BCL9 stable knockdown cells. LF: left flank of mouse, RF: right flank. n=6 for shS1 vs. shB1, n=5 for shS1 vs. shB2.Immunohistochemical staining of the xenograft; the red arrows indicate the representative mitotic cells. Scale bars represent 200 μm.


Figure 2Immunoblotting analysis of BCL9 and cell cycle protein expression in double thymidine synchronised cells ; the black arrow indicates the delayed band of mitotic BCL9 . Immunofluorescence confocal analysis of BCL9 localization on the spindle by antibody staining . Yellow arrows indicate the spindle pole . Scale bars represent : 2 μm for interphase and anaphase ; 5 μm for prophase , metaphase and telophase . Immunofluorescence confocal analysis of BCL9 localization on the centrosome . Yellow arrows indicate the centrosome . Scale bars represent : 5 μm for control treatment , 10 μm for BCL9 peptide treatment . Confocal microscope 3D - analysis and radial histograms of the spindle angle in the cells with siRNA treatment ; yellow arrows indicate the centrosome . Scale bars represent 5 μm . Confocal ( left panel ) and live cell imaging ( right panel ) analysis of chromosome segregation in H2BGFP - cells following siRNA treatment ; yellow arrows indicate the typical abnormal chromosomes by BCL9 knockdown . 23 H2BGFP - mitotic cells were monitored for each time experiment ; data are shown from three independent experiments . Scale bars represent 5 μm . Quantification of aberrant chromosome segregation rate by live cell imaging of H2BGFP cells upon Wnt3a or DKK1 treatment . 23 H2BGFP - mitotic cells were monitored for each time experiment ; data are shown from three independent experiments . Quantification of the duration of NEB to ANA ( NEB : nuclear envelope breakdown , ANA : anaphase ) , NEB to cell death in interphase after cell division ( blue colour bar ) or in mitosis ( dark red bar ) , or growth well in 30 hours ( pink bar ) by live cell imaging analysis in monitored H2BGFP cells with different treatments .

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Figure 2Immunoblotting analysis of BCL9 and cell cycle protein expression in double thymidine synchronised cells; the black arrow indicates the delayed band of mitotic BCL9.Immunofluorescence confocal analysis of BCL9 localization on the spindle by antibody staining. Yellow arrows indicate the spindle pole. Scale bars represent: 2 μm for interphase and anaphase; 5 μm for prophase, metaphase and telophase.Immunofluorescence confocal analysis of BCL9 localization on the centrosome. Yellow arrows indicate the centrosome. Scale bars represent: 5 μm for control treatment, 10 μm for BCL9 peptide treatment.Confocal microscope 3D-analysis and radial histograms of the spindle angle in the cells with siRNA treatment; yellow arrows indicate the centrosome. Scale bars represent 5 μm.Confocal (left panel) and live cell imaging (right panel) analysis of chromosome segregation in H2BGFP-cells following siRNA treatment; yellow arrows indicate the typical abnormal chromosomes by BCL9 knockdown. 23 H2BGFP-mitotic cells were monitored for each time experiment; data are shown from three independent experiments. Scale bars represent 5 μm. Quantification of aberrant chromosome segregation rate by live cell imaging of H2BGFP cells upon Wnt3a or DKK1 treatment. 23 H2BGFP-mitotic cells were monitored for each time experiment; data are shown from three independent experiments.Quantification of the duration of NEB to ANA (NEB: nuclear envelope breakdown, ANA: anaphase), NEB to cell death in interphase after cell division (blue colour bar) or in mitosis (dark red bar), or growth well in 30 hours (pink bar) by live cell imaging analysis in monitored H2BGFP cells with different treatments.


Figure 3Volcano plot of the BCL9 mitotic interactome by the SILAC proteomics approach . Red dashed line indicates a strict cut off by a normalised SILAC H / L ratio > 2 . An independent immunoprecipitation assay for validation of potential BCL9 interactors in both asynchronised and mitotic cells . The relative binding of CLTC with BCL9 is quantified and labelled on top of the CLTC blot . Immunoprecipitation assay of the endogenous BCL9 and Axin1 interaction in synchronised HeLa cells , from G1S to G2M . P - H3S10 , a mitotic marker indicating mitosis .

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Figure 3Volcano plot of the BCL9 mitotic interactome by the SILAC proteomics approach. Red dashed line indicates a strict cut off by a normalised SILAC H/L ratio > 2.An independent immunoprecipitation assay for validation of potential BCL9 interactors in both asynchronised and mitotic cells. The relative binding of CLTC with BCL9 is quantified and labelled on top of the CLTC blot.Immunoprecipitation assay of the endogenous BCL9 and Axin1 interaction in synchronised HeLa cells, from G1S to G2M. P-H3S10, a mitotic marker indicating mitosis.


Figure 4Duolink analysis ( A ) and its quantification ( B ) of the interaction by assessing the PLA complex in mitotic cells after BCL9 or Axin1 knockdown ( n = 20 ) . Scale bars represent 5 μm . Duolink analysis of the interaction of BCL9 and CLTC in mitotic cells . Scale bars represent 5 μm . Immunoprecipitation analysis of the interaction of truncated BCL9 forms in mitosis ; the purple stars indicate the domain required for clathrin complex binding , and the green stars indicate the domains for β - catenin complex binding . The domain required for both clathrin and β - catenin complexes binding is indicated by a red line . Quantification of the relative binding of the truncated forms with the clathrin complex or β - catenin complex ; the red frame indicates the common domains of BCL9 for binding of both complexes . Immunoprecipitation analysis of the BCL9 - MF interaction in CLTC knockdown mitotic cells . Immunoprecipitation analysis of the endogenous clathrin interaction with LRP6 signalosome components in BCL9 MF - overexpressing mitotic cells .

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Figure 4Duolink analysis (A) and its quantification (B) of the interaction by assessing the PLA complex in mitotic cells after BCL9 or Axin1 knockdown (n=20). Scale bars represent 5 μm.Duolink analysis of the interaction of BCL9 and CLTC in mitotic cells. Scale bars represent 5 μm.Immunoprecipitation analysis of the interaction of truncated BCL9 forms in mitosis; the purple stars indicate the domain required for clathrin complex binding, and the green stars indicate the domains for β-catenin complex binding. The domain required for both clathrin and β-catenin complexes binding is indicated by a red line.Quantification of the relative binding of the truncated forms with the clathrin complex or β-catenin complex; the red frame indicates the common domains of BCL9 for binding of both complexes.Immunoprecipitation analysis of the BCL9-MF interaction in CLTC knockdown mitotic cells.Immunoprecipitation analysis of the endogenous clathrin interaction with LRP6 signalosome components in BCL9 MF-overexpressing mitotic cells.


Figure 5Immunoblotting analysis of signalosome components by immunoblotting in BCL9 , CLTC or in both BCL9 and CLTC knockdown mitotic cells with CHX . Quantification of the stability of several LRP6 signalosome components by immunoblotting of mitotic cells with CHX . Immunofluorescence confocal analysis of Ser1490 - phosphorylated LRP6 and CLTC co - localization in mitotic HeLa cells ; white arrows indicate the colocalization . Scale bars represent 5 μm . Immunofluorescence analysis of α - tubulin , Axin1 and CLTC triple - staining in mitotic cells . The white arrows indicate the colocalization . Scale bars represent 5 μm . Immunofluorescence analysis of colocalization of LRP6CAGFP and CLTC or CD63 in interphase cells . The white arrows indicate the colocalization . Scale bars represent 5 μm . Quantification of signalosome components and mitotic Wnt target expression level by immunoblotting in natural mitotic cells treated for 1 hour with DMSO or MDC , an inhibitor of clathrin - mediated - endocytosis . A representative image of soft agar oncogenic colony formation assays for stable knockdown cells . A representative image of the mouse xenograft tumours for stable knockdown cells at the end point of the experiment .

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Figure 5Immunoblotting analysis of signalosome components by immunoblotting in BCL9, CLTC or in both BCL9 and CLTC knockdown mitotic cells with CHX. Quantification of the stability of several LRP6 signalosome components by immunoblotting of mitotic cells with CHX.Immunofluorescence confocal analysis of Ser1490-phosphorylated LRP6 and CLTC co-localization in mitotic HeLa cells; white arrows indicate the colocalization. Scale bars represent 5 μm.Immunofluorescence analysis of α-tubulin, Axin1 and CLTC triple-staining in mitotic cells. The white arrows indicate the colocalization. Scale bars represent 5 μm.Immunofluorescence analysis of colocalization of LRP6CAGFP and CLTC or CD63 in interphase cells. The white arrows indicate the colocalization. Scale bars represent 5 μm.Quantification of signalosome components and mitotic Wnt target expression level by immunoblotting in natural mitotic cells treated for 1 hour with DMSO or MDC, an inhibitor of clathrin-mediated-endocytosis.A representative image of soft agar oncogenic colony formation assays for stable knockdown cells.A representative image of the mouse xenograft tumours for stable knockdown cells at the end point of the experiment.


Figure 6In vitro CDK1 / cyclin B1 kinase assay for immunoprecipitation - purified BCL9 - Flag or T172A - Flag proteins . Immunoprecipitation analysis of transiently - expressed BCL9 - MF or T172A - MF interactors in synchronised cells , from G1S to G2 M phase . The relative level of BCL9 - MF or T172A - MF immunoprecipitation is determined ; red numbers indicate up - regulation , and green numbers indicate down - regulation compared to the starting time point . Immunoprecipitation analysis of the transiently - expressed BCL9 - MF interaction in mitotic cells treated with RO3306 . Immunoprecipitation analysis of the interaction of the T172 inactive mutant T172A , the activation mutant T172D , and the double inactive mutant T172 \ T144 ( 2A ) or activation mutant ( 2D ) transiently - expressed in mitotic cells . Quantification for the inactive or activation mutant interactions indicated in Fig 6F . The wild type BCL9 interaction is normalised to 1 , indicated by a green dashed line with an arrow .

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Figure 6In vitro CDK1/cyclin B1 kinase assay for immunoprecipitation-purified BCL9-Flag or T172A-Flag proteins.Immunoprecipitation analysis of transiently-expressed BCL9-MF or T172A-MF interactors in synchronised cells, from G1S to G2 M phase. The relative level of BCL9-MF or T172A-MF immunoprecipitation is determined; red numbers indicate up-regulation, and green numbers indicate down-regulation compared to the starting time point.Immunoprecipitation analysis of the transiently-expressed BCL9-MF interaction in mitotic cells treated with RO3306.Immunoprecipitation analysis of the interaction of the T172 inactive mutant T172A, the activation mutant T172D, and the double inactive mutant T172\T144 (2A) or activation mutant (2D) transiently-expressed in mitotic cells.Quantification for the inactive or activation mutant interactions indicated in Fig 6F. The wild type BCL9 interaction is normalised to 1, indicated by a green dashed line with an arrow.


Figure 7Immunohistochemical staining of p - T172 - BCL9 expression in a human HCC tissue . MN : matched normal tissues , T : tumour tissues . Scale bar represents1000 μm . IHC staining and the expression scored in a tissue array of 75 pairs of human lung cancer patient tissues . Scale bar represents 1000 μm . A representative figure of p - T172 - BCL9 IHC staining in HCC to indicate the expression in mitotic or in interphase cells . Scale bar represents 100 μm . Representative figures of p - T172 - BCL9 IHC staining in interphase cells of breast cancer ( D ) or lung cancer ( E ) patient tissue . Scale bars represent 100 μm . Immunoprecipitation analysis of the interaction in 5 pairs of human HCC patient tissues by a p - T172 - BCL9 antibody or a BCL9 antibody . CLTC relative binding with BCL9 was determined . Log - rank ( Mantel - Cox ) survival analysis based on high and low p - T172 - BCL9 expression scored by immunohistochemical staining in two tissue arrays with ( G ) 75 lung cancer samples or ( H ) 145 breast cancer samples .

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Figure 7Immunohistochemical staining of p-T172-BCL9 expression in a human HCC tissue. MN: matched normal tissues, T: tumour tissues. Scale bar represents1000 μm.IHC staining and the expression scored in a tissue array of 75 pairs of human lung cancer patient tissues. Scale bar represents 1000 μm.A representative figure of p-T172-BCL9 IHC staining in HCC to indicate the expression in mitotic or in interphase cells. Scale bar represents 100 μm.Representative figures of p-T172-BCL9 IHC staining in interphase cells of breast cancer (D) or lung cancer (E) patient tissue. Scale bars represent 100 μm.Immunoprecipitation analysis of the interaction in 5 pairs of human HCC patient tissues by a p-T172-BCL9 antibody or a BCL9 antibody. CLTC relative binding with BCL9 was determined.Log-rank (Mantel-Cox) survival analysis based on high and low p-T172-BCL9 expression scored by immunohistochemical staining in two tissue arrays with (G) 75 lung cancer samples or (H) 145 breast cancer samples.


Figure 1WT or SLC15A4feeble BMDCs unstimulated or infected with flagellin - expressing STm Cell pellets collected at the indicated time points after STm infection were lysed , fractionated by SDS - PAGE and immunoblotted for caspase - 1 ( E , F ) ( E Representative immunoblots showing pro - caspase - 1 ( pro - casp . - 1 ) and cleaved p20 ( casp . - 1 p20 ) bands ( E ) ( F , Quantification of band intensities for caspase - 1 p20 normalized to pro - caspase - 1 and actin ( E ) from three independent experiments are shown as fold change ( F Data information : Data represent mean ± SD . * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 . Two - tailed Student ' s t - test . WT or SLC15A4feeble BMDCs unstimulated or infected with flagellin - expressing STm Cell pellets collected at the indicated time points after STm infection were lysed , fractionated by SDS - PAGE and immunoblotted for GSDMD or LC - 3 G ) . Representative immunoblots showing GSDMD full length ( GSDMD FL ) , cleaved GSDMD N - terminal fragment ( GSDMD N - ter ) , LC3 - II and LC3 - I bands in cell lysates ( G ) . H , Quantification of band intensities for GSDMD N - ter normalized to actin ( G ) and LC3 - II normalized to LC3 - I and actin ( H ) from three independent experiments are shown as fold change , H ) Data information : Data represent mean ± SD . * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 . Two - tailed Student ' s t - test .

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Figure 1WT or SLC15A4feeble BMDCs unstimulated or infected with flagellin-expressing STm Cell pellets collected at the indicated time points after STm infection were lysed, fractionated by SDS-PAGE and immunoblotted for caspase-1 (E, F) (E Representative immunoblots showing pro-caspase-1 (pro-casp.-1) and cleaved p20 (casp.-1 p20) bands (E) (F, Quantification of band intensities for caspase-1 p20 normalized to pro-caspase-1 and actin (E) from three independent experiments are shown as fold change (F Data information: Data represent mean ± SD. * p<0.05; **p<0.01; ***p<0.001. Two-tailed Student's t-test.WT or SLC15A4feeble BMDCs unstimulated or infected with flagellin-expressing STm Cell pellets collected at the indicated time points after STm infection were lysed, fractionated by SDS-PAGE and immunoblotted for GSDMD or LC-3 G). Representative immunoblots showing GSDMD full length (GSDMD FL), cleaved GSDMD N-terminal fragment (GSDMD N-ter), LC3-II and LC3-I bands in cell lysates (G). H, Quantification of band intensities for GSDMD N-ter normalized to actin (G) and LC3-II normalized to LC3-I and actin (H) from three independent experiments are shown as fold change , H) Data information: Data represent mean ± SD. * p<0.05; **p<0.01; ***p<0.001. Two-tailed Student's t-test.


Figure 3Intestinal CD11c + DCs were isolated from colons of WT and SLC15A4feeble mice on day 16 and cultured overnight . B . Cell pellets from three independent experiments were immunoblotted for LC3 and actin . Upper panel . Representative immunoblot . Lower panel . Quantification of band intensities for LC3 - II normalized to LC3 - I and actin are shown as fold induction relative to samples from untreated mice . Note that LC3 - I signal from ex vivo samples is almost undetectable . Data information : Mean is indicated . * * p < 0 . 01 . Mann - Whitney non - parametric statistical test .

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Figure 3Intestinal CD11c+ DCs were isolated from colons of WT and SLC15A4feeble mice on day 16 and cultured overnight. B. Cell pellets from three independent experiments were immunoblotted for LC3 and actin. Upper panel. Representative immunoblot. Lower panel. Quantification of band intensities for LC3-II normalized to LC3-I and actin are shown as fold induction relative to samples from untreated mice. Note that LC3-I signal from ex vivo samples is almost undetectable. Data information: Mean is indicated. **p<0.01. Mann-Whitney non-parametric statistical test.


Figure 4WT or SLC15A4feeble BMDCs expressing ASC - GFP and mCherry - LC3 ( A , B were infected with flagellin - expressing STm and fixed A . Cells were analyzed by fluorescence microscopy . Representative images showing ASC speck ( green ) relative to mCherry - LC3 ( red ) in two infected WT and SLC15A4feeble DCs each . B . Quantification of LC3 fluorescence per unit area in a radius of 1 μm surrounding the ASC speck in 20 cells per cell type in each of 3 independent experiments . N , nucleus . Corresponding DIC images show nuclear position . Scale bar , 8 μm . Data information : Data represent mean ± SD . * * p < 0 . 01 . Two - tailed Student ' s t - test . WT or SLC15A4feeble BMDCs non - transduced ( C ) were infected with flagellin - expressing STm and fixed C . Cells were stained for endogenous ASC and p62 , labeled with DAPI and analyzed by fluorescence microscopy . Representative images showing ASC speck ( red ) relative to p62 ( green ) in two infected WT and SLC15A4feeble DCs each . Note p62 staining surrounding ASC specks in SLC15A4feeble DCs . D . Quantification of perinuclear ( within a radius of 3 μm from the nucleus ) ASC specks in at least 30 cells per cell type in each of 3 independent experiments . N , nucleus . Corresponding DIC images show nuclear position . Scale bar , 8 μm . Data information : Data represent mean ± SD . * * p < 0 . 01 . Two - tailed Student ' s t - test . SLC15A4feeble BMDCs transduced with SLC15A4 - GFP ( E were infected with flagellin - expressing STm and fixed ( E , Cells were stained for endogenous ASC , labeled with DAPI and analyzed by fluorescence microscopy . Representative images showing ASC speck ( red ) and SLC15A4 - GFP ( green ) in SLC15A4feeble DCs together with non - transduced SLC15A4feeble DCs . Note the perinuclear positioning of ASC specks in the transduced cells ( E ) . N , nucleus . Dotted white lines , cell outlines . Corresponding DIC images show nuclear position . Scale bar , 8 μm . SLC15A4feeble BMDCs transduced with SLC15A4 - GFP F , were infected with flagellin - expressing STm and fixed Quantification of perinuclear ( within a radius of 3 μm from the nucleus ) and non - perinuclear ASC specks in at least 30 cells per cell type in each of three independent experiments ( F ) . Data information : Data represent mean ± SD . * * p < 0 . 01 . Two - tailed Student ' s t - test . SLC15A4feeble BMDCs transduced with SLC15A4 - GFP G ) were infected with flagellin - expressing STm and lysed ( G ) 1 h after infection . G . Representative immunoblots showing pro - caspase - 1 ( pro - casp . - 1 ) and cleaved p20 ( casp . - 1 p20 ) , LC3 - II and LC3 - I , and actin bands .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4WT or SLC15A4feeble BMDCs expressing ASC-GFP and mCherry-LC3 (A, B were infected with flagellin-expressing STm and fixed A. Cells were analyzed by fluorescence microscopy. Representative images showing ASC speck (green) relative to mCherry-LC3 (red) in two infected WT and SLC15A4feeble DCs each. B. Quantification of LC3 fluorescence per unit area in a radius of 1 μm surrounding the ASC speck in 20 cells per cell type in each of 3 independent experiments. N, nucleus. Corresponding DIC images show nuclear position. Scale bar, 8 μm. Data information: Data represent mean ± SD. **p<0.01. Two-tailed Student's t-test.WT or SLC15A4feeble BMDCs non-transduced (C) were infected with flagellin-expressing STm and fixed C. Cells were stained for endogenous ASC and p62, labeled with DAPI and analyzed by fluorescence microscopy. Representative images showing ASC speck (red) relative to p62 (green) in two infected WT and SLC15A4feeble DCs each. Note p62 staining surrounding ASC specks in SLC15A4feeble DCs. D. Quantification of perinuclear (within a radius of 3 μm from the nucleus) ASC specks in at least 30 cells per cell type in each of 3 independent experiments. N, nucleus. Corresponding DIC images show nuclear position. Scale bar, 8 μm. Data information: Data represent mean ± SD. **p<0.01. Two-tailed Student's t-test.SLC15A4feeble BMDCs transduced with SLC15A4-GFP (E were infected with flagellin-expressing STm and fixed (E, Cells were stained for endogenous ASC, labeled with DAPI and analyzed by fluorescence microscopy. Representative images showing ASC speck (red) and SLC15A4-GFP (green) in SLC15A4feeble DCs together with non-transduced SLC15A4feeble DCs. Note the perinuclear positioning of ASC specks in the transduced cells (E). N, nucleus. Dotted white lines, cell outlines. Corresponding DIC images show nuclear position. Scale bar, 8 μm.SLC15A4feeble BMDCs transduced with SLC15A4-GFP F, were infected with flagellin-expressing STm and fixed Quantification of perinuclear (within a radius of 3 μm from the nucleus) and non-perinuclear ASC specks in at least 30 cells per cell type in each of three independent experiments (F). Data information: Data represent mean ± SD. **p<0.01. Two-tailed Student's t-test.SLC15A4feeble BMDCs transduced with SLC15A4-GFP G) were infected with flagellin-expressing STm and lysed (G) 1 h after infection. G. Representative immunoblots showing pro-caspase-1 (pro-casp.-1) and cleaved p20 (casp.-1 p20), LC3-II and LC3-I, and actin bands.


Figure 5WT or SLC15A4feeble BMDCs transduced with constitutively active RagB ( RagBQ99L ) or metap2 ( control ) were infected with flagellin - expressing STm . ( A , B ) . Cell pellets collected 1 h after STm infection were lysed , fractionated by SDS - PAGE and immunoblotted for phospho ( P ) and total mTOR , ULK1 kinase , p70 kinase and S6 , FLAG and actin . Representative immunoblots . Non - specific band right above FLAG - RagBQ99L is indicated with an asterisk ( A ) . Quantification of band intensities for P - mTOR normalized to total mTOR , P - ULK1 normalized to total ULK1 , P - p70 normalized to total p70 and P - S6 normalized to S6 , in transduced cells from 3 independent experiments are shown as fold change relative to WT control ( B ) . Data information : Data represent mean ± SD . * * p < 0 . 01 ; * * * p < 0 . 001 . Two - tailed Student ' s t - test . WT or SLC15A4feeble BMDCs transduced with constitutively active RagB ( RagBQ99L ) or metap2 ( control ) ( E , Cell pellets were probed for caspase - 1 , GSDMD , actin ( E , Representative immunoblots showing pro - caspase - 1 ( pro - casp . - 1 ) and cleaved p20 ( casp . - 1 p20 ) , GSDMD full length ( GSDMD FL ) and cleaved GSDMD N - terminal fragment ( GSDMD N - ter ) bands . WT or SLC15A4feeble BMDCs transduced with non - target control , ATG5 or ATG7 shRNAs G ) G ) . Cell pellets were probed for caspase - 1 , GSDMD , actin , ATG5 and ATG 7 ( G ) . Representative immunoblots showing pro - caspase - 1 ( pro - casp . - 1 ) and cleaved p20 ( casp . - 1 p20 ) , GSDMD full length ( GSDMD FL ) and cleaved GSDMD N - terminal fragment ( GSDMD N - ter ) bands . WT or SLC15A4feeble BMDCs transduced with constitutively active RagB ( RagBQ99L ) or metap2 ( control ) H , I ) and mcherry - LC3 ( H , I ) , ( H , I ) . Cells were stained for endogenous ASC , labeled with DAPI and analyzed by fluorescence microscopy . Representative images showing ASC speck ( green ) and LC3 ( red ) in SLC15A4feeble DCs ( H ) . Quantification of perinuclear ( within a radius of 3 μm from the nucleus ) in at least 30 cells per cell type in each of three independent experiments ( I ) . N , nucleus . Corresponding DIC images show nuclear position . Scale bar , 8 μ Data information : Data represent mean ± SD . * * p < 0 . 01 ; * * * p < 0 . 001 . Two - tailed Student ' s t - test .

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Figure 5WT or SLC15A4feeble BMDCs transduced with constitutively active RagB (RagBQ99L) or metap2 (control) were infected with flagellin-expressing STm. (A, B). Cell pellets collected 1 h after STm infection were lysed, fractionated by SDS-PAGE and immunoblotted for phospho (P) and total mTOR, ULK1 kinase, p70 kinase and S6, FLAG and actin. Representative immunoblots. Non-specific band right above FLAG-RagBQ99L is indicated with an asterisk (A). Quantification of band intensities for P-mTOR normalized to total mTOR, P-ULK1 normalized to total ULK1, P-p70 normalized to total p70 and P-S6 normalized to S6, in transduced cells from 3 independent experiments are shown as fold change relative to WT control (B). Data information: Data represent mean ± SD. **p<0.01; ***p<0.001. Two-tailed Student's t-test.WT or SLC15A4feeble BMDCs transduced with constitutively active RagB (RagBQ99L) or metap2 (control) (E, Cell pellets were probed for caspase-1, GSDMD, actin (E, Representative immunoblots showing pro-caspase-1 (pro-casp.-1) and cleaved p20 (casp.-1 p20), GSDMD full length (GSDMD FL) and cleaved GSDMD N-terminal fragment (GSDMD N-ter) bands.WT or SLC15A4feeble BMDCs transduced with non-target control, ATG5 or ATG7 shRNAs G) G). Cell pellets were probed for caspase-1, GSDMD, actin , ATG5 and ATG 7 (G). Representative immunoblots showing pro-caspase-1 (pro-casp.-1) and cleaved p20 (casp.-1 p20), GSDMD full length (GSDMD FL) and cleaved GSDMD N-terminal fragment (GSDMD N-ter) bands.WT or SLC15A4feeble BMDCs transduced with constitutively active RagB (RagBQ99L) or metap2 (control) H, I) and mcherry-LC3 (H, I), (H, I). Cells were stained for endogenous ASC, labeled with DAPI and analyzed by fluorescence microscopy. Representative images showing ASC speck (green) and LC3 (red) in SLC15A4feeble DCs (H). Quantification of perinuclear (within a radius of 3 μm from the nucleus) in at least 30 cells per cell type in each of three independent experiments (I). N, nucleus. Corresponding DIC images show nuclear position. Scale bar, 8 μ Data information: Data represent mean ± SD. **p<0.01; ***p<0.001. Two-tailed Student's t-test.


Figure 1F . Microscopic images of the Q97 - GFP fluorescence . Bars , 5 µm . G . The percentage of cells containing or lacking detectable aggregates was quantified . Cell boundaries are indicated by dashed lines .

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Figure 1F. Microscopic images of the Q97-GFP fluorescence. Bars, 5 µm. G. The percentage of cells containing or lacking detectable aggregates was quantified. Cell boundaries are indicated by dashed lines.


Figure 2E . Q97 - GFP was expressed in mia40 - 3 and corresponding wild type cells for the times indicated . Cells were harvested , washed and survivors were counted after plating on glucose plates . Mean values and standard deviations of three replicates are shown .

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Figure 2E. Q97-GFP was expressed in mia40-3 and corresponding wild type cells for the times indicated. Cells were harvested, washed and survivors were counted after plating on glucose plates. Mean values and standard deviations of three replicates are shown.


Figure 3B . Cell extracts were analyzed by Western blotting after shifting cultures to galactose for 16 h . 25 , Q25 - GFP ; 97 , Q97 - GFP ; ev , empty vector . C . Microscopy images of the indicated strains 12 h after shifting them to galactose . Note that in GAL - Mia40 cells the form and number of aggregates is very different to WT cells . Bar , 5 µm . E . PolyQ proteins were expressed for 24 h before survivors were counted . Mean values and standard deviations from three independent experiments are shown .

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Figure 3B. Cell extracts were analyzed by Western blotting after shifting cultures to galactose for 16 h. 25, Q25-GFP; 97, Q97-GFP; ev, empty vector.C. Microscopy images of the indicated strains 12 h after shifting them to galactose. Note that in GAL-Mia40 cells the form and number of aggregates is very different to WT cells. Bar, 5 µm.E. PolyQ proteins were expressed for 24 h before survivors were counted. Mean values and standard deviations from three independent experiments are shown.


Figure 4B . Still frames after different times of growth indicate a uniform fluorescence in Q25 - GFP - expressing cells . Q97 - GFP expression in wild type leads to many small and scattered aggregates per cell . In GAL - Mia40 cells , Q97 - GFP accumulates to much larger intensities but then suddenly collapses into one single aggregate per cell . The arrow depicts aggregate formation . Bars , 4 µm . C . Quantified signal intensities in cells of the indicated mutants . In top and middle graphs , each curve corresponds to a single cell . Averaged signals ( mean + SEM ) in bottom graph ( N = 20 for WT , N = 17 cells for GAL - Mia40 ) .

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Figure 4B. Still frames after different times of growth indicate a uniform fluorescence in Q25-GFP-expressing cells. Q97-GFP expression in wild type leads to many small and scattered aggregates per cell. In GAL-Mia40 cells, Q97-GFP accumulates to much larger intensities but then suddenly collapses into one single aggregate per cell. The arrow depicts aggregate formation. Bars, 4 µm. C. Quantified signal intensities in cells of the indicated mutants. In top and middle graphs, each curve corresponds to a single cell. Averaged signals (mean + SEM) in bottom graph (N=20 for WT, N=17 cells for GAL-Mia40).


Figure 5B . Q97 - GFP was expressed for 4 . 5 h in wild type and GAL - Mia40 cells in two biological replicates each . Cells were lysed with NP - 40 before soluble proteins were quantified . Mitochondrial proteins ( mito . ) and members of the mitochondrial carrier family ( carrier ) are visualized in a volcano plot . P - values were derived from a moderated t - test . Mitochondrial ( mito . ) and carrier proteins are indicated by yellow and red dots , respectively . Rnq1 is indicated in blue . I . Cells of the newly made strains were grown on galactose for 1 or 7 days , respectively . Cells were lysed before the distribution of Rnq1 in the soluble and pellet fractions was quantified . Shown are mean values and standard deviations of three independent replicates .

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Figure 5B. Q97-GFP was expressed for 4.5 h in wild type and GAL-Mia40 cells in two biological replicates each. Cells were lysed with NP-40 before soluble proteins were quantified. Mitochondrial proteins (mito.) and members of the mitochondrial carrier family (carrier) are visualized in a volcano plot. P-values were derived from a moderated t-test. Mitochondrial (mito.) and carrier proteins are indicated by yellow and red dots, respectively. Rnq1 is indicated in blue.I. Cells of the newly made strains were grown on galactose for 1 or 7 days, respectively. Cells were lysed before the distribution of Rnq1 in the soluble and pellet fractions was quantified. Shown are mean values and standard deviations of three independent replicates.


Figure 6C . ∆ ura3 cells expressing the Oxa1 - Ura3 reporter for the cytosolic accumulation of mitochondrial precursor proteins ( Hansen et al . , 2018 ) were transformed with plasmids expressing the proteins TDP - 43 , the TDP - 43Q331K mutant , Q25 - GFP , Q97 - GFP , α - synuclein , FUS or GFP from a constitutive glyceraldehyde - 3 - phosphate dehydrogenase ( GPD ) promoter . Note that the expression of all aggregation - prone proteins ( indicated by red arrows ) allows cells to grow on uracil - deficient plates indicating the cytosolic accumulation of the Oxa1 - Ura3 fusion protein . E . Wild type cells were transformed with plasmids for the expression of the indicated proteins . Q97 - GFPnF is a non - fluorescent version of Q97 - GFP . Close proximity of the two split GFP parts results in fluorescence which was visualized by microscopy . Shown are mean values and standard deviations of three replicates . Bar , 5 µm .

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Figure 6C. ∆ura3 cells expressing the Oxa1-Ura3 reporter for the cytosolic accumulation of mitochondrial precursor proteins (Hansen et al., 2018) were transformed with plasmids expressing the proteins TDP-43, the TDP-43Q331K mutant, Q25-GFP, Q97-GFP, α-synuclein, FUS or GFP from a constitutive glyceraldehyde-3-phosphate dehydrogenase (GPD) promoter. Note that the expression of all aggregation-prone proteins (indicated by red arrows) allows cells to grow on uracil-deficient plates indicating the cytosolic accumulation of the Oxa1-Ura3 fusion protein.E. Wild type cells were transformed with plasmids for the expression of the indicated proteins. Q97-GFPnF is a non-fluorescent version of Q97-GFP. Close proximity of the two split GFP parts results in fluorescence which was visualized by microscopy. Shown are mean values and standard deviations of three replicates. Bar, 5 µm.


Figure 7B . The open reading frames of the indicated proteins were cloned downstream of a GAL promoter in plasmids and transformed into cells harboring the Q97 - GFP expression plasmid . Cells harboring an empty vector ( ev ) were used for control . Cells were shifted to galactose medium overnight before they were dropped onto glucose or galactose plates . If cells are directly dropped from lactate medium onto galactose plates , only individual cells ( indicated by red protein names ) escaped the polyQ toxicity ( see Fig . EV4C ) . D . Cells expressing the indicated carrier proteins from GAL promoters were dropped onto galactose plates . Cell growth was quantified by densitometry from one representative plate . OM refers to carrier proteins that were expressed as fusions with an N - terminal outer membrane anchor . See Fig . EV4D , E for scans of the respective plates .

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Figure 7B. The open reading frames of the indicated proteins were cloned downstream of a GAL promoter in plasmids and transformed into cells harboring the Q97-GFP expression plasmid. Cells harboring an empty vector (ev) were used for control. Cells were shifted to galactose medium overnight before they were dropped onto glucose or galactose plates. If cells are directly dropped from lactate medium onto galactose plates, only individual cells (indicated by red protein names) escaped the polyQ toxicity (see Fig. EV4C).D. Cells expressing the indicated carrier proteins from GAL promoters were dropped onto galactose plates. Cell growth was quantified by densitometry from one representative plate. OM refers to carrier proteins that were expressed as fusions with an N-terminal outer membrane anchor. See Fig. EV4D, E for scans of the respective plates.