Figure 3B . The EBV infection efficiency in HK1 cells transfected with the indicated siRNAs was analyzed by FACS . The cells were infected with EBV - GFP virus at 24 h after siRNA transfection . The ratio of GFP - positive cells was quantified by FACS at 24 hpi . The mean value of the GFP - positive rates in the siNC transfected HK1 cells was normalized to 1 . M . The relative EBV production in C666 cells transfected with YTHDF1 - specific siRNAs ( siY1 - 1 and siY1 - 2 ) or the siNC control was measured by qPCR . The DNA from the cell - free culture supernatant was harvested at 24 h after siRNA transfection . The number of EBV copies in the supernatant of the cells was quantified by qPCR using specific primers targeting BamHI - W fragment region of EBV . The mean value of EBV copies in the siNC cells was normalized to 1 .

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Figure 3B. The EBV infection efficiency in HK1 cells transfected with the indicated siRNAs was analyzed by FACS. The cells were infected with EBV-GFP virus at 24 h after siRNA transfection. The ratio of GFP-positive cells was quantified by FACS at 24 hpi. The mean value of the GFP-positive rates in the siNC transfected HK1 cells was normalized to 1.M. The relative EBV production in C666 cells transfected with YTHDF1-specific siRNAs (siY1-1 and siY1-2) or the siNC control was measured by qPCR. The DNA from the cell-free culture supernatant was harvested at 24 h after siRNA transfection. The number of EBV copies in the supernatant of the cells was quantified by qPCR using specific primers targeting BamHI-W fragment region of EBV. The mean value of EBV copies in the siNC cells was normalized to 1.


Figure 4B . The relative mRNA levels of BZLF1 and BRLF1 in the latently infected or reactivated ( 24 h post NaB treatment ) CNE2EBV cells transfected with the YTHDF1 - specific siRNAs ( siY1 - 1 and siY1 - 2 ) or the siNC control . After 24 h of siRNA transfection , CNE2EBV cells were treated with NaB ( 2 mM ) or mock for 24 h , followed by qRT - PCR analysis . All the gene expression levels were normalized to the housekeeping gene ACTB . F . The protein level of YTHDF1 , BZLF1 and ACTB in CNE2EBV cells following EBV reactivation ( 24 h post - NaB treatment ) , EBV infected HK1 or NPEC2 - Bmi1 cells at 24 hpi . The whole - cell protein was extracted at same time as the RNA .

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Figure 4B. The relative mRNA levels of BZLF1 and BRLF1 in the latently infected or reactivated (24 h post NaB treatment) CNE2EBV cells transfected with the YTHDF1-specific siRNAs (siY1-1 and siY1-2) or the siNC control. After 24 h of siRNA transfection, CNE2EBV cells were treated with NaB (2 mM) or mock for 24 h, followed by qRT-PCR analysis. All the gene expression levels were normalized to the housekeeping gene ACTB.F. The protein level of YTHDF1, BZLF1 and ACTB in CNE2EBV cells following EBV reactivation (24 h post-NaB treatment), EBV infected HK1 or NPEC2-Bmi1 cells at 24 hpi. The whole-cell protein was extracted at same time as the RNA.


Figure 5D . The relative enrichment of the BZLF1 and BRLF1 mRNA in CNE2EBV cells with transfected with siYTHDF2 , siYTHDF3 or siNC siRNA following EBV reactivation . After 24 h of siRNA transfection , CNE2EBV cells were treated with TPA ( 30 ng / ml ) and NaB ( 2 mM ) for 24 h . The RIP assays were performed using YTHDF1 - specific antibody or IgG control in these cells . Fold enrichment was determined by calculating the 2 - ΔCt of the RIP sample relative to the input sample . E , F . The relative m6A and YTHDF1 enrichment of the BZLF1 ( E ) or BRLF1 ( F ) mRNAs measured in CNE2EBV cells transfected with the METTL14 - specific siRNAs ( siM14 ) or siNC control following EBV reactivation . After 24 h of siRNA transfection , CNE2EBV cells were treated with TPA ( 30 ng / ml ) and NaB ( 2 mM ) for 24 h , followed by MeRIP and RIP assays . The fold enrichment was determined by calculating the 2 - ΔCt of the RIP sample relative to the input sample .

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 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

Figure 5D. The relative enrichment of the BZLF1 and BRLF1 mRNA in CNE2EBV cells with transfected with siYTHDF2, siYTHDF3 or siNC siRNA following EBV reactivation. After 24 h of siRNA transfection, CNE2EBV cells were treated with TPA (30 ng/ml) and NaB (2 mM) for 24 h. The RIP assays were performed using YTHDF1-specific antibody or IgG control in these cells. Fold enrichment was determined by calculating the 2-ΔCt of the RIP sample relative to the input sample.E, F. The relative m6A and YTHDF1 enrichment of the BZLF1 (E) or BRLF1 (F) mRNAs measured in CNE2EBV cells transfected with the METTL14-specific siRNAs (siM14) or siNC control following EBV reactivation. After 24 h of siRNA transfection, CNE2EBV cells were treated with TPA (30 ng/ml) and NaB (2 mM) for 24 h, followed by MeRIP and RIP assays. The fold enrichment was determined by calculating the 2-ΔCt of the RIP sample relative to the input sample.


Figure 6D . The interaction between YTHDF1 and DCP2 was detected by co - IP in 293T cells . F . The interactions between endogenous YTHDF1 and ZAP , DDX17 or DCP2 were detected by endogenous IP assays in CNE2EBV cells transfected with YTHDF2 - , YTHDF3 - specific siRNAs or the siNC control . YTHDF1 , YTHDF2 , YTHDF3 , ZAP , DDX17 , and DCP2 were detected in the immunoprecipitated complexes using specific antibodies , respectively .

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Figure 6D. The interaction between YTHDF1 and DCP2 was detected by co-IP in 293T cells.F. The interactions between endogenous YTHDF1 and ZAP, DDX17 or DCP2 were detected by endogenous IP assays in CNE2EBV cells transfected with YTHDF2-, YTHDF3-specific siRNAs or the siNC control. YTHDF1, YTHDF2, YTHDF3, ZAP, DDX17, and DCP2 were detected in the immunoprecipitated complexes using specific antibodies, respectively.


Figure 7F . The relative mRNA levels of BZLF1 and BRLF1 in C666 cells transfected with YTHDF1 - specific siRNAs and XRN1 - specific siRNAs . The C666 cells were co - transfected with YTHDF1 - specific siRNAs and XRN1 - specific siRNAs for 24 h . The mRNA levels were determined using qPCR . G . The relative enrichment of BZLF1 and BRLF1 mRNAs by ZAP , DDX17 , or DCP2 in CNE2EBV cells following EBV reactivation . CNE2EBV cells were transfected with plasmids encoding myc - ZAP , myc - DDX17 , myc - DCP2 , or vector control . At 24 h post transfection , the cells were treated with TPA ( 30 ng / ml ) and NaB ( 2 mM ) for 24 h . The RIP assays were performed using Myc - beads . The fold enrichment was determined by calculating the 2 - ΔCt of the RIP sample relative to the input sample . H . Determination of the EBV infection efficiency in HK1 cells transfected with the XRN1 - specific siRNA or the siNC control . After 24 h of siRNA transfection , HK1 cells were infected with EBV and analyzed by FACS at 24 hpi .

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 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 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

Figure 7F. The relative mRNA levels of BZLF1 and BRLF1 in C666 cells transfected with YTHDF1-specific siRNAs and XRN1-specific siRNAs. The C666 cells were co-transfected with YTHDF1-specific siRNAs and XRN1-specific siRNAs for 24 h. The mRNA levels were determined using qPCR.G. The relative enrichment of BZLF1 and BRLF1 mRNAs by ZAP, DDX17, or DCP2 in CNE2EBV cells following EBV reactivation. CNE2EBV cells were transfected with plasmids encoding myc-ZAP, myc-DDX17, myc-DCP2, or vector control. At 24 h post transfection, the cells were treated with TPA (30 ng/ml) and NaB (2 mM) for 24 h. The RIP assays were performed using Myc-beads. The fold enrichment was determined by calculating the 2-ΔCt of the RIP sample relative to the input sample.H. Determination of the EBV infection efficiency in HK1 cells transfected with the XRN1-specific siRNA or the siNC control. After 24 h of siRNA transfection, HK1 cells were infected with EBV and analyzed by FACS at 24 hpi.


figf1 ( a ) Subcellular compartments from U937 cell extracts were separated by sedimentation velocity on discontinuous sucrose gradient and probed for the indicated proteins by immunoblotting . Numbers represent fractions from the gradient ( 1 , top ; 5 , bottom ) . ( b ) Immunoblot analysis of cellular compartments after purification of mitochondria by Qproteome Mitochondria Isolation Kit . ( c ) Immunoblot analysis of KDEL and IRGM distribution in different cellular fractions after purification of mitochondria as in b . d ) Intracellular localization of IRGM analysed by confocal microscopy . Cells were stained with antibodies against the indicated proteins or vitally with MitoTracker Red ( MTR ) where indicated . Images on the right are merged from images on the left . A - L ; endogenous IRGM localization in HeLa cells relative to calnexin , MTR , cytochrome c and COX IV . M - O ; IRGM localization in primary human macrophages relative to mitochondria visualized with cytochrome c antibody . Scale bars , 5 μm . ( e ) Analysis of IRGM ( green ) and COX IV ( red ) intensity as a function of distance along the line shown in the inset . Image in inset corresponds to panels J - L in d . Uncropped images of blots are shown in Supplementary Information , Fig . S8 .

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figf1(a) Subcellular compartments from U937 cell extracts were separated by sedimentation velocity on discontinuous sucrose gradient and probed for the indicated proteins by immunoblotting. Numbers represent fractions from the gradient (1, top; 5, bottom).(b) Immunoblot analysis of cellular compartments after purification of mitochondria by Qproteome Mitochondria Isolation Kit.(c) Immunoblot analysis of KDEL and IRGM distribution in different cellular fractions after purification of mitochondria as in b.d) Intracellular localization of IRGM analysed by confocal microscopy. Cells were stained with antibodies against the indicated proteins or vitally with MitoTracker Red (MTR) where indicated. Images on the right are merged from images on the left. A-L; endogenous IRGM localization in HeLa cells relative to calnexin, MTR, cytochrome c and COX IV. M-O; IRGM localization in primary human macrophages relative to mitochondria visualized with cytochrome c antibody. Scale bars, 5 μm.(e) Analysis of IRGM (green) and COX IV (red) intensity as a function of distance along the line shown in the inset. Image in inset corresponds to panels J-L in d. Uncropped images of blots are shown in Supplementary Information, Fig. S8.


figf2 ( a ) Purified mitochondria were left untreated , or were treated with Proteinase K ( Pr . K ) following osmotic shock ( OS ) , or solubilization of the total protein using Triton X - 100 ( TX - 100 ) , as indicated . Samples were analysed by immunoblotting with antibodies against the indicated proteins . ( b ) Mitochondrial preparations were subjected to membrane disruption by freeze - thaw cycles as indicated and accessibility of the indicated proteins to Proteinase K was examined by immunoblotting . ( c ) Immunoblot analysis of membranous organelles from U937 cells separated by isopycnic sucrose density - gradient centrifugation . Numbers represent fractions from the gradient ( 1 , top ; 12 , bottom ) . Uncropped images of blots are shown in Supplementary Information , Fig . S8 .

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figf2(a) Purified mitochondria were left untreated, or were treated with Proteinase K (Pr.K) following osmotic shock (OS), or solubilization of the total protein using Triton X-100 (TX-100), as indicated. Samples were analysed by immunoblotting with antibodies against the indicated proteins.(b) Mitochondrial preparations were subjected to membrane disruption by freeze-thaw cycles as indicated and accessibility of the indicated proteins to Proteinase K was examined by immunoblotting.(c) Immunoblot analysis of membranous organelles from U937 cells separated by isopycnic sucrose density-gradient centrifugation. Numbers represent fractions from the gradient (1, top; 12, bottom). Uncropped images of blots are shown in Supplementary Information, Fig. S8.


figf3 ( a ) Cells were transfected with control siRNA or with siRNA specific to IRGM ( top ) and DRP1 ( bottom ) for 48 h and protein samples from these cells were then analysed by immunoblotting with anti - IRGM and anti - DRP1 antibodies . Actin was used as a loading control . ( b ) HeLa cells were treated with siRNA specific to either IRGM or DRP1 as indicated and were then labelled with MTR and analysed by live - cell confocal microscopy . Scale bars , 5 μm . ( c ) Cells were treated with either control siRNA or with DRP1 , IRGM , ATG7 or BECN1 ( Beclin 1 ) siRNAs , labelled with MTR and the percentage of cells with mitochondrial morphologies ranging from normal , punctiform ( dots ) and elongated were quantified . Definition of mitochondrial morphologies and quantification criteria are given in Supplementary Information , Figure S2 . ( d ) Cells were transfected with either control siRNA or with siRNA specific to MFN1 / MFN2 , IRGM or IRGM and MFN1 / MFN2 , and then labelled with MTR and analysed for mitochondrial morphologies . Data are means ± s . e . m . ( c , n = 3 ; d , n = 4 ) . Asterisk indicates P 0 . 05 , double asterisks indicate P 0 . 01 and dagger indicates P > 0 . 05 ( t - test ) .

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figf3(a) Cells were transfected with control siRNA or with siRNA specific to IRGM (top) and DRP1 (bottom) for 48 h and protein samples from these cells were then analysed by immunoblotting with anti-IRGM and anti-DRP1 antibodies. Actin was used as a loading control.(b) HeLa cells were treated with siRNA specific to either IRGM or DRP1 as indicated and were then labelled with MTR and analysed by live-cell confocal microscopy. Scale bars, 5 μm.(c) Cells were treated with either control siRNA or with DRP1, IRGM, ATG7 or BECN1 (Beclin 1) siRNAs, labelled with MTR and the percentage of cells with mitochondrial morphologies ranging from normal, punctiform (dots) and elongated were quantified. Definition of mitochondrial morphologies and quantification criteria are given in Supplementary Information, Figure S2.(d) Cells were transfected with either control siRNA or with siRNA specific to MFN1/MFN2, IRGM or IRGM and MFN1/MFN2, and then labelled with MTR and analysed for mitochondrial morphologies. Data are means ± s.e.m. (c, n = 3; d, n = 4). Asterisk indicates P 0.05, double asterisks indicate P 0.01 and dagger indicates P > 0.05 (t-test).


figf4 ( a ) U937 cells were transfected with siRNA specific to FIS1 , DRP1 or IRGM ( or control oligonucleotides ) as indicated , followed by plasmid encoding GFP - LC3 . Autophagy was induced by treatment with hIFN - γ . GFP - LC3 puncta in cells were imaged by confocal microscopy . Scale bars , 5 μm . ( b ) LC3 puncta per cell were quantified from cells treated as in a . ( c ) U937 cells were transfected with siRNA specific to FIS1 , DRP1 or IRGM ( or control oligonucleotides ) as indicated , followed by plasmid encoding GFP - LC3 . Autophagy was induced by starvation . GFP - LC3 puncta in cells were imaged by confocal microscopy . Scale bars , 5 μm . ( d ) U937 cells were transfected with either control siRNA or siRNA specific to MFN1 / MFN2 , endogenous LC3 puncta per cell were quantified . ( e ) U937 cells were transfected with the indicated siRNA and infected with BCG . Autophagy was induced by starvation and phagosome maturation was analysed using LysoTracker probe ( LTR ) as a reporter of acidification . ( f ) U937 cells were treated with the indicated siRNA and infected with virulent M . tuberculosis H37Rv . Autophagy was induced by starvation , or cells were kept in full medium , as indicated . Colony forming units were counted , and expressed as a percentage of the number of plated bacterial colonies from untreated cells , to assess bacterial survival . Data are means ± s . e . m . ( n = 6 ; two independent transfections , 6 independent infections ) . Asterisk indicates P 0 . 05 , and dagger indicates P > 0 . 05 ( t - test ) for the indicated data .

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figf4(a) U937 cells were transfected with siRNA specific to FIS1, DRP1 or IRGM (or control oligonucleotides) as indicated, followed by plasmid encoding GFP-LC3. Autophagy was induced by treatment with hIFN-γ. GFP-LC3 puncta in cells were imaged by confocal microscopy. Scale bars, 5 μm. (b) LC3 puncta per cell were quantified from cells treated as in a.(c) U937 cells were transfected with siRNA specific to FIS1, DRP1 or IRGM (or control oligonucleotides) as indicated, followed by plasmid encoding GFP-LC3. Autophagy was induced by starvation. GFP-LC3 puncta in cells were imaged by confocal microscopy. Scale bars, 5 μm.(d) U937 cells were transfected with either control siRNA or siRNA specific to MFN1/MFN2, endogenous LC3 puncta per cell were quantified.(e) U937 cells were transfected with the indicated siRNA and infected with BCG. Autophagy was induced by starvation and phagosome maturation was analysed using LysoTracker probe (LTR) as a reporter of acidification.(f) U937 cells were treated with the indicated siRNA and infected with virulent M. tuberculosis H37Rv. Autophagy was induced by starvation, or cells were kept in full medium, as indicated. Colony forming units were counted, and expressed as a percentage of the number of plated bacterial colonies from untreated cells, to assess bacterial survival. Data are means ± s.e.m. (n = 6; two independent transfections, 6 independent infections). Asterisk indicates P 0.05, and dagger indicates P > 0.05 (t-test) for the indicated data.


figf5 ( b ) HeLa cells were transfected with plasmids encoding fluorescent protein - tagged IRGMb , IRGMd WT ( wild - type IRGMd ) or IRGMS47N mutant ( control cells ; vector expressing GFP only ) , and after 48 h were labelled with MTR and imaged by live - cell confocal microscopy . Scale bars , 5 μm . ( c ) Fluorescence intensity analysis of green and red channels along a line drawn through two adjacent cells , one GFP - IRGMd positive and one GFP - IRGMd negative , as indicated in the inset . ( d ) Quantification of GFP + MTR + cells from an experiment performed as in b . ( e ) Nitrocellulose filters spotted with the indicated lipids were incubated with GST or GST - IRGM . Binding was detected by immunoblotting . ( f ) Agarose beads ( control beads ) and cardiolipin - bound agarose beads ( CL beads ) were incubated with GST or GST - IRGMd . Proteins were eluted from the beads and immunoblotted . Two lanes on the left indicate immunoblotting of samples used to incubate beads . ( g ) Nitrocellulose filters spotted with the indicated lipids ( defined in e ) were incubated with wild - type IRGMd , IRGMdS47N , and the IRGM isoform IRGMb at the indicated concentrations . GST control is shown in Supplementary Information , Figure S1d . Membranes in e - g were probed with anti - GST antibody .

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figf5(b) HeLa cells were transfected with plasmids encoding fluorescent protein-tagged IRGMb, IRGMd WT (wild-type IRGMd) or IRGMS47N mutant (control cells; vector expressing GFP only), and after 48 h were labelled with MTR and imaged by live-cell confocal microscopy. Scale bars, 5 μm.(c) Fluorescence intensity analysis of green and red channels along a line drawn through two adjacent cells, one GFP-IRGMd positive and one GFP-IRGMd negative, as indicated in the inset.(d) Quantification of GFP+ MTR+ cells from an experiment performed as in b.(e) Nitrocellulose filters spotted with the indicated lipids were incubated with GST or GST-IRGM. Binding was detected by immunoblotting.(f) Agarose beads (control beads) and cardiolipin-bound agarose beads (CL beads) were incubated with GST or GST-IRGMd. Proteins were eluted from the beads and immunoblotted. Two lanes on the left indicate immunoblotting of samples used to incubate beads.(g) Nitrocellulose filters spotted with the indicated lipids (defined in e) were incubated with wild-type IRGMd, IRGMdS47N, and the IRGM isoform IRGMb at the indicated concentrations. GST control is shown in Supplementary Information, Figure S1d. Membranes in e-g were probed with anti-GST antibody.


figf6 ( a ) HeLa cells transfected with plasmids encoding GFP or GFP - IRGMd were stained with MTR and imaged by live - cell confocal microscopy . Scale bars , 5 μm . ( b - d ) Quantification of mitochondrial morphologies in cells transfected with IRGMd , IRGMdS47N or IRGMb GFP fusions ( control cells express GFP only ) . ( e ) HeLa were transfected with the indicated siRNAs and vector encoding GFP - IRGMd , and labelled with MTR . The percentage of cells with mitochondrial morphologies ranging from normal , punctiform ( dots ) and elongated were quantified . ( f ) HeLa were transfected with the indicated siRNAs and vector encoding GFP - IRGMd ( or vector encoding GFP only , as a control ) and labelled with MTR . Percentages of GFP + cells with MTR staining at 48 h were quantified . ( g ) Confocal microscopy images of HeLa cells transfected with vector encoding GFP - DRP1 ( A ) or GFP - IRGMd ( B - G ) and labelled with MTR ( A - D ) or antibodies against COX - IV ( F ) . A , steady - state DRP1 distribution ; B - D , translocation of IRGMd from the cytosol to mitochondria at indicated times post - transfection ; E - G , co - localization of IRGMd and COX - IV ( G is merged from E and F ) . Scale bars , 5 μm . ( h ) HeLa cells , co - transfected with GFP - IRGMd , and control , BECN1 or ATG7 siRNA , were labelled with MTR 48 h post - transfection , imaged by live - cell microscopy , and percentages of GFP + cells that were also MTR + were quantified . ( i ) Cells from Atg7 wild - type ( WT ) or Atg7 − / − MEFs transfected with vectors encoding GFP - IRGMd for 48 h were stained with MTR , imaged , and percentage of GFP + cells that were also MTR + cells was quantified . ( j ) Wild - type W2 ( Bax / Bak + / + ) or mutant D3 ( Bax / Bak − / − ) BMK cells were transfected with vectors encoding GFP - IRGMd and after 48 h were labelled with MTR . Cells were imaged by live - cell microscopy , and percentages of GFP + cells that were also MTR + were quantified . ( k ) HeLa cells were transfected with vectors encoding GFP - IRGMd , treated with z - VAD as indicated , and after 48 h stained with MTR . Cells were imaged by live - cell microscopy , and percentages of GFP + cells that were also MTR + were quantified . Data are means ± s . e . m . ( n = 3 ) . Dagger indicates P ≥ 0 . 05 , asterisk indicates P 0 . 05 and double asterisks indicate P 0 . 01 ( t - test ) for the indicated data .

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 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 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 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 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 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 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

figf6(a) HeLa cells transfected with plasmids encoding GFP or GFP-IRGMd were stained with MTR and imaged by live-cell confocal microscopy. Scale bars, 5 μm. (b-d) Quantification of mitochondrial morphologies in cells transfected with IRGMd, IRGMdS47N or IRGMb GFP fusions (control cells express GFP only).(e)HeLa were transfected with the indicated siRNAs and vector encoding GFP-IRGMd, and labelled with MTR. The percentage of cells with mitochondrial morphologies ranging from normal, punctiform (dots) and elongated were quantified.(f) HeLa were transfected with the indicated siRNAs and vector encoding GFP-IRGMd (or vector encoding GFP only, as a control) and labelled with MTR. Percentages of GFP+ cells with MTR staining at 48 h were quantified.(g) Confocal microscopy images of HeLa cells transfected with vector encoding GFP-DRP1 (A) or GFP-IRGMd (B-G) and labelled with MTR (A-D) or antibodies against COX-IV (F). A, steady-state DRP1 distribution; B-D, translocation of IRGMd from the cytosol to mitochondria at indicated times post-transfection; E-G, co-localization of IRGMd and COX-IV (G is merged from E and F). Scale bars, 5 μm.(h) HeLa cells, co-transfected with GFP-IRGMd, and control, BECN1 or ATG7 siRNA, were labelled with MTR 48 h post-transfection, imaged by live-cell microscopy, and percentages of GFP+ cells that were also MTR+ were quantified.(i) Cells from Atg7 wild-type (WT) or Atg7−/− MEFs transfected with vectors encoding GFP-IRGMd for 48 h were stained with MTR, imaged, and percentage of GFP+ cells that were also MTR+ cells was quantified.(j) Wild-type W2 (Bax/Bak+/+) or mutant D3 (Bax/Bak−/−) BMK cells were transfected with vectors encoding GFP-IRGMd and after 48 h were labelled with MTR. Cells were imaged by live-cell microscopy, and percentages of GFP+ cells that were also MTR+ were quantified.(k) HeLa cells were transfected with vectors encoding GFP-IRGMd, treated with z-VAD as indicated, and after 48 h stained with MTR. Cells were imaged by live-cell microscopy, and percentages of GFP+ cells that were also MTR+ were quantified. Data are means ± s.e.m. (n = 3). Dagger indicates P ≥ 0.05, asterisk indicates P 0.05 and double asterisks indicate P 0.01 (t-test) for the indicated data.


figf7 ( a ) Percentage of HeLa cells transfected with vectors encoding GFP or GFP - IRGMd that were rounded 48 h post - transfection . ( b ) HeLa cells transfected with plasmids encoding GFP or GFP - IRGMd were stained with 7 - AAD after 48 h and percentages of GFP + cells that were also 7 - AAD + were quantified . ( c ) HeLa cells transfected with vectors encoding GFP - IRGMd versus GFP - IRGMdS47N and YFP - IRGMb ( vector encoding GFP only was transfected as a control ) were stained with 7 - AAD after 48 h , and percentages of GFP + ( for IRGMd and IRGMS47N ) or YFP + ( for IRGMb ) cells that were also 7 - AAD + were quantified . ( d ) HeLa cells were transfected with plamsids encoding GFP - IRGMd , GFP - IRGMDS47N or YFP - IRGMb ( vector encoding GFP only was transfected as a control ) , and after 48 h were stained with propidium iodide ( PI ) . Percentages of GFP + or YFP + cells that were also PI + were quantified . ( e ) Images of HeLa cells transfected with vectors encoding GFP - IRGMd ( bottom ) were stained for active caspases 3 and 7 using FLICA dye . Staurosporine ( STS ; middle images ) was used as a positive control . Cells were transfected with vectors encoding GFP only as a control ( top ) . ( f ) HeLa cells were transfected with plasmids encoding GFP - IRGMd and were stained with antibodies against HMGB1 ( absence of nuclear HMGB1 stain is a marker of HMGB1 release ) or DAPI . Top : images indicate GFP - IRGMd ( left ) , HMGB1 ( middle ) and DAPI ( right ) localization . Bottom : phase - contrast microscopy image of cells ( left ) and image merged from other panels ( right ) . Scale bars , 5 μm . ( g - i ) Cells were transfected with plasmids encoding GFP or GFP - IRGMd , and control or DRP1 siRNA , as indicated . After 48 h , ( g ) cells were stained with 7 - AAD and percentages of GFP + cells that were also 7 - AAD + were quantified , ( h ) rounded cells were quantified , or ( i ) cells were stained with HMGB1 and HMGB1 + nuclei were quantified . Data are means ± s . e . m . ( n = 3 ) . Asterisk indicates P 0 . 05 and double asterisk indicate P 0 . 01 ( t - test ) .

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 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 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 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 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 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

figf7(a) Percentage of HeLa cells transfected with vectors encoding GFP or GFP-IRGMd that were rounded 48 h post-transfection.(b) HeLa cells transfected with plasmids encoding GFP or GFP-IRGMd were stained with 7-AAD after 48 h and percentages of GFP+ cells that were also 7-AAD+ were quantified.(c) HeLa cells transfected with vectors encoding GFP-IRGMd versus GFP-IRGMdS47N and YFP-IRGMb (vector encoding GFP only was transfected as a control) were stained with 7-AAD after 48 h, and percentages of GFP+ (for IRGMd and IRGMS47N) or YFP+ (for IRGMb) cells that were also 7-AAD+ were quantified.(d) HeLa cells were transfected with plamsids encoding GFP-IRGMd, GFP-IRGMDS47N or YFP-IRGMb (vector encoding GFP only was transfected as a control), and after 48 h were stained with propidium iodide (PI). Percentages of GFP+ or YFP+ cells that were also PI+ were quantified.(e) Images of HeLa cells transfected with vectors encoding GFP-IRGMd (bottom) were stained for active caspases 3 and 7 using FLICA dye. Staurosporine (STS; middle images) was used as a positive control. Cells were transfected with vectors encoding GFP only as a control (top).(f) HeLa cells were transfected with plasmids encoding GFP-IRGMd and were stained with antibodies against HMGB1 (absence of nuclear HMGB1 stain is a marker of HMGB1 release) or DAPI. Top: images indicate GFP-IRGMd (left), HMGB1 (middle) and DAPI (right) localization. Bottom: phase-contrast microscopy image of cells (left) and image merged from other panels (right). Scale bars, 5 μm.(g-i) Cells were transfected with plasmids encoding GFP or GFP-IRGMd, and control or DRP1 siRNA, as indicated. After 48 h, (g) cells were stained with 7-AAD and percentages of GFP+ cells that were also 7-AAD+ were quantified, (h) rounded cells were quantified, or (i) cells were stained with HMGB1 and HMGB1+ nuclei were quantified. Data are means ± s.e.m. (n = 3). Asterisk indicates P 0.05 and double asterisk indicate P 0.01 (t-test).


figf8 ( a , b ) HeLa cells were transfected with plasmids encoding GFP , CFP - IRGMa , GFP - IRGMc or GFP - IRGMd , and after 24 h ( a ) or 48 h ( b ) , stained with MTR . Percentages of positively transfected cells that were also MTR + were then quantified . ( c , d ) Cells were transfected as in a , b , and after 24 h ( c ) or 48 h ( d ) the percentages of positively transfected cells that were also rounded by morphology were quantified . ( b ) HeLa cells transfected with plasmids encoding GFP or GFP - IRGMd were stained with 7 - AAD after 48 h and percentages of GFP + cells that were also 7 - AAD + were quantified .

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

figf8(a, b) HeLa cells were transfected with plasmids encoding GFP, CFP-IRGMa, GFP-IRGMc or GFP-IRGMd, and after 24 h (a) or 48 h (b), stained with MTR. Percentages of positively transfected cells that were also MTR+ were then quantified. (c, d) Cells were transfected as in a, b, and after 24 h (c) or 48 h (d) the percentages of positively transfected cells that were also rounded by morphology were quantified.(b) HeLa cells transfected with plasmids encoding GFP or GFP-IRGMd were stained with 7-AAD after 48 h and percentages of GFP+ cells that were also 7-AAD+ were quantified.


Figure 1B Quantified fluorescence of UbG76V - GFP normalized to mRFP in the hypodermis from animals of the indicated timepoint ( in hours ) after the L4 stage . Animals have been exposed to the indicated knockdown RNAi bacterial strains or a strain that only contains the empty RNAi vector . * P < 0 . 001 , Student t - test corrected for multiple comparisons at each time point using the Holm - Sidek method . N = 20 animals per genotype and timepoint . Error bars indicate SEM . C , D Co - expression via the col - 19 promoter of UbG76V - GFP ( green ) and mRFP ( red ) in C . elegans hypodermis from a single integrated transgene at L4 + 48 hours . Wild - type animals are shown . Bar , 50 microns . UbG76V - GFP fluorescence is barely detectable . E , F Co - expression via the sur - 5 promoter of UbG76V - GFP ( green ) and mCherry ( red ) in C . elegans from two separate integrated transgenes ( with focus on the intestine ) at L4 + 48 hours . Wild - type animals are shown . Bar , 50 microns . UbG76V - GFP fluorescence is barely detectable . G , H Co - expression of UbG76V - GFP ( green ) and mRFP ( red ) in C . elegans hypodermis at L4 + 48 hours in dop - 1 ( vs100 ) mutants . Bar , 50 microns . Abundant , stable UbG76V - GFP fluorescence is observed . I , J Co - expression of UbG76V - GFP ( green ) and mCherry ( red ) in C . elegans from the sur - 5 promoter at L4 + 48 hours in dop - 1 ( vs100 ) mutants . Bar , 50 microns . Abundant , stable UbG76V - GFP fluorescence is observed , particularly in the intestine . K Quantified fluorescence of UbG76V - GFP normalized to mRFP in the hypodermis from animals of the indicated timepoint ( in hours ) after the L4 stage and of the indicated genotype . * P < 0 . 001 , Student t - test corrected for multiple comparisons at each time point using the Holm - Sidek method . N = 20 animals per genotype and timepoint . Error bars indicate SEM . L Quantified fluorescence of UbG76V - GFP normalized to mRFP in the hypodermis of animals at L4 + 48 hours and of then indicated genotypes . * * * P < 0 . 001 , ANOVA with Dunnetts posthoc comparison to the wild - type control . N = 20 animals per genotype and timepoint . Error bars indicate SEM . M Quantified fluorescence of UbG76V - GFP normalized to mCherry ( both expressed by the sur - 5 promoter ) in the intestine of animals at L4 + 48 hours and of then indicated genotypes . * * * P < 0 . 001 , * * P < 0 . 01 , * P < 0 . 05 , ANOVA with Dunnetts posthoc comparison to the wild - type control . N = 20 animals per genotype and timepoint . Error bars indicate SEM .

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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 1B Quantified fluorescence of UbG76V-GFP normalized to mRFP in the hypodermis from animals of the indicated timepoint (in hours) after the L4 stage. Animals have been exposed to the indicated knockdown RNAi bacterial strains or a strain that only contains the empty RNAi vector. *P<0.001, Student t-test corrected for multiple comparisons at each time point using the Holm-Sidek method. N=20 animals per genotype and timepoint. Error bars indicate SEM.C,D Co-expression via the col-19 promoter of UbG76V-GFP (green) and mRFP (red) in C. elegans hypodermis from a single integrated transgene at L4+48 hours. Wild-type animals are shown. Bar, 50 microns. UbG76V-GFP fluorescence is barely detectable.E,F Co-expression via the sur-5 promoter of UbG76V-GFP (green) and mCherry (red) in C. elegans from two separate integrated transgenes (with focus on the intestine) at L4+48 hours. Wild-type animals are shown. Bar, 50 microns. UbG76V-GFP fluorescence is barely detectable.G,H Co-expression of UbG76V-GFP (green) and mRFP (red) in C. elegans hypodermis at L4+48 hours in dop-1(vs100) mutants. Bar, 50 microns. Abundant, stable UbG76V-GFP fluorescence is observed.I,J Co-expression of UbG76V-GFP (green) and mCherry (red) in C. elegans from the sur-5 promoter at L4+48 hours in dop-1(vs100) mutants. Bar, 50 microns. Abundant, stable UbG76V-GFP fluorescence is observed, particularly in the intestine.K Quantified fluorescence of UbG76V-GFP normalized to mRFP in the hypodermis from animals of the indicated timepoint (in hours) after the L4 stage and of the indicated genotype. *P<0.001, Student t-test corrected for multiple comparisons at each time point using the Holm-Sidek method. N=20 animals per genotype and timepoint. Error bars indicate SEM.L Quantified fluorescence of UbG76V-GFP normalized to mRFP in the hypodermis of animals at L4+48 hours and of then indicated genotypes. ***P<0.001, ANOVA with Dunnetts posthoc comparison to the wild-type control. N=20 animals per genotype and timepoint. Error bars indicate SEM.M Quantified fluorescence of UbG76V-GFP normalized to mCherry (both expressed by the sur-5 promoter) in the intestine of animals at L4+48 hours and of then indicated genotypes. ***P<0.001, **P<0.01, *P<0.05, ANOVA with Dunnetts posthoc comparison to the wild-type control. N=20 animals per genotype and timepoint. Error bars indicate SEM.


Figure 2A Western blot of lysed nematodes at the L4 + 48 hour stage , probed with antibodies recognizing , ubiquitin , GFP , RPN - 11 , multiple 20S proteasome subunits , or actin as a loading control . Animals express UbG76V - GFP protein in their hypodermis . The position of molecular weight markers is shown on the left of each blot . The position of UbG76V - GFP protein , as well as UbG76V - GFP with the indicated number of additional ubiquitin moieties , based on molecular weight , is indicated on the right of each blot . Twenty animals were loaded per lane for each indicated genotype ( WT indicates wild type ) . B Western blot of lysed nematodes at the L4 + 48 hour stage , probed and annotated as in ( A ) . Animals express UbG76V - GFP protein from the sur - 5 promoter . Thirty animals were loaded per lane for each indicated genotype ( WT indicates wild type ) . C Quantified fluorescence of UbG76V - GFP normalized to mRFP from lysates of transgenic nematodes at L4 + 48 hours that express these reporters in the hypodermis . Animals were exposed to bacteria containing the indicated RNAi clone or an empty RNAi vector as a control . * P < 0 . 05 , ANOVA with Dunnetts posthoc comparison to the empty RNAi vector control . N = 3 trials . Error bars indicate SEM . D Quantified epoxomicin - sensitive proteasome activity ( as measured through the turnover of fluorescent chymotrypsin substrate ) from the same lysates and exposed to the same RNAi as in ( C ) . * P < 0 . 05 , ANOVA with Dunnetts posthoc comparison to the empty RNAi vector control . N = 3 trials . Error bars indicate SEM . E Quantified fluorescence of UbG76V - GFP normalized to mRFP from lysates of transgenic nematodes at L4 + 48 hours that express these reporters in the hypodermis . Animals were of the indicated genotype . * * * P < 0 . 001 , * * P < 0 . 01 , * P < 0 . 05 , ANOVA with Dunnetts posthoc comparison to wild type . N = 4 trials . Error bars indicate SEM . F Quantified epoxomicin - sensitive proteasome activity ( as measured through the turnover of fluorescent chymotrypsin substrate ) from the same lysates as in ( E ) . No statistical difference ( P < 0 . 05 cutoff ) was detected by ANOVA . N = 4 trials . G Percentage of live animals of the indicated genotype assayed after 6 - hour exposure to heat shock . 100 % of control animals of the same genotype but not given heat shock survived . * P < 0 . 01 , Student t - test . N = 3 trials . Error bars indicate SEM . H Percent of animals of the indicated genotype containing aggregates of Poly ( Q ) 44 : : YFP at the indicated time ( in days ) after L4 stage . * P < 0 . 01 , Student t - test . N = 3 trials , 50 - 80 animals per trial per day . Error bars indicate SEM .

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 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 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 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 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 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 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

Figure 2A Western blot of lysed nematodes at the L4+48 hour stage, probed with antibodies recognizing, ubiquitin, GFP, RPN-11, multiple 20S proteasome subunits, or actin as a loading control. Animals express UbG76V-GFP protein in their hypodermis. The position of molecular weight markers is shown on the left of each blot. The position of UbG76V-GFP protein, as well as UbG76V-GFP with the indicated number of additional ubiquitin moieties, based on molecular weight, is indicated on the right of each blot. Twenty animals were loaded per lane for each indicated genotype (WT indicates wild type).B Western blot of lysed nematodes at the L4+48 hour stage, probed and annotated as in (A). Animals express UbG76V-GFP protein from the sur-5 promoter. Thirty animals were loaded per lane for each indicated genotype (WT indicates wild type).C Quantified fluorescence of UbG76V-GFP normalized to mRFP from lysates of transgenic nematodes at L4+48 hours that express these reporters in the hypodermis. Animals were exposed to bacteria containing the indicated RNAi clone or an empty RNAi vector as a control. *P<0.05, ANOVA with Dunnetts posthoc comparison to the empty RNAi vector control. N=3 trials. Error bars indicate SEM.D Quantified epoxomicin-sensitive proteasome activity (as measured through the turnover of fluorescent chymotrypsin substrate) from the same lysates and exposed to the same RNAi as in (C). *P<0.05, ANOVA with Dunnetts posthoc comparison to the empty RNAi vector control. N=3 trials. Error bars indicate SEM.E Quantified fluorescence of UbG76V-GFP normalized to mRFP from lysates of transgenic nematodes at L4+48 hours that express these reporters in the hypodermis. Animals were of the indicated genotype. ***P<0.001, **P<0.01, *P<0.05, ANOVA with Dunnetts posthoc comparison to wild type. N=4 trials. Error bars indicate SEM.F Quantified epoxomicin-sensitive proteasome activity (as measured through the turnover of fluorescent chymotrypsin substrate) from the same lysates as in (E). No statistical difference (P<0.05 cutoff) was detected by ANOVA. N=4 trials.G Percentage of live animals of the indicated genotype assayed after 6-hour exposure to heat shock. 100% of control animals of the same genotype but not given heat shock survived. *P<0.01, Student t-test. N=3 trials. Error bars indicate SEM.H Percent of animals of the indicated genotype containing aggregates of Poly(Q)44::YFP at the indicated time (in days) after L4 stage. *P<0.01, Student t-test. N=3 trials, 50-80 animals per trial per day. Error bars indicate SEM.


Figure 3A Relative abundance of mRNA for the indicated xenobiotic stress response gene ( or UbG76V - GFP or mRFP ) detected in wild type versus dop - 1 mutants . Values are based on RNA - seq / EdgeR analysis , with the relative levels for each gene normalized to the value observed in the wild - type control . * * * * P < 0 . 0001 , * * P < 0 . 01 , * P < 0 . 05 FDR from EdgeR analysis using a Benjamini and Hochberg correction . B , C Quantified fluorescence of UbG76V - GFP normalized to ( B ) mRFP in the hypodermis from L4 + 48 hour animals exposed to feeding RNAi for either an empty vector control or the indicated xenobiotic stress gene . * * * * P < 0 . 0001 , * * * P < 0 . 001 , * * P < 0 . 01 , ANOVA , Tukey ' s multiple comparison test compared to control . N = 20 animals per genotype and timepoint . Error bars indicate SEM . B , C Quantified fluorescence of UbG76V - GFP normalized to ( C ) mCherry in the intestine from L4 + 48 hour animals exposed to feeding RNAi for either an empty vector control or the indicated xenobiotic stress gene . * * * * P < 0 . 0001 , * * * P < 0 . 001 , * * P < 0 . 01 , ANOVA , Tukey ' s multiple comparison test compared to control . N = 20 animals per genotype and timepoint . Error bars indicate SEM . D Percentage of live animals raised on the indicated feeding RNAi ( or empty vector control ) bacteria assayed after 6 - hour exposure to heat shock . 100 % of control animals of the same genotype but not given heat shock survived . * * * P < 0 . 001 , * * P < 0 . 01 , * P < 0 . 05 , ANOVA , Tukey ' s multiple comparison test compared to wild type . N = 3 trials . Error bars indicate SEM . E Quantified fluorescence of UbG76V - GFP normalized to mRFP in the hypodermis from L4 + 48 hour animals of the indicated genotype . * * * * P < 0 . 0001 , ANOVA , Tukey ' s multiple comparison test compared to wild type . N = 20 animals per genotype and timepoint . Error bars indicate SEM . F Percentage of live animals of the indicated genotype assayed after 6 - hour exposure to heat shock . 100 % of control animals of the same genotype but not given heat shock survived . * * P < 0 . 01 , * P < 0 . 05 , ANOVA , Tukey ' s multiple comparison test compared to wild type . N = 3 trials . Error bars indicate SEM . G Percent of animals of the indicated genotype containing aggregates of Poly ( Q ) 44 : : YFP at the indicated time ( in days ) after L4 stage . * P < 0 . 001 , Student t - test corrected for multiple comparisons at each time point using the Holm - Sidek method . N = 3 trials , 50 - 80 animals per trial per day . Error bars indicate SEM .

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 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 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 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 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 O O O O O O O O O O O O O O O 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

Figure 3A Relative abundance of mRNA for the indicated xenobiotic stress response gene (or UbG76V-GFP or mRFP) detected in wild type versus dop-1 mutants. Values are based on RNA-seq/EdgeR analysis, with the relative levels for each gene normalized to the value observed in the wild-type control. ****P<0.0001, **P<0.01, *P<0.05 FDR from EdgeR analysis using a Benjamini and Hochberg correction.B,C Quantified fluorescence of UbG76V-GFP normalized to (B) mRFP in the hypodermis from L4+48 hour animals exposed to feeding RNAi for either an empty vector control or the indicated xenobiotic stress gene. ****P<0.0001, ***P<0.001, **P<0.01, ANOVA, Tukey's multiple comparison test compared to control. N=20 animals per genotype and timepoint. Error bars indicate SEM.B,C Quantified fluorescence of UbG76V-GFP normalized to (C) mCherry in the intestine from L4+48 hour animals exposed to feeding RNAi for either an empty vector control or the indicated xenobiotic stress gene. ****P<0.0001, ***P<0.001, **P<0.01, ANOVA, Tukey's multiple comparison test compared to control. N=20 animals per genotype and timepoint. Error bars indicate SEM.D Percentage of live animals raised on the indicated feeding RNAi (or empty vector control) bacteria assayed after 6-hour exposure to heat shock. 100% of control animals of the same genotype but not given heat shock survived. ***P<0.001, **P<0.01, *P<0.05, ANOVA, Tukey's multiple comparison test compared to wild type. N=3 trials. Error bars indicate SEM.E Quantified fluorescence of UbG76V-GFP normalized to mRFP in the hypodermis from L4+48 hour animals of the indicated genotype. ****P<0.0001, ANOVA, Tukey's multiple comparison test compared to wild type. N=20 animals per genotype and timepoint. Error bars indicate SEM.F Percentage of live animals of the indicated genotype assayed after 6-hour exposure to heat shock. 100% of control animals of the same genotype but not given heat shock survived. **P<0.01, *P<0.05, ANOVA, Tukey's multiple comparison test compared to wild type. N=3 trials. Error bars indicate SEM.G Percent of animals of the indicated genotype containing aggregates of Poly(Q)44::YFP at the indicated time (in days) after L4 stage. *P<0.001, Student t-test corrected for multiple comparisons at each time point using the Holm-Sidek method. N=3 trials, 50-80 animals per trial per day. Error bars indicate SEM.


Figure 4A Quantified fluorescence of UbG76V - GFP normalized to mRFP in the hypodermis from L4 + 48 hour animals of the indicated genotype . * * * * P < 0 . 0001 , ANOVA , Tukey ' s multiple comparison test compared to wild type . N = 20 animals per genotype . Error bars indicate SEM . B Quantified fluorescence of UbG76V - GFP normalized to mRFP in the hypodermis from either L4 + 24 hour animals ( left half of graph ) or L4 + 48 hour animals ( right half of graph ) of the indicated genotype . Animals were either continually grown on regular OP50 food ( gray bars ) or switched to PA14 food at the L4 stage ( orange bars ) . * * * * P < 0 . 0001 , * * * P < 0 . 001 , * * P < 0 . 01 , ANOVA , Tukey ' s multiple comparison test compared to wild type . N = 20 animals per genotype . Error bars indicate SEM . C Survival curves ( percentage of live animals assayed each hour after PA14 exposure ) for the indicated mutants at 20oC . Plog - rank < 0 . 0001 . Median survival time was 90 hours for wildtype ( N = 304 ) , 72 hours for dop - 1 ( vs100 ) ( N = 180 ) , 78 hours for elt - 3 ( gk121 ) ( N = 120 ) , 78 hours for pqm - 1 ( ok485 ) ( N = 116 ) , 78 hours for nhr - 28 ( gk568 ) ( N = 123 ) , and 42 hours for skn - 1 ( tm3411 ) ( N = 33 ) . Eight wild - type animals and six dop - 1 mutants were censored ( the animals crawled up the side of the plate and dessicated ) . The media contained 50 µg / ml of 5 - fluoro - 2 ′ - deoxyuridine ( FUdR to prevent the growth of egg offspring during the experiment . D Quantified fluorescence of UbG76V - GFP normalized to mRFP in the hypodermis from either L4 + 48 hour animals ( left half of graph ) or L4 + 72 hour animals ( right half of graph ) of the indicated genotype . Animals were either continually grown on regular OP50 food ( gray bars ) or switched to NGM plates containing 1 mM paraquat at the L4 stage ( orange bars ) . * * * * P < 0 . 0001 , * * * P < 0 . 001 , ANOVA , Tukey ' s multiple comparison test compared to wild type . # P < 0 . 001 ANOVA , Bonferoni multicomparison as indicated by the bracketed lines . N = 20 animals per genotype . Error bars indicate SEM . No UbG76V - GFP fluorescence was detected in wild type at L4 + 72 hours on either OP50 or switched to NGM plates containing 1mM paraquat .

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 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 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 O O O 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 O O O O O O O O O O O O O

Figure 4A Quantified fluorescence of UbG76V-GFP normalized to mRFP in the hypodermis from L4+48 hour animals of the indicated genotype. ****P<0.0001, ANOVA, Tukey's multiple comparison test compared to wild type. N=20 animals per genotype. Error bars indicate SEM.B Quantified fluorescence of UbG76V-GFP normalized to mRFP in the hypodermis from either L4+24 hour animals (left half of graph) or L4+48 hour animals (right half of graph) of the indicated genotype. Animals were either continually grown on regular OP50 food (gray bars) or switched to PA14 food at the L4 stage (orange bars). ****P<0.0001, ***P<0.001, **P<0.01, ANOVA, Tukey's multiple comparison test compared to wild type. N=20 animals per genotype. Error bars indicate SEM.C Survival curves (percentage of live animals assayed each hour after PA14 exposure) for the indicated mutants at 20oC. Plog-rank<0.0001. Median survival time was 90 hours for wildtype (N=304), 72 hours for dop-1(vs100) (N=180), 78 hours for elt-3(gk121) (N=120), 78 hours for pqm-1(ok485) (N=116), 78 hours for nhr-28(gk568) (N=123), and 42 hours for skn-1(tm3411) (N=33). Eight wild-type animals and six dop-1 mutants were censored (the animals crawled up the side of the plate and dessicated). The media contained 50 µg/ml of 5-fluoro-2′-deoxyuridine (FUdR to prevent the growth of egg offspring during the experiment.D Quantified fluorescence of UbG76V-GFP normalized to mRFP in the hypodermis from either L4+48 hour animals (left half of graph) or L4+72 hour animals (right half of graph) of the indicated genotype. Animals were either continually grown on regular OP50 food (gray bars) or switched to NGM plates containing 1 mM paraquat at the L4 stage (orange bars). ****P<0.0001, ***P<0.001, ANOVA, Tukey's multiple comparison test compared to wild type. #P<0.001 ANOVA, Bonferoni multicomparison as indicated by the bracketed lines. N=20 animals per genotype. Error bars indicate SEM. No UbG76V-GFP fluorescence was detected in wild type at L4+72 hours on either OP50 or switched to NGM plates containing 1mM paraquat.


Figure 1Lgr5fl / fl / Axin2CreErt2 ( Lgr5 - / - ) and control mice were infected for 2 months with H . pylori . A Representative H & E staining from antral glands ( upper row ) . The dotted line marks the gland base cell area , which expands upon infection in Lgr5 - / - and control mice . Immunofluorescence labeling for GSII and Ki67 ( middle row ) and GIF and Muc5ac ( lower row ) of antral epithelium from Lgr5 - / - and control mice uninfected and after 2 months of infection . Scale bars : 50 µm . Lgr5fl / fl / Axin2CreErt2 ( Lgr5 - / - ) and control mice were infected for 2 months with H . pylori . B Quantification of antral gland height in Lgr5 - / - compared to control mice . Uninfected control ( n = 5 ) versus uninfected Lgr5 - / - ( n = 4 ) versus infected control ( n = 4 ) versus infected Lgr5 - / - ( n = 4 ) . Data information : B , data are mean + SD . * = p < 0 . 05 ; * * = p < 0 . 01 ; * * * = p < 0 . 001 ; ns = not significant ( one - way ANOVA + Tukey ´ s multiple comparison test for B , Lgr5fl / fl / Axin2CreErt2 ( Lgr5 - / - ) and control mice were infected for 2 months with H . pylori . C Quantifications of GSII + area ( n = 4 - 5 biological replicates per group , n = 10 glands per mouse ) , Ki67 as percentage of total DAPI - stained cells ( n = 3 - 4 biological replicates per group , n = 20 glands per mouse ) , GIF + area ( n = 4 - 5 biological replicates per group , n = 10 glands per mouse ) , Muc5ac + area ( n = 4 - 5 biological replicates per group , n = 10 glands per mouse ) . Data information : For ( one - way ANOVA + Tukey ´ s multiple comparison test for C , Lgr5fl / fl / Axin2CreErt2 ( Lgr5 - / - ) and control mice were infected for 2 months with H . pylori . D H . pylori CFUs in Lgr5 - / - ( n = 5 ) and control mice ( n = 5 ) . Data information : For D , data are mean + SD . * = p < 0 . 05 ; * * = p < 0 . 01 ; * * * = p < 0 . 001 ; ns = not significant t - test for DLgr5fl / fl / Axin2CreErt2 ( Lgr5 - / - ) and control mice were infected for 2 months with H . pylori . E qPCR expression levels of Lgr4 , Lgr5 , Axin2 , gland base cell markers ( Gif , Pgc , Tff2 , Muc6 ) , pit cell marker Muc5ac , and Itln1 in uninfected Lgr5 - / - ( n = 6 - 10 ) and control mice ( n = 4 - 10 ) . F qPCR expression levels of Lgr4 , Lgr5 , gland base cell markers ( Gif , Pgc , Muc6 ) , and Itln1 in Lgr5 - / - ( n = 5 ) and control mice ( n = 5 ) after 2 months of infection . Data information : For E and F , data are mean + SD . * = p < 0 . 05 ; * * = p < 0 . 01 ; * * * = p < 0 . 001 ; ns = not significant t - test for E and F ) .

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 O O O O O O 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 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 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 O O O O O O O O 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 1Lgr5fl/fl/Axin2CreErt2 (Lgr5-/-) and control mice were infected for 2 months with H. pylori. A Representative H&E staining from antral glands (upper row). The dotted line marks the gland base cell area, which expands upon infection in Lgr5-/- and control mice. Immunofluorescence labeling for GSII and Ki67 (middle row) and GIF and Muc5ac (lower row) of antral epithelium from Lgr5-/- and control mice uninfected and after 2 months of infection. Scale bars: 50 µm.Lgr5fl/fl/Axin2CreErt2 (Lgr5-/-) and control mice were infected for 2 months with H. pylori. B Quantification of antral gland height in Lgr5-/- compared to control mice. Uninfected control (n = 5) versus uninfected Lgr5-/- (n = 4) versus infected control (n = 4) versus infected Lgr5-/- (n = 4). Data information: B, data are mean + SD. * = p < 0.05; ** = p < 0.01; *** = p < 0.001; ns = not significant (one-way ANOVA + Tukey´s multiple comparison test for B,Lgr5fl/fl/Axin2CreErt2 (Lgr5-/-) and control mice were infected for 2 months with H. pylori. C Quantifications of GSII+ area (n = 4-5 biological replicates per group, n = 10 glands per mouse), Ki67 as percentage of total DAPI-stained cells (n = 3-4 biological replicates per group, n = 20 glands per mouse), GIF+ area (n = 4-5 biological replicates per group, n = 10 glands per mouse), Muc5ac+ area (n = 4-5 biological replicates per group, n = 10 glands per mouse). Data information: For (one-way ANOVA + Tukey´s multiple comparison test for C,Lgr5fl/fl/Axin2CreErt2 (Lgr5-/-) and control mice were infected for 2 months with H. pylori. D H. pylori CFUs in Lgr5-/- (n = 5) and control mice (n = 5). Data information: For D, data are mean + SD. * = p < 0.05; ** = p < 0.01; *** = p < 0.001; ns = not significant t-test for DLgr5fl/fl/Axin2CreErt2 (Lgr5-/-) and control mice were infected for 2 months with H. pylori. E qPCR expression levels of Lgr4, Lgr5, Axin2, gland base cell markers (Gif, Pgc, Tff2, Muc6), pit cell marker Muc5ac, and Itln1 in uninfected Lgr5-/- (n = 6-10) and control mice (n = 4-10). F qPCR expression levels of Lgr4, Lgr5, gland base cell markers (Gif, Pgc, Muc6), and Itln1 in Lgr5-/- (n = 5) and control mice (n = 5) after 2 months of infection. Data information: For E and F, data are mean + SD. * = p < 0.05; ** = p < 0.01; *** = p < 0.001; ns = not significant t-test for E and F).


Figure 2Lgr4fl / fl / Axin2CreErt2 ( Lgr4 - / - ) and control mice were infected for 2 months with H . pylori . A Representative H & E staining from antral glands ( upper row ) . The dotted line marks the gland base cell area , which does not expand in infected Lgr4 - / - but does expand in the corresponding control mice . Immunofluorescence labeling for GSII and Ki67 ( middle row ) and GIF and Muc5ac ( bottom row ) of antral epithelium from Lgr4 - / - and control mice uninfected and after 2 months of infection . Scale bars : 50 µm . Lgr4fl / fl / Axin2CreErt2 ( Lgr4 - / - ) and control mice were infected for 2 months with H . pylori . B Quantification of antral gland height in Lgr4 - / - compared to control mice . Uninfected control ( n = 2 ) versus uninfected Lgr4 - / - ( n = 4 ) versus infected control ( n = 4 ) versus infected Lgr4 - / - ( n = 4 ) . Data information : For B , , data are mean + SD . * = p < 0 . 05 ; * * = p < 0 . 01 ; * * * = p < 0 . 001 ; * * * * = p < 0 . 0001 ; ns = not significant ( one - way ANOVA + Tukey ´ s multiple comparison test for BLgr4fl / fl / Axin2CreErt2 ( Lgr4 - / - ) and control mice were infected for 2 months with H . pylori . C Quantifications of GSII + area ( n = 2 - 4 biological replicates per group , n = 10 glands per mouse ) , Ki67 as percentage of total DAPI - stained cells ( n = 2 - 4 biological replicates per group , n = 20 glands per mouse ) , GIF + area ( n = 2 - 4 biological replicates per group , n = 10 glands per mouse ) , Muc5ac + area ( n = 4 - 5 biological replicates per group , n = 10 glands per mouse ) . Data information : For C , data are mean + SD . * = p < 0 . 05 ; * * = p < 0 . 01 ; * * * = p < 0 . 001 ; * * * * = p < 0 . 0001 ; ns = not significant ( one - way ANOVA + Tukey ´ s multiple comparison test for C , Lgr4fl / fl / Axin2CreErt2 ( Lgr4 - / - ) and control mice were infected for 2 months with H . pylori . D H . pylori CFUs show higher colonization in Lgr4 - / - ( n = 5 ) compared to control mice ( n = 4 ) . Data information : For D , , data are mean + SD . * = p < 0 . 05 ; * * = p < 0 . 01 ; * * * = p < 0 . 001 ; * * * * = p < 0 . 0001 ; ns = not significant t - test for DLgr4fl / fl / Axin2CreErt2 ( Lgr4 - / - ) and control mice were infected for 2 months with H . pylori . E qPCR expression levels of Lgr4 , Lgr5 , Axin2 , gland base cell markers ( Gif , Pgc , Tff2 , Muc6 ) , pit cell marker Muc5ac , and Itln1 in uninfected Lgr4 - / - ( n = 4 - 10 ) and control mice ( n = 4 - 6 ) . F qPCR expression levels of Lgr4 , Lgr5 , gland base cell markers ( Gif , Pgc , Muc6 ) , and Itln1 in Lgr4 - / - ( n = 5 ) and control mice ( n = 5 ) after 2 months of infection . Data information : For E and F , data are mean + SD . * = p < 0 . 05 ; * * = p < 0 . 01 ; * * * = p < 0 . 001 ; * * * * = p < 0 . 0001 ; ns = not significant t - test for E and F ) .

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 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 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 O O O O O O O O O O O O O O O O O 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 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 O O O O O O O O O O O O O O O O O O O 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 2Lgr4fl/fl/Axin2CreErt2 (Lgr4-/-) and control mice were infected for 2 months with H. pylori. A Representative H&E staining from antral glands (upper row). The dotted line marks the gland base cell area, which does not expand in infected Lgr4-/- but does expand in the corresponding control mice. Immunofluorescence labeling for GSII and Ki67 (middle row) and GIF and Muc5ac (bottom row) of antral epithelium from Lgr4-/- and control mice uninfected and after 2 months of infection. Scale bars: 50 µm.Lgr4fl/fl/Axin2CreErt2 (Lgr4-/-) and control mice were infected for 2 months with H. pylori. B Quantification of antral gland height in Lgr4-/- compared to control mice. Uninfected control (n = 2) versus uninfected Lgr4-/- (n = 4) versus infected control (n = 4) versus infected Lgr4-/- (n = 4). Data information: For B, , data are mean + SD. * = p < 0.05; ** = p < 0.01; *** = p < 0.001; **** = p < 0.0001; ns = not significant (one-way ANOVA + Tukey´s multiple comparison test for BLgr4fl/fl/Axin2CreErt2 (Lgr4-/-) and control mice were infected for 2 months with H. pylori. C Quantifications of GSII+ area (n = 2-4 biological replicates per group, n = 10 glands per mouse), Ki67 as percentage of total DAPI-stained cells (n = 2-4 biological replicates per group, n = 20 glands per mouse), GIF+ area (n = 2-4 biological replicates per group, n = 10 glands per mouse), Muc5ac+ area (n = 4-5 biological replicates per group, n = 10 glands per mouse). Data information: For C, data are mean + SD. * = p < 0.05; ** = p < 0.01; *** = p < 0.001; **** = p < 0.0001; ns = not significant (one-way ANOVA + Tukey´s multiple comparison test for C,Lgr4fl/fl/Axin2CreErt2 (Lgr4-/-) and control mice were infected for 2 months with H. pylori. D H. pylori CFUs show higher colonization in Lgr4-/- (n = 5) compared to control mice (n = 4). Data information: For D, , data are mean + SD. * = p < 0.05; ** = p < 0.01; *** = p < 0.001; **** = p < 0.0001; ns = not significant t-test for DLgr4fl/fl/Axin2CreErt2 (Lgr4-/-) and control mice were infected for 2 months with H. pylori. E qPCR expression levels of Lgr4, Lgr5, Axin2, gland base cell markers (Gif, Pgc, Tff2, Muc6), pit cell marker Muc5ac, and Itln1 in uninfected Lgr4-/- (n = 4-10) and control mice (n = 4-6). F qPCR expression levels of Lgr4, Lgr5, gland base cell markers (Gif, Pgc, Muc6), and Itln1 in Lgr4-/- (n = 5) and control mice (n = 5) after 2 months of infection. Data information: For E and F, data are mean + SD. * = p < 0.05; ** = p < 0.01; *** = p < 0.001; **** = p < 0.0001; ns = not significant t-test for E and F).


Figure 3A , B GSEA comparing microarray data from 2 - month infected Lgr4fl / fl / Axin2CreErt2 ( Lgr4 - / - ) mice with publicly available data demonstrating downregulation of antral stem cell signature ( A ) and beta - catenin targets ( B ) . C qPCR expression levels for beta catenin targets Axin2 and Sox9 in uninfected and infected Lgr4 - / - and control mice ( n = 2 - 9 ) . Data information : For C , data are mean + SD . * = p < 0 . 05 ; * * = p < 0 . 01 ; * * * = p < 0 . 001 ( one - way ANOVA + Tukey ´ s multiple comparison test ) . D GSEA comparing microarray data from 2 - month infected Lgr4 - / - mice with publicly available data demonstrating downregulation of the TNF - α signaling via NF - κB . E Immunofluorescence labeling for p65 , Ki67 and GSII in the mouse antrum tissue from uninfected and infected Lgr4 - / - and control mice . Insets show magnified images . Scale bars : 50 µm . F Quantification of p65 + area as percentage of total DAPI - stained cells of uninfected and infected Lgr4 - / - and control mice ( n = 4 biological replicates per group , n = 20 glands per mouse ) . Data information : For data are mean + SD . * = p < 0 . 05 ; * * = p < 0 . 01 ; * * * = p < 0 . 001 ( one - way ANOVA + Tukey ´ s multiple comparison test ) . G Immunofluorescence co - localization of p65 acetyl K310 ( activated form of NF - κB ) in the mouse antrum tissue of infected Lgr4 - / - and in control mice . Insets show magnified images . Scale bars : 50µm . Data information : For G images represent findings that were reproduced at least twice in the laboratory in independent biological replicates . H qPCR expression levels of Cxcl1 , Cxcl2 , Cxcl10 , Ccl2 in uninfected and infected Lgr4 - / - mice ( n = 4 - 5 ) and control mice ( n = 2 - 5 ) . Data information : For H , data are mean + SD . * = p < 0 . 05 ; * * = p < 0 . 01 ; * * * = p < 0 . 001 ( one - way ANOVA + Tukey ´ s multiple comparison test ) . I Immunofluorescence labeling for Cxcl1 in the mouse antrum tissue in infected Lgr4 - / - and in control mice . Scale bars : 50 µm . Data information : For I images represent findings that were reproduced at least twice in the laboratory in independent biological replicates .

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 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 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 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 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 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 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

Figure 3A, B GSEA comparing microarray data from 2-month infected Lgr4fl/fl/Axin2CreErt2 (Lgr4-/-) mice with publicly available data demonstrating downregulation of antral stem cell signature (A) and beta-catenin targets (B).C qPCR expression levels for beta catenin targets Axin2 and Sox9 in uninfected and infected Lgr4-/- and control mice (n = 2-9). Data information: For C, data are mean + SD. * = p < 0.05; ** = p < 0.01; *** = p < 0.001 (one-way ANOVA + Tukey´s multiple comparison test).D GSEA comparing microarray data from 2-month infected Lgr4-/- mice with publicly available data demonstrating downregulation of the TNF-α signaling via NF-κB.E Immunofluorescence labeling for p65, Ki67 and GSII in the mouse antrum tissue from uninfected and infected Lgr4-/- and control mice. Insets show magnified images. Scale bars: 50 µm.F Quantification of p65+ area as percentage of total DAPI-stained cells of uninfected and infected Lgr4-/- and control mice (n = 4 biological replicates per group, n = 20 glands per mouse). Data information: For data are mean + SD. * = p < 0.05; ** = p < 0.01; *** = p < 0.001 (one-way ANOVA + Tukey´s multiple comparison test).G Immunofluorescence co-localization of p65 acetyl K310 (activated form of NF-κB) in the mouse antrum tissue of infected Lgr4-/- and in control mice. Insets show magnified images. Scale bars: 50µm. Data information: For G images represent findings that were reproduced at least twice in the laboratory in independent biological replicates.H qPCR expression levels of Cxcl1, Cxcl2, Cxcl10, Ccl2 in uninfected and infected Lgr4-/- mice (n = 4-5) and control mice (n = 2-5). Data information: For H, data are mean + SD. * = p < 0.05; ** = p < 0.01; *** = p < 0.001 (one-way ANOVA + Tukey´s multiple comparison test).I Immunofluorescence labeling for Cxcl1 in the mouse antrum tissue in infected Lgr4-/- and in control mice. Scale bars: 50 µm. Data information: For I images represent findings that were reproduced at least twice in the laboratory in independent biological replicates.


Figure 4A Immunofluorescence labeling for p65 and E - cadherin in the mouse antrum tissue of 2 weeks infected B6 - Myh11CreErt2 / Rspondin3fl / fl ( Rspo3 - / - ) and control mice . Arrows in infected control mice indicate nuclear p65 that is not observed in Rspo3 - / - mice . Scale bars : 50 µm . B Quantification of p65 + area as percentage of total DAPI - stained cells of infected Rspo3 - / - and control mice ( n = 3 biological replicates per group , n = 20 glands per mouse ) . Data information : For B , data are mean + SD . * = p < 0 . 05 ; * * = p < 0 . 01 ; ns = not significant ( t - test for BC qPCR expression level of Cxcl1 and Cxcl2 in 2 - week infected Rspo3 - / - mice ( n = 3 ) versus infected control mice ( n = 4 ) . D qPCR expression level of Cxcl1 and Cxcl2 in 2 - month infected Rspo3 - / - mice ( n = 5 ) versus infected control mice ( n = 6 ) . Data information : For C , D , data are mean + SD . * = p < 0 . 05 ; * * = p < 0 . 01 ; ns = not significant ( t - test for C and DE Myh11CreERT2 / Rosa26Sor6 ( CAG - Rspo3 ) ( Rspo3 KI ) mice were infected for 6 weeks with H . pylori . Immunofluorescence labeling for p65 , GSII and KI67 in uninfected and infected Rspo3 KI and control mice . Scale bars : 50 µm . F Quantification of p65 + area as percentage of total DAPI - stained cells of uninfected and infected Rspo3 KI and control mice ( n = 2 - 3 biological replicates per group , n = 20 glands per mouse ) . G Quantification of GSII + area of uninfected and infected Rspo3 KI and control mice ( n = 2 - 3 biological replicates per group , n = 20 glands per mouse ) . H Quantification of Ki67 as percentage of total DAPI - stained cells of uninfected and infected Rspo3 KI and control mice ( n = 2 - 3 biological replicates per group , n = 20 glands per mouse ) . Data information : For , F , G and H data are mean + SD . * = p < 0 . 05 ; * * = p < 0 . 01 ; ns = not significant one - way ANOVA + Tukey ´ s multiple comparison test for F , G and H ) . I Immunofluorescence labeling for p65 acetyl K310 in infected Rspo3 KI and control mice . Insets show magnified images . Scale bars : 50 µm .

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 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 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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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

Figure 4A Immunofluorescence labeling for p65 and E-cadherin in the mouse antrum tissue of 2 weeks infected B6-Myh11CreErt2/Rspondin3fl/fl (Rspo3-/-) and control mice. Arrows in infected control mice indicate nuclear p65 that is not observed in Rspo3-/- mice. Scale bars: 50 µm.B Quantification of p65+ area as percentage of total DAPI-stained cells of infected Rspo3-/- and control mice (n = 3 biological replicates per group, n = 20 glands per mouse). Data information: For B, data are mean + SD. * = p < 0.05; ** = p < 0.01; ns = not significant (t-test for BC qPCR expression level of Cxcl1 and Cxcl2 in 2-week infected Rspo3-/- mice (n = 3) versus infected control mice (n = 4). D qPCR expression level of Cxcl1 and Cxcl2 in 2-month infected Rspo3-/- mice (n = 5) versus infected control mice (n = 6). Data information: For C, D, data are mean + SD. * = p < 0.05; ** = p < 0.01; ns = not significant (t-test for C and DE Myh11CreERT2/Rosa26Sor6(CAG-Rspo3) (Rspo3 KI) mice were infected for 6 weeks with H. pylori. Immunofluorescence labeling for p65, GSII and KI67 in uninfected and infected Rspo3 KI and control mice. Scale bars: 50 µm. F Quantification of p65+ area as percentage of total DAPI-stained cells of uninfected and infected Rspo3 KI and control mice (n = 2-3 biological replicates per group, n = 20 glands per mouse). G Quantification of GSII+ area of uninfected and infected Rspo3 KI and control mice (n = 2-3 biological replicates per group, n = 20 glands per mouse). H Quantification of Ki67 as percentage of total DAPI-stained cells of uninfected and infected Rspo3 KI and control mice (n = 2-3 biological replicates per group, n = 20 glands per mouse). Data information: For , F, G and H data are mean + SD. * = p < 0.05; ** = p < 0.01; ns = not significant one-way ANOVA + Tukey´s multiple comparison test for F, G and H).I Immunofluorescence labeling for p65 acetyl K310 in infected Rspo3 KI and control mice. Insets show magnified images. Scale bars: 50 µm.


Figure 5A Immunofluorescence labelling for MPO in uninfected and 2 - month infected Lgr4 - / - and control mice ( top panel ) and 2 - month infected Lgr5 - / - and control mice ( bottom panel ) . Asterisks indicate nonspecific staining . Scale bars : 50 µm . B Quantification of MPO + cells in uninfected and 2 - month infected Lgr4 - / - and control mice ( n = 2 biological replicates per group , n = 5 - 10 FOVs per mouse ) ( left panel ) and 2 - month infected Lgr5 - / - and control mice ( n = 2 biological replicates per group , n = 6 - 8 FOVs per mouse ) ( right panel ) . Data information : For B data are mean + SD . * * = p < 0 . 01 ; * * * = p < 0 . 001 ; ns = not significant ( one - way ANOVA + Tukey ´ s multiple comparison test ) .

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 O O O 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 Immunofluorescence labelling for MPO in uninfected and 2-month infected Lgr4-/- and control mice (top panel) and 2-month infected Lgr5-/- and control mice (bottom panel). Asterisks indicate nonspecific staining. Scale bars: 50 µm. B Quantification of MPO+ cells in uninfected and 2-month infected Lgr4-/- and control mice (n = 2 biological replicates per group, n = 5-10 FOVs per mouse) (left panel) and 2-month infected Lgr5-/- and control mice (n = 2 biological replicates per group, n = 6-8 FOVs per mouse) (right panel). Data information: For B data are mean + SD. ** = p < 0.01; *** = p < 0.001; ns = not significant (one-way ANOVA + Tukey´s multiple comparison test).


Figure 6Antrum organoids from wildtype mice were grown in full medium ( FM ) for 2 - 3 days , followed by respective medium conditions for 3 - 5 days ( FM , - Rspo , - Rspo / - Wnt , + BMP2 ) . 0 . 5 µM ADP heptose or 10 ng / ml TNF - α was added for 2 h to all organoid conditions prior to analysis . A Bright - field images of organoids in respective conditions before treatment with ADP heptose or TNF - α . Scale bars : 200 µm . Antrum organoids from wildtype mice were grown in full medium ( FM ) for 2 - 3 days , followed by respective medium conditions for 3 - 5 days ( FM , - Rspo , - Rspo / - Wnt , + BMP2 ) . 0 . 5 µM ADP heptose or 10 ng / ml TNF - α was added for 2 h to all organoid conditions prior to analysis . B Immunofluorescence labeling for GSII and Muc5ac of organoids in respective conditions before treatment with ADP heptose or TNF - α . Scale bars : 50 µm . Antrum organoids from wildtype mice were grown in full medium ( FM ) for 2 - 3 days , followed by respective medium conditions for 3 - 5 days ( FM , - Rspo , - Rspo / - Wnt , + BMP2 ) . C qPCR expression level of Lgr5 in untreated organoids in respective medium conditions ( n = 4 - 5 biological replicates ) . D qPCR expression level of Gif in untreated organoids in respective medium conditions ( n = 4 - 5 biological replicates ) . Data information : For C , D , data are mean + SD . * = p < 0 . 05 ; * * = p < 0 . 01 ; * * * * = p < 0 . 0001 ( one - way ANOVA + Tukey ´ s multiple comparison test for C and Genes without asterisks are not significantly altered . Antrum organoids from wildtype mice were grown in full medium ( FM ) for 2 - 3 days , followed by respective medium conditions for 3 - 5 days ( FM , - Rspo , - Rspo / - Wnt , + BMP2 ) . 0 . 5 µM ADP heptose or 10 ng / ml TNF - α was added for 2 h to all organoid conditions prior to analysis . E qPCR expression level of Cxcl1 , Cxcl2 , Cxcl10 , Ccl2 in organoids after treatment with ADP heptose compared to untreated controls within the respective medium conditions ( n = 6 biological replicates per condition ) . F qPCR expression level of Cxcl1 , Cxcl2 , Cxcl10 , Ccl2 in organoids after treatment with TNF - α compared to untreated controls within the respective medium conditions ( n = 2 - 3 biological replicates per condition ) . Data information : For E and F data are mean + SD . * = p < 0 . 05 ; * * = p < 0 . 01 ; * * * * = p < 0 . 0001 ; t - test for E and F ) . Genes without asterisks are not significantly altered .

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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 6Antrum organoids from wildtype mice were grown in full medium (FM) for 2-3 days, followed by respective medium conditions for 3-5 days (FM, -Rspo, -Rspo/-Wnt, +BMP2). 0.5 µM ADP heptose or 10 ng/ml TNF-α was added for 2 h to all organoid conditions prior to analysis. A Bright-field images of organoids in respective conditions before treatment with ADP heptose or TNF-α. Scale bars: 200 µm.Antrum organoids from wildtype mice were grown in full medium (FM) for 2-3 days, followed by respective medium conditions for 3-5 days (FM, -Rspo, -Rspo/-Wnt, +BMP2). 0.5 µM ADP heptose or 10 ng/ml TNF-α was added for 2 h to all organoid conditions prior to analysis. B Immunofluorescence labeling for GSII and Muc5ac of organoids in respective conditions before treatment with ADP heptose or TNF-α. Scale bars: 50 µm.Antrum organoids from wildtype mice were grown in full medium (FM) for 2-3 days, followed by respective medium conditions for 3-5 days (FM, -Rspo, -Rspo/-Wnt, +BMP2). C qPCR expression level of Lgr5 in untreated organoids in respective medium conditions (n = 4-5 biological replicates). D qPCR expression level of Gif in untreated organoids in respective medium conditions (n = 4-5 biological replicates). Data information: For C, D, data are mean + SD. * = p < 0.05; ** = p < 0.01; **** = p < 0.0001 (one-way ANOVA + Tukey´s multiple comparison test for C and Genes without asterisks are not significantly altered.Antrum organoids from wildtype mice were grown in full medium (FM) for 2-3 days, followed by respective medium conditions for 3-5 days (FM, -Rspo, -Rspo/-Wnt, +BMP2). 0.5 µM ADP heptose or 10 ng/ml TNF-α was added for 2 h to all organoid conditions prior to analysis. E qPCR expression level of Cxcl1, Cxcl2, Cxcl10, Ccl2 in organoids after treatment with ADP heptose compared to untreated controls within the respective medium conditions (n = 6 biological replicates per condition). F qPCR expression level of Cxcl1, Cxcl2, Cxcl10, Ccl2 in organoids after treatment with TNF-α compared to untreated controls within the respective medium conditions (n = 2-3 biological replicates per condition). Data information: For E and F data are mean + SD. * = p < 0.05; ** = p < 0.01; **** = p < 0.0001 ; t-test for E and F). Genes without asterisks are not significantly altered.


Figure 7Antrum organoids from wildtype mice were grown in full medium ( FM ) for 2 - 3 days , followed by respective medium conditions for 3 - 5 days ( FM , - Rspo , - Rspo / - Wnt , + BMP2 ) . 0 . 5 µM ADP heptose was added for 2 h to all organoid conditions prior to analysis . A Electrophoretic mobility shift assay ( EMSA ) and SDS - PAGE western blot from whole cell protein ( WCL ) lysates from gastric organoids untreated or treated with ADP heptose , and maintained either in FM in medium without Rspo , without Wnt and Rspo , or + BMP2 . Representative images from n = 3 biological replicates . B Quantification of EMSA based on n = 3 biological replicates using ImageJ with normalization to loading ( ɑ - Tubulin ) . Data information : For B data are mean + SD . Significance is calculated by one - way ANOVA , F = 9 . 21 . C EMSA supershift from FM + ADP heptose organoids with the antibodies indicated . Blue arrow indicates NF - κB binding . Red arrow points to gel expected locations of supershifts from antibodies against p65 , cRel , or RelB . Grey arrow points to supershift location on the gel from antibodies against p52 or p50 . IgG lane serves as a negative control .

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 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

Figure 7Antrum organoids from wildtype mice were grown in full medium (FM) for 2-3 days, followed by respective medium conditions for 3-5 days (FM, -Rspo, -Rspo/-Wnt, +BMP2). 0.5 µM ADP heptose was added for 2 h to all organoid conditions prior to analysis. A Electrophoretic mobility shift assay (EMSA) and SDS-PAGE western blot from whole cell protein (WCL) lysates from gastric organoids untreated or treated with ADP heptose, and maintained either in FM in medium without Rspo, without Wnt and Rspo, or +BMP2. Representative images from n = 3 biological replicates. B Quantification of EMSA based on n = 3 biological replicates using ImageJ with normalization to loading (ɑ-Tubulin). Data information: For B data are mean + SD. Significance is calculated by one-way ANOVA, F = 9.21.C EMSA supershift from FM + ADP heptose organoids with the antibodies indicated. Blue arrow indicates NF-κB binding. Red arrow points to gel expected locations of supershifts from antibodies against p65, cRel, or RelB. Grey arrow points to supershift location on the gel from antibodies against p52 or p50. IgG lane serves as a negative control.


Figure 8A Bright - field images of antrum organoids from 129 ; 129P2 - ctnnb1tm ( NFKBIAΔN ) 1Rsu ( ΔN ) mice and control mice , passage 0 , day 6 . Scale bars : 500 µm . B , C qPCR expression levels of Lgr4 , Lgr5 , Gif , Pgc , Muc6 ( B ) and Cxcl1 , Cxcl2 ( C ) of organoids ( n = 3 biological replicates ) . Data information : For B , C , data are mean + SD . * = p < 0 . 05 ; * * = p < 0 . 01 ; ns = not significant ( t - test ) . D Representative H & E staining and immunofluorescence labeling for GSII , Ki67 and MUC5AC in uninfected ΔN and control mice . Scale bars : 50 µm . E Quantification of GSII and Ki67 as percentage of total DAPI - stained cells in ΔN and control mice ( n = 5 biological replicates per group , n = 20 glands per mouse ) . Data information : For E data are mean + SD . * = p < 0 . 05 ; * * = p < 0 . 01 ; ns = not significant ( t - test ) .

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 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 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

Figure 8A Bright-field images of antrum organoids from 129;129P2-ctnnb1tm(NFKBIAΔN)1Rsu (ΔN) mice and control mice, passage 0, day 6. Scale bars: 500 µm.B, C qPCR expression levels of Lgr4, Lgr5, Gif, Pgc, Muc6 (B) and Cxcl1, Cxcl2 (C) of organoids (n = 3 biological replicates). Data information: For B, C, data are mean + SD. * = p < 0.05; ** = p < 0.01; ns = not significant (t-test).D Representative H&E staining and immunofluorescence labeling for GSII, Ki67 and MUC5AC in uninfected ΔN and control mice. Scale bars: 50 µm. E Quantification of GSII and Ki67 as percentage of total DAPI-stained cells in ΔN and control mice (n = 5 biological replicates per group, n = 20 glands per mouse). Data information: For E data are mean + SD. * = p < 0.05; ** = p < 0.01; ns = not significant (t-test).


Figure 1 ( B - C ) Survival ( B ) and weight loss ( C ) . N = 10 mice for each group . The P - value of survival curve by Gehan - Breslow - Wilcoxon test is shown . ( E ) Cytokine expression levels of Tnfa , Ifng , Il1a , and Il1b were measured by RT - qPCR normalized by Ppia . Data are presented as mean ± SEM , N = 4 mice per group . * , p < 0 . 05 by unpaired Student ' s t - test with two sided .

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Figure 1(B-C) Survival (B) and weight loss (C). N = 10 mice for each group. The P-value of survival curve by Gehan-Breslow-Wilcoxon test is shown.(E) Cytokine expression levels of Tnfa, Ifng, Il1a, and Il1b were measured by RT-qPCR normalized by Ppia. Data are presented as mean ± SEM, N = 4 mice per group. *, p < 0.05 by unpaired Student's t-test with two sided.