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 .

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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 .

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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.


A 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 ) .

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A 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 ) .

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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 .

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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 .

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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 .

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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 .

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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 .

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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 .

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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.


A 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 .

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A 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 .

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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 .

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 .

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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 .

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 .

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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 .

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.


A 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 .

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A 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 .

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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 .

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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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR O

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.


Lgr5fl / 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 .

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Lgr5fl/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 ,

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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 ,

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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

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 D


Lgr5fl / 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 ) .

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Lgr5fl/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).


Lgr4fl / 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 .

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Lgr4fl/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

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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 B


Lgr4fl / 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 ,

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Lgr4fl/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

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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 D


Lgr4fl / 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 ) .

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Lgr4fl/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).


A , 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 ) .

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A, 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 ) .

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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 .

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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 .

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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 ) .

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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 .

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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 ) .

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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 .

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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.


A 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 .

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A 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

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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 B


C 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

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C 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 D


E 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 ) .

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E 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 .

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I Immunofluorescence labeling for p65 acetyl K310 in infected Rspo3 KI and control mice. Insets show magnified images. Scale bars: 50 µm.


A 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 ) .

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A 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).


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 . A Bright - field images of organoids in respective conditions before treatment with ADP heptose or TNF - α . Scale bars : 200 µm .

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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. 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 .

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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 .

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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 O O O O O O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O 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-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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.


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 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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 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 .

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.


A 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 .

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

A 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 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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).


( 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 .

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(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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(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.


( B - C ) Catalytically active sACE22 . v2 . 4 - IgG1 ( B ) and catalytically dead sACE22 . v2 . 4 ( NN ) - IgG1 ( C ) were immobilized on BLI biosensors that were transferred to solutions of gamma RBD as the soluble analyte ( 0 - 60 s ) and returned to buffer to measure dissociation ( 60 - 360 s ) . RBD concentrations are indicated on the right of the sensorgrams .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-C) Catalytically active sACE22.v2.4-IgG1 (B) and catalytically dead sACE22.v2.4(NN)-IgG1 (C) were immobilized on BLI biosensors that were transferred to solutions of gamma RBD as the soluble analyte (0-60 s) and returned to buffer to measure dissociation (60-360 s). RBD concentrations are indicated on the right of the sensorgrams.


( D - E ) Catalytically active sACE22 . v2 . 4 - IgG1 and catalytically dead sACE22 . v2 . 4 ( NN ) - IgG1 were aerosolized ( 7 . 5 ml protein at 8 . 3 mg / ml in 25 minutes ) and delivered by inhalation to K18 - hACE2 transgenic mice at 12 h , 48 h , and 84 h post - inoculation with SARS - CoV - 2 gamma variant . 10 mice in each group were observed for survival ( D ) and weight loss ( E ) . The P - value of survival curve by Gehan - Breslow - Wilcoxon test is shown . Error bars for mouse weight are centered on the mean and show SEM . Catalytically active and inactive proteins were tested in the same experiment versus PBS control shown in Figure 1 .

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

(D-E) Catalytically active sACE22.v2.4-IgG1 and catalytically dead sACE22.v2.4(NN)-IgG1 were aerosolized (7.5 ml protein at 8.3 mg/ml in 25 minutes) and delivered by inhalation to K18-hACE2 transgenic mice at 12 h, 48 h, and 84 h post-inoculation with SARS-CoV-2 gamma variant. 10 mice in each group were observed for survival (D) and weight loss (E). The P-value of survival curve by Gehan-Breslow-Wilcoxon test is shown. Error bars for mouse weight are centered on the mean and show SEM. Catalytically active and inactive proteins were tested in the same experiment versus PBS control shown in Figure 1.


( B - C ) Survival ( B ) and weight loss ( C ) following treatment with an IgG1 isotype control ( red ) versus catalytically active ( blue ) and dead ( green ) sACE22 . v2 . 4 - IgG1 . N = 8 mice for IgG1 group and catalytic active group , N = 9 for catalytic dead group . Error bars for mouse weight are centered on the mean and show SEM . Gehan - Breslow - Wilcoxon test was applied to analyze the survival curves . P = 0 . 0099 for catalytic active group compared to catalytic dead group . P = 0 . 017 for catalytic dead group compared to IgG1 control .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-C) Survival (B) and weight loss (C) following treatment with an IgG1 isotype control (red) versus catalytically active (blue) and dead (green) sACE22.v2.4-IgG1. N = 8 mice for IgG1 group and catalytic active group, N = 9 for catalytic dead group. Error bars for mouse weight are centered on the mean and show SEM. Gehan-Breslow-Wilcoxon test was applied to analyze the survival curves. P = 0.0099 for catalytic active group compared to catalytic dead group. P = 0.017 for catalytic dead group compared to IgG1 control.


( D - E ) Catalytically active sACE22 . v2 . 4 - IgG1 ( D ) and catalytically dead sACE22 . v2 . 4 ( NN ) - IgG1 ( E ) were immobilized on BLI biosensors and transferred to solutions of RBD ( original variant ) as the soluble analyte ( 0 - 60 s ) and returned to buffer to measure dissociation ( 60 - 360 s ) . RBD concentrations are indicated on the right of the BLI sensorgrams .

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

(D-E) Catalytically active sACE22.v2.4-IgG1 (D) and catalytically dead sACE22.v2.4(NN)-IgG1 (E) were immobilized on BLI biosensors and transferred to solutions of RBD (original variant) as the soluble analyte (0-60 s) and returned to buffer to measure dissociation (60-360 s). RBD concentrations are indicated on the right of the BLI sensorgrams.


( A ) Binding of monomeric sACE2 - 8his proteins to Expi293F cells expressing S of BA . 1 omicron was measured by flow cytometry .

(A) Binding of monomeric sACE2-8his proteins to Expi293F cells expressing S of BA.1 omicron was measured by flow cytometry.


( B ) Binding of dimeric sACE22 - IgG1 proteins was compared to the binding of antibodies authorized for therapeutic use in COVID - 19 patients . Binding to Expi293F cells expressing BA . 1 omicron S was measured by flow cytometry .

O O O O O O O O O O O O O O O O O O O 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) Binding of dimeric sACE22-IgG1 proteins was compared to the binding of antibodies authorized for therapeutic use in COVID-19 patients. Binding to Expi293F cells expressing BA.1 omicron S was measured by flow cytometry.


( C ) Neutralization of BA . 1 omicron pseudovirus . sACE22 - IgG1 ( grey ) or sACE22 . v2 . 4 - IgG1 ( blue ) were incubated with pseudovirus for 1 h before adding to HeLa - hACE2 - 11 cells . Infection 48 h later was measured by luciferase reporter gene expression . Data are mean ± SD , N = 3 independent biological replicates .

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

(C) Neutralization of BA.1 omicron pseudovirus. sACE22-IgG1 (grey) or sACE22.v2.4-IgG1 (blue) were incubated with pseudovirus for 1 h before adding to HeLa-hACE2-11 cells. Infection 48 h later was measured by luciferase reporter gene expression. Data are mean ± SD, N = 3 independent biological replicates.


( D - E ) Authentic BA . 1 omicron virus ( isolate USA / MD - HP20874 / 2021 ) was incubated with sACE22 - IgG1 ( D ) or sACE22 . v2 . 4 - IgG1 ( E ) for 1 h and added to Calu - 3 cells . Infection 48 h later was measured by RT - qPCR for the viral N gene . 3 µM remdesivir ( black columns ) is a positive neutralization control .

(D-E) Authentic BA.1 omicron virus (isolate USA/MD-HP20874/2021) was incubated with sACE22-IgG1 (D) or sACE22.v2.4-IgG1 (E) for 1 h and added to Calu-3 cells. Infection 48 h later was measured by RT-qPCR for the viral N gene. 3 µM remdesivir (black columns) is a positive neutralization control.


( B - C ) Survival ( B ) and weight loss ( C ) . N = 8 ( IgG1 control ) or 9 ( sACE22 . v2 . 4 - IgG1 treated ) mice . Error bars for mouse weight are centered on the mean and show SEM .

O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-C) Survival (B) and weight loss (C). N = 8 (IgG1 control) or 9 (sACE22.v2.4-IgG1 treated) mice. Error bars for mouse weight are centered on the mean and show SEM.


( E ) Viral Spike , Nsp , and Rdrp mRNA levels in lung tissue were measured by RT - qPCR at Days 8 and 14 , normalized to Ppia transcript levels . Data are presented as mean ± SEM , N = 4 mice per group . ns , not significant ; * * , p < 0 . 01 by two - ways ANOVA , corrected by Tukey .

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

(E) Viral Spike, Nsp, and Rdrp mRNA levels in lung tissue were measured by RT-qPCR at Days 8 and 14, normalized to Ppia transcript levels. Data are presented as mean ± SEM, N = 4 mice per group. ns, not significant; **, p < 0.01 by two-ways ANOVA, corrected by Tukey.


( A ) Using flow cytometry , binding to Expi293F cells expressing S of BA . 2 omicron was measured for monomeric sACE2 - 8his proteins . ( B ) Binding of antibodies and dimeric sACE22 - IgG1 proteins to Expi293F cells expressing BA . 2 omicron S measured by flow cytometry .

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

(A) Using flow cytometry, binding to Expi293F cells expressing S of BA.2 omicron was measured for monomeric sACE2-8his proteins. (B) Binding of antibodies and dimeric sACE22-IgG1 proteins to Expi293F cells expressing BA.2 omicron S measured by flow cytometry.


( C ) BA . 2 omicron pseudovirus was incubated with sACE22 - IgG1 ( grey ) or sACE22 . v2 . 4 - IgG1 ( blue ) for 1 h and added to HeLa - hACE2 - 11 cells . Infection was measured after 48 h .

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

(C) BA.2 omicron pseudovirus was incubated with sACE22-IgG1 (grey) or sACE22.v2.4-IgG1 (blue) for 1 h and added to HeLa-hACE2-11 cells. Infection was measured after 48 h.


( D - E ) Binding of monomeric sACE2 - 8his proteins ( D ) and dimeric IgG1 proteins ( E ) to Expi293F cells expressing BA . 4 / BA . 5 omicron S as measured by flow cytometry .

(D-E) Binding of monomeric sACE2-8his proteins (D) and dimeric IgG1 proteins (E) to Expi293F cells expressing BA.4/BA.5 omicron S as measured by flow cytometry.


Mouse bone marrow - derived macrophages ( BMDMs ) infected with Mtb Erd - tdTomato and imaged by time - lapse microscopy at 1 - or 2 - hour intervals for 132 hours BMDMs infected with aggregates of Mtb Erd - tdTomato from axenic culture and imaged by time - lapse microscopy at 1 - hour intervals for 60 hours ( A - B ) Percentage of macrophages surviving after interaction with an extracellular Mtb aggregate originating from the debris of dead macrophages ( A ) or from axanic culture ( B ) . Each line represents an independent experimental replicate ( n = 3 replicates with ≥ 100 cells per replicate ) .

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

Mouse bone marrow-derived macrophages (BMDMs) infected with Mtb Erd-tdTomato and imaged by time-lapse microscopy at 1- or 2-hour intervals for 132 hours BMDMs infected with aggregates of Mtb Erd-tdTomato from axenic culture and imaged by time-lapse microscopy at 1-hour intervals for 60 hours (A-B) Percentage of macrophages surviving after interaction with an extracellular Mtb aggregate originating from the debris of dead macrophages (A) or from axanic culture (B). Each line represents an independent experimental replicate (n = 3 replicates with ≥ 100 cells per replicate).


BMDMs infected with aggregates of Mtb Erd - tdTomato from axenic culture and imaged by time - lapse microscopy at 1 - hour intervals for 60 hours E , F ( E ) Example of a macrophage that interacts with an Mtb aggregate ( 0h00 ) , fragments it ( 3h00 ) , redistributes the bacteria in a " bullseye " pattern around the nucleus ( 12h00 ) , and dies ( 16h00 ) . Scale bar , 20 μm . ( F ) Example of a macrophage that stably interacts with an Mtb aggregate without fragmenting it ( 0h00 - 09h00 ) and ultimately dies ( 12h00 ) . In ( E and F ) , Draq7 staining of the nucleus ( in blue ) is used as a marker for cell death . Scale bar , 20 μm .

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

BMDMs infected with aggregates of Mtb Erd-tdTomato from axenic culture and imaged by time-lapse microscopy at 1-hour intervals for 60 hours E, F (E) Example of a macrophage that interacts with an Mtb aggregate (0h00), fragments it (3h00), redistributes the bacteria in a "bullseye" pattern around the nucleus (12h00), and dies (16h00). Scale bar, 20 μm. (F) Example of a macrophage that stably interacts with an Mtb aggregate without fragmenting it (0h00-09h00) and ultimately dies (12h00). In (E and F), Draq7 staining of the nucleus (in blue) is used as a marker for cell death. Scale bar, 20 μm.


BMDMs infected with aggregates of Mtb Erd - tdTomato from axenic culture and imaged by time - lapse microscopy at 1 - hour intervals for 60 hours ( G - H ) BMDMs infected with aggregates of Mtb were imaged by time - lapse microscopy ( every 30 min for up to 13 . 5h ) followed by fixation , immunostaining ( nuclei stained with Hoechst , membrane staining with anti - CD45 antibody ) and imaging by confocal microscopy . White arrows indicate intracellular bacteria , cyan arrows indicate extracellular bacteria . All scale bars , 10 μM . ( G . I , H . I ) Time - lapse microscopy image - series of macrophages that interacts with Mtb aggregates and fragment ( G ) or do not fragment ( H ) them . ( G . II , H . II ) Max intensity projection of confocal microscopy images of the same macrophages shown in panels I . ( G . III , H . III ) 3 - D reconstruction of the cells imaged in panels II , images are cropped in x or y in the position indicated by the white dotted lines in panels II to show the inside of the cell .

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

BMDMs infected with aggregates of Mtb Erd-tdTomato from axenic culture and imaged by time-lapse microscopy at 1-hour intervals for 60 hours (G-H) BMDMs infected with aggregates of Mtb were imaged by time-lapse microscopy (every 30 min for up to 13.5h) followed by fixation, immunostaining (nuclei stained with Hoechst, membrane staining with anti-CD45 antibody) and imaging by confocal microscopy. White arrows indicate intracellular bacteria, cyan arrows indicate extracellular bacteria. All scale bars, 10 μM. (G.I,H.I) Time-lapse microscopy image-series of macrophages that interacts with Mtb aggregates and fragment (G) or do not fragment (H) them. (G.II,H.II) Max intensity projection of confocal microscopy images of the same macrophages shown in panels I. (G.III,H.III) 3-D reconstruction of the cells imaged in panels II, images are cropped in x or y in the position indicated by the white dotted lines in panels II to show the inside of the cell.


BMDMs infected with aggregates of Mtb Erd - tdTomato from axenic culture and imaged by time - lapse microscopy at 1 - hour intervals for 60 hours ( I - M ) BMDMs infected with aggregates of Mtb were imaged by time - lapse fluorescence microscopy followed by SEM . ( I ) Example of a macrophage showing the typical " bullseye " pattern of bacterial redistribution around the cell nucleus . Scale bar , 10 μm . ( J ) Correlative SEM image of ( I ) . ( K ) Example of a macrophage interacting with an extracellular Mtb aggregate without complete fragmentation and redistribution of bacteria . White arrows indicate intracellular bacteria that have been detached from the main aggregate and internalized . Scale bar , 10 μm . Time - lapse microscopy image - series of these macrophages are shown in Appendix Fig S4A . ( L - M ) Correlative SEM image of ( K ) . Scale bar , 2 μm ( M ) .

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

BMDMs infected with aggregates of Mtb Erd-tdTomato from axenic culture and imaged by time-lapse microscopy at 1-hour intervals for 60 hours (I-M) BMDMs infected with aggregates of Mtb were imaged by time-lapse fluorescence microscopy followed by SEM. (I) Example of a macrophage showing the typical "bullseye" pattern of bacterial redistribution around the cell nucleus. Scale bar, 10 μm. (J) Correlative SEM image of (I). (K) Example of a macrophage interacting with an extracellular Mtb aggregate without complete fragmentation and redistribution of bacteria. White arrows indicate intracellular bacteria that have been detached from the main aggregate and internalized. Scale bar, 10 μm. Time-lapse microscopy image-series of these macrophages are shown in Appendix Fig S4A. (L-M) Correlative SEM image of (K). Scale bar, 2 μm (M).


Mouse bone marrow - derived macrophages ( BMDMs ) infected with Mtb Erd - tdTomato and imaged by time - lapse microscopy at 1 - or 2 - hour intervals for 132 hours ( O ) Percentage of macrophages surviving over time after uptake and fragmentation of Mtb aggregates or after contact without fragmentation . Each line represents an independent biological replicate . The vertical dotted line marks the 12h post - interaction time - point . BMDMs were treated with cytochalasin D to prevent bacterial uptake , infected with aggregates of Mtb and imaged by time - lapse microscopy at 1 - hour intervals for 60 hours . ( P ) Percentage survival over time for macrophages in contact with Mtb aggregates . Time 0 represents the time when stable contact with an aggregate begins . Each line represents an independent biological replicate ( n = 3 replicates with ≥ 100 cells per replicate ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Mouse bone marrow-derived macrophages (BMDMs) infected with Mtb Erd-tdTomato and imaged by time-lapse microscopy at 1- or 2-hour intervals for 132 hours (O) Percentage of macrophages surviving over time after uptake and fragmentation of Mtb aggregates or after contact without fragmentation. Each line represents an independent biological replicate. The vertical dotted line marks the 12h post-interaction time-point. BMDMs were treated with cytochalasin D to prevent bacterial uptake, infected with aggregates of Mtb and imaged by time-lapse microscopy at 1-hour intervals for 60 hours. (P) Percentage survival over time for macrophages in contact with Mtb aggregates. Time 0 represents the time when stable contact with an aggregate begins. Each line represents an independent biological replicate (n = 3 replicates with ≥ 100 cells per replicate).


BMDMs were treated with cytochalasin D and infected with aggregated or non - aggregated Mtb Erd - tdTomato and imaged by time - lapse microscopy at 1 - hour intervals for 60 hours . ( B - E ) Examples of macrophages infected with low ( B ) , medium ( C ) , or high ( D ) doses of non - aggregated bacteria or with bacterial aggregates ( E ) . Scale bars , 20 μm .

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

BMDMs were treated with cytochalasin D and infected with aggregated or non-aggregated Mtb Erd-tdTomato and imaged by time-lapse microscopy at 1-hour intervals for 60 hours. (B-E) Examples of macrophages infected with low (B), medium (C), or high (D) doses of non-aggregated bacteria or with bacterial aggregates (E). Scale bars, 20 μm.


BMDMs were treated with cytochalasin D and infected with aggregated or non - aggregated Mtb Erd - tdTomato and imaged by time - lapse microscopy at 1 - hour intervals for 60 hours . ( F ) Percentage survival over time for macrophages in contact with increasing doses of non - aggregated bacteria or with bacterial aggregates . Time 0 for cells " in contact " represents the time when stable contact with the bacteria begins . Each line represents a biological replicate ( n ≥ 100 cells per condition ) .

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

BMDMs were treated with cytochalasin D and infected with aggregated or non-aggregated Mtb Erd-tdTomato and imaged by time-lapse microscopy at 1-hour intervals for 60 hours. (F) Percentage survival over time for macrophages in contact with increasing doses of non-aggregated bacteria or with bacterial aggregates. Time 0 for cells "in contact" represents the time when stable contact with the bacteria begins. Each line represents a biological replicate (n ≥ 100 cells per condition).


( A ) BMDMs expressing a membrane - targeted tdTomato were treated with cytochalasin D , infected with aggregates of Mtb expressing GFP , and imaged by time - lapse microscopy at 20 - minute intervals for 24 hours . Example of a BMDM interacting with an extracellular Mtb aggregate . Top panels show the macrophage plasma membrane and the bacterial aggregate . Intimate interaction between the macrophage plasma membrane and the bacterial aggregate begins at 4h00 ( indicated by the white arrow ) . Bottom panels show the Annexin V - 647 positive plasma membrane domain that appears at 5 hours 20 minutes and co - localizes with the bacterial aggregate . At 8h00 , plasma membrane integrity is lost ( top panel ) and Annexin V accumulates throughout the dead cell ( bottom panel ) .

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

(A) BMDMs expressing a membrane-targeted tdTomato were treated with cytochalasin D, infected with aggregates of Mtb expressing GFP, and imaged by time-lapse microscopy at 20-minute intervals for 24 hours. Example of a BMDM interacting with an extracellular Mtb aggregate. Top panels show the macrophage plasma membrane and the bacterial aggregate. Intimate interaction between the macrophage plasma membrane and the bacterial aggregate begins at 4h00 (indicated by the white arrow). Bottom panels show the Annexin V-647 positive plasma membrane domain that appears at 5 hours 20 minutes and co-localizes with the bacterial aggregate. At 8h00, plasma membrane integrity is lost (top panel) and Annexin V accumulates throughout the dead cell (bottom panel).


( E - G ) Cytochalasin D - treated BMDMs were infected with aggregates of Mtb Erd - tdTomato in the presence of Annexin V - FITC . Selected cells were imaged by time - lapse fluorescence microscopy followed by SEM . Arrows in top panels indicate the cell imaged by SEM . Top left : brightfield image of macrophage . Top left and right : fluorescence image of the Mtb aggregate . Top right : fluorescence image of Annexin V staining . Bottom : correlative SEM image . Scale bars , 20 μm in top panels and 1 μm in bottom panels . ( E ) Example of a bystander BMDM that is not in contact with a bacterial aggregate . ( F ) Example of an Annexin V - negative BMDM interacting with a Mtb aggregate . Arrows in bottom panel indicate areas of intact plasma membrane interacting with bacterial aggregates . ( G ) Example of a BMDM with blebbed Annexin V - positive membrane domains interacting with an Mtb aggregate .

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

(E-G) Cytochalasin D-treated BMDMs were infected with aggregates of Mtb Erd-tdTomato in the presence of Annexin V-FITC. Selected cells were imaged by time-lapse fluorescence microscopy followed by SEM. Arrows in top panels indicate the cell imaged by SEM. Top left: brightfield image of macrophage. Top left and right: fluorescence image of the Mtb aggregate. Top right: fluorescence image of Annexin V staining. Bottom: correlative SEM image. Scale bars, 20 μm in top panels and 1 μm in bottom panels. (E) Example of a bystander BMDM that is not in contact with a bacterial aggregate. (F) Example of an Annexin V-negative BMDM interacting with a Mtb aggregate. Arrows in bottom panel indicate areas of intact plasma membrane interacting with bacterial aggregates. (G) Example of a BMDM with blebbed Annexin V-positive membrane domains interacting with an Mtb aggregate.


Cytochalasin D - treated BMDMs were stained with the membrane - permeable dye Oregon Green 488 Bapta - 1 AM to visualize intracellular Ca2 + , infected with aggregates of Mtb Erdman WT and imaged by time - lapse microscopy at 20 - minute intervals for 24 hours . Oregon Green 488 Bapta - 1 AM fluorescence values at each time point were normalized to time 0 for uninfected bystander cells and to the time of first contact with an Mtb aggregate for infected cells . ( A ) Examples of BMDMs dying with ( yellow outline ) or without ( white outline ) interacting with extracellular Mtb aggregates . Cell outlines are based on brightfield images Cell death is indicated by Draq7 nuclear staining . Scale bar , 10 μm . ( B ) Oregon Green 488 Bapta - 1 AM fluorescence over time in dying bystander macrophages ( n = 15 ) and in dying macrophages in contact with an Mtb aggregates ( n = 63 ) . Lines represent median fluorescence values for all cells , error bars represent interquartile ranges . The distributions of the fluorescence values at time of death in bystander and dying macrophages are significantly different , p value < 0 . 0001 , calculated using a Welch ' s t test .

O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Cytochalasin D-treated BMDMs were stained with the membrane-permeable dye Oregon Green 488 Bapta-1 AM to visualize intracellular Ca2+, infected with aggregates of Mtb Erdman WT and imaged by time-lapse microscopy at 20-minute intervals for 24 hours. Oregon Green 488 Bapta-1 AM fluorescence values at each time point were normalized to time 0 for uninfected bystander cells and to the time of first contact with an Mtb aggregate for infected cells. (A) Examples of BMDMs dying with (yellow outline) or without (white outline) interacting with extracellular Mtb aggregates. Cell outlines are based on brightfield images Cell death is indicated by Draq7 nuclear staining. Scale bar, 10 μm. (B) Oregon Green 488 Bapta-1 AM fluorescence over time in dying bystander macrophages (n=15) and in dying macrophages in contact with an Mtb aggregates (n=63). Lines represent median fluorescence values for all cells, error bars represent interquartile ranges. The distributions of the fluorescence values at time of death in bystander and dying macrophages are significantly different, p value <0.0001, calculated using a Welch's t test.


( E ) BMDMs treated with cytochalasin D were infected with aggregates of Mtb Erd - tdTomato and imaged by time - lapse microscopy at 1 - hour intervals for 60 hours . Percentage of macrophages that die within the first 12 hours after stable contact with an Mtb aggregate without ( untr . ) or with supplementation of BAPTA - AM . Each symbol represents the percentage of dead macrophages for a single biological replicate ( n ≥ 60 cells per replicate ) . Bars represent means and standard deviations , P value calculated using a t test .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(E) BMDMs treated with cytochalasin D were infected with aggregates of Mtb Erd-tdTomato and imaged by time-lapse microscopy at 1-hour intervals for 60 hours. Percentage of macrophages that die within the first 12 hours after stable contact with an Mtb aggregate without (untr.) or with supplementation of BAPTA-AM. Each symbol represents the percentage of dead macrophages for a single biological replicate (n ≥ 60 cells per replicate). Bars represent means and standard deviations, P value calculated using a t test.


BMDMs treated with cytochalasin D were infected with aggregates of Mtb Erd - tdTomato , fixed at 24 hours post infection and processed for immunofluorescence with antibodies targeting cellular markers for cell death pathways . Controls and representative microscopy images for all the antibodies used are provided in Macrophages are defined as " in contact " when the body of a macrophage identified in brightfield images overlaps with a bacterial aggregate identified in the fluorescence channel ( A ) Percentage of dead uninfected bystander macrophages ( byst . ) or dead macrophages in contact ( cont . ) with an extracellular Mtb aggregate that stain positive for cleaved Caspase - 8 ( cCasp8 ) . Each symbol represents the percentage of positive dead macrophages for a single biological replicate ( n ≥ 50 cells per replicate ) . Bars represent means and standard deviations . P values were calculated using a t test . ( B ) Percentage of dead uninfected bystander macrophages ( byst . ) or dead macrophages in contact with an extracellular Mtb aggregate ( cont . ) that display ASC - specks . Each symbol represents the percentage of positive dead macrophages for a single biological replicate ( n ≥ 80 cells per replicate ) . Bars represent means and standard deviations . P values were calculated using a t test .

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

BMDMs treated with cytochalasin D were infected with aggregates of Mtb Erd-tdTomato, fixed at 24 hours post infection and processed for immunofluorescence with antibodies targeting cellular markers for cell death pathways. Controls and representative microscopy images for all the antibodies used are provided in Macrophages are defined as "in contact" when the body of a macrophage identified in brightfield images overlaps with a bacterial aggregate identified in the fluorescence channel (A) Percentage of dead uninfected bystander macrophages (byst.) or dead macrophages in contact (cont.) with an extracellular Mtb aggregate that stain positive for cleaved Caspase-8 (cCasp8). Each symbol represents the percentage of positive dead macrophages for a single biological replicate (n ≥ 50 cells per replicate). Bars represent means and standard deviations. P values were calculated using a t test. (B) Percentage of dead uninfected bystander macrophages (byst.) or dead macrophages in contact with an extracellular Mtb aggregate (cont.) that display ASC-specks. Each symbol represents the percentage of positive dead macrophages for a single biological replicate (n ≥ 80 cells per replicate). Bars represent means and standard deviations. P values were calculated using a t test.


BMDMs treated with cytochalasin D were infected with aggregates of Mtb Erd - tdTomato , fixed at 24 hours post infection and processed for immunofluorescence with antibodies targeting cellular markers for cell death pathways . Controls and representative microscopy images for all the antibodies used are provided Macrophages are defined as " in contact " when the body of a macrophage identified in brightfield images overlaps with a bacterial aggregate identified in the fluorescence channel ( C ) Representative example of a dead macrophage in contact with an extracellular Mtb aggregate that display a ASC - speck ( white arrow ) . Nuclei stained with Hoechst . Scale bar , 10 μm .

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

BMDMs treated with cytochalasin D were infected with aggregates of Mtb Erd-tdTomato, fixed at 24 hours post infection and processed for immunofluorescence with antibodies targeting cellular markers for cell death pathways. Controls and representative microscopy images for all the antibodies used are provided Macrophages are defined as "in contact" when the body of a macrophage identified in brightfield images overlaps with a bacterial aggregate identified in the fluorescence channel (C) Representative example of a dead macrophage in contact with an extracellular Mtb aggregate that display a ASC-speck (white arrow). Nuclei stained with Hoechst. Scale bar, 10 μm.


BMDMs treated with cytochalasin D were infected with aggregates of Mtb Erd - tdTomato , fixed at 24 hours post infection and processed for immunofluorescence with antibodies targeting cellular markers for cell death pathways . Controls and representative microscopy images for all the antibodies used are provided Macrophages are defined as " in contact " when the body of a macrophage identified in brightfield images overlaps with a bacterial aggregate identified in the fluorescence channel Median fluorescence values for bystander macrophages ( byst . ) or macrophages in contact with an extracellular Mtb aggregate ( cont . ) stained with anti - cleaved Caspase - 1 antibody ( cCasp1 ) , an ( E ) anti - phosphorylated RIP3 antibody ( pRIP3 ) or ( F ) anti - phosphorylated MLKL antibody ( pMLKL ) . In ( G ) BMDMs were treated with MCC950 ( NLRP3 inhibitor ) or with VX765 ( Caspase - 1 inhibitor ) during the course of the infection . Each symbol represents a single macrophage . Black bars represent median and interquartile range . P - value were calculated using an unpaired Mann - Whitney test ; ns , P values > 0 . 05 . E : n = 182 , 112 respectively ; F : n = 186 , 84 respectively ; G : n = 117 , 121 , 111 , 129 respectively ; n represents the number of individual cells analyzed per condition ) .

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

BMDMs treated with cytochalasin D were infected with aggregates of Mtb Erd-tdTomato, fixed at 24 hours post infection and processed for immunofluorescence with antibodies targeting cellular markers for cell death pathways. Controls and representative microscopy images for all the antibodies used are provided Macrophages are defined as "in contact" when the body of a macrophage identified in brightfield images overlaps with a bacterial aggregate identified in the fluorescence channel Median fluorescence values for bystander macrophages (byst.) or macrophages in contact with an extracellular Mtb aggregate (cont.) stained with anti-cleaved Caspase-1 antibody (cCasp1), an (E) anti-phosphorylated RIP3 antibody (pRIP3) or (F) anti-phosphorylated MLKL antibody (pMLKL). In (G) BMDMs were treated with MCC950 (NLRP3 inhibitor) or with VX765 (Caspase-1 inhibitor) during the course of the infection. Each symbol represents a single macrophage. Black bars represent median and interquartile range. P-value were calculated using an unpaired Mann-Whitney test; ns, P values > 0.05. E: n=182, 112 respectively; F: n=186, 84 respectively; G: n=117, 121, 111, 129 respectively; n represents the number of individual cells analyzed per condition).


( A - D ) BMDMs treated with cytochalasin D were infected with aggregates of different Mtb strains and imaged by time - lapse microscopy at 1 - hour intervals for 60 hours . The plots represent the percentage of macrophages that die within the first 12 hours after stable contact with an Mtb aggregate . Each symbol represents the percentage of dead macrophages for a single biological replicate ( n ≥ 3 replicates with ≥ 70 cells per replicate ) . Bars represent means and standard deviations . P - values were calculated using a one - way ANOVA test ( A , C , D ) or t test ( B ) comparing each strain to the wild - type reference strain ( A - D ) ; ns , P values > 0 . 05 . ( A ) Macrophages in contact with aggregates of Mtb H37Rv wild - type ( WT ) , ∆ RD1 mutant and complemented ∆ RD1 mutant ( ∆ RD1 : : 2F9 ) . ( B ) Macrophages in contact with aggregates of Mtb Erdman wild - type ( WT ) or PDIM - deficient ( fadD26 ) strains . ( C ) Macrophages in contact with aggregates of Mtb Erdman wild - type ( WT ) or mutant strains ( espI eccD1 , esxA , espA ) and the complemented strains ( esxA : : esxBA , espA : : espA ) . ( D ) Macrophages in contact with aggregates of Mtb Erdman wild - type ( WT ) or mutant strains ( espB , espA espB ) and the espB - complemented strains ( espB : : espB , espA espB : : espB ) .

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

(A-D) BMDMs treated with cytochalasin D were infected with aggregates of different Mtb strains and imaged by time-lapse microscopy at 1-hour intervals for 60 hours. The plots represent the percentage of macrophages that die within the first 12 hours after stable contact with an Mtb aggregate. Each symbol represents the percentage of dead macrophages for a single biological replicate (n ≥ 3 replicates with ≥ 70 cells per replicate). Bars represent means and standard deviations. P-values were calculated using a one-way ANOVA test (A,C,D) or t test (B) comparing each strain to the wild-type reference strain (A-D); ns, P values > 0.05. (A) Macrophages in contact with aggregates of Mtb H37Rv wild-type (WT), ∆RD1 mutant and complemented ∆RD1 mutant (∆RD1::2F9). (B) Macrophages in contact with aggregates of Mtb Erdman wild-type (WT) or PDIM-deficient (fadD26) strains. (C) Macrophages in contact with aggregates of Mtb Erdman wild-type (WT) or mutant strains (espI eccD1, esxA, espA) and the complemented strains (esxA::esxBA, espA::espA). (D) Macrophages in contact with aggregates of Mtb Erdman wild-type (WT) or mutant strains (espB, espA espB) and the espB-complemented strains (espB::espB, espA espB::espB).


( F ) BMDMs treated with cytochalasin D were infected with aggregates of different Mtb strains , incubated with Annexin V and imaged by time - lapse microscopy at 1 - hour intervals for 48 hours . Percentage of macrophages that show Annexin V - positive membrane domains within the first 12 hours after entering in contact with Mtb aggregates . Each symbol represents a single biological replicate ( n ≥ 3 replicates with ≥ 90 cells per replicate ) . Bars represent means and standard deviations .

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

(F) BMDMs treated with cytochalasin D were infected with aggregates of different Mtb strains, incubated with Annexin V and imaged by time-lapse microscopy at 1-hour intervals for 48 hours. Percentage of macrophages that show Annexin V - positive membrane domains within the first 12 hours after entering in contact with Mtb aggregates. Each symbol represents a single biological replicate (n ≥ 3 replicates with ≥ 90 cells per replicate). Bars represent means and standard deviations.


( G ) Cytochalasin D - treated BMDMs were infected with aggregates of Mtb Erdman WT , stained with the membrane - permeable dye Oregon Green 488 Bapta - 1 AM to visualize cytosolic Ca2 + and imaged by time - lapse microscopy at 20 - minute intervals for 24 hours . Oregon Green 488 Bapta - 1 AM fluorescence values at each time point were normalized to the time of first contact with an Mtb aggregate for infected cells . Values in the plot correspond to the time of death after first contact or 16 hours post - contact for cells that survive . Each symbol represents a single macrophage . ( n = 123 , 64 , 48 , 71 , 78 , 55 respectively ) . Black bars represent median and interquartile range . P - value were calculated using a one sample Wilcoxon test ; ns , P values > 0 . 05 .

O O O O O O O O O O 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-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(G) Cytochalasin D-treated BMDMs were infected with aggregates of Mtb Erdman WT, stained with the membrane-permeable dye Oregon Green 488 Bapta-1 AM to visualize cytosolic Ca2+ and imaged by time-lapse microscopy at 20-minute intervals for 24 hours. Oregon Green 488 Bapta-1 AM fluorescence values at each time point were normalized to the time of first contact with an Mtb aggregate for infected cells. Values in the plot correspond to the time of death after first contact or 16 hours post-contact for cells that survive. Each symbol represents a single macrophage. (n=123, 64, 48, 71, 78, 55 respectively). Black bars represent median and interquartile range. P-value were calculated using a one sample Wilcoxon test; ns, P values > 0.05.