A . Infection by a L . pneumophila strain overexpressing MvcA but not the inactive mutant MvcAC83A interferes with UBE2N modification . Raw264 . 7 cells were infected with the indicated bacterial strains at an MOI of 5 for 2 h . Saponin soluble proteins resolved by SDS - PAGE were probed with a UBE2N - specific antibody ( top panel ) . The delivery of MavC and MvcA into infected cells was probed with antibodies specific to each of these two proteins . Note that MavC but not MvcA is detectable in cells infected with wild type bacteria ( middle panels labeled as " translocation " ) . Tubulin was probed as a loading control . Protein levels of MavC and MvcA associated with the bacteria were similarly probed in the saponin insoluble fraction ( lower panels labeled as " expression " ) . The bacterial metabolism enzyme isocitrate dehydrogenase ( ICDH ) was probed as a loading control . Note that endogenous MvcA was not detectable in bacteria grown in bacteriological medium ( the middle panel of the lower portion ) .

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A. Infection by a L. pneumophila strain overexpressing MvcA but not the inactive mutant MvcAC83A interferes with UBE2N modification. Raw264.7 cells were infected with the indicated bacterial strains at an MOI of 5 for 2 h. Saponin soluble proteins resolved by SDS-PAGE were probed with a UBE2N-specific antibody (top panel). The delivery of MavC and MvcA into infected cells was probed with antibodies specific to each of these two proteins. Note that MavC but not MvcA is detectable in cells infected with wild type bacteria (middle panels labeled as "translocation"). Tubulin was probed as a loading control. Protein levels of MavC and MvcA associated with the bacteria were similarly probed in the saponin insoluble fraction (lower panels labeled as "expression"). The bacterial metabolism enzyme isocitrate dehydrogenase (ICDH) was probed as a loading control. Note that endogenous MvcA was not detectable in bacteria grown in bacteriological medium (the middle panel of the lower portion).


B . Coexpression of MvcA with MavC causes accumulation of unmodified UBE2N in mammalian cells . HEK293T cells were transfected with combinations of empty vector and plasmid directing the expression of MavC or MvcA . 16 h after transfection , modification of endogenous UBE2N was detected by immunoblotting ( upper level ) . The canonical ubiquitin deamidase CifYpt from Yersinia pseudotuberculosis ( Ypt ) , which cannot modify UBE2N , was included as a control . Note that coexpression of MvcA led to accumulation of unmodified UBE2N ( 4th lane ) in samples that expressed MavC . The expression of MavC MvcA and Cif was detected using a Flag - specific antibody ( lower panel ) . Tubulin was probed as a loading control ( lower panel ) .

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B. Coexpression of MvcA with MavC causes accumulation of unmodified UBE2N in mammalian cells. HEK293T cells were transfected with combinations of empty vector and plasmid directing the expression of MavC or MvcA. 16 h after transfection, modification of endogenous UBE2N was detected by immunoblotting (upper level). The canonical ubiquitin deamidase CifYpt from Yersinia pseudotuberculosis (Ypt), which cannot modify UBE2N, was included as a control. Note that coexpression of MvcA led to accumulation of unmodified UBE2N (4th lane) in samples that expressed MavC. The expression of MavC MvcA and Cif was detected using a Flag-specific antibody (lower panel). Tubulin was probed as a loading control (lower panel).


C . MvcA interferes with MavC - induced UBE2N ubiquitination . Reactions containing the indicated combinations of proteins were allowed to proceed for 30 min and samples resolved by SDS - PAGE were detected by Coomassie Brilliant blue ( CBB ) staining . Note that MvcA ( inclusion of MvcA with MavC , 7th lane . Compare to MavC alone in 3rd lane ) but not the MvcAC83A mutant ( 8th lane ) abolished MavC - induced UBE2N modification .

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C. MvcA interferes with MavC-induced UBE2N ubiquitination. Reactions containing the indicated combinations of proteins were allowed to proceed for 30 min and samples resolved by SDS-PAGE were detected by Coomassie Brilliant blue (CBB) staining. Note that MvcA (inclusion of MvcA with MavC, 7th lane. Compare to MavC alone in 3rd lane) but not the MvcAC83A mutant (8th lane) abolished MavC-induced UBE2N modification.


D . Dose - dependent modification of UBE2N by MavC and MvcA . A series of reactions containing MavC and MvcA at the indicated molar ratios were established and the reactions were allowed to proceed for 30 min . Samples resolved by SDS - PAGE were detected by CBB staining .

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D. Dose-dependent modification of UBE2N by MavC and MvcA. A series of reactions containing MavC and MvcA at the indicated molar ratios were established and the reactions were allowed to proceed for 30 min. Samples resolved by SDS-PAGE were detected by CBB staining.


A . The hydrolytic cleavage of UBE2N - Ub by MvcA into UBE2N and ubiquitin ( Q40E variant ) . Reactions containing the indicated proteins were allowed to proceed for 30 min and samples resolved by SDS - PAGE were detected by CBB staining ( left ) . Mass spectrometric analysis of the protein band corresponding to ubiquitin produced by MvcA from UBE2N - Ub ( right ) . The ubiquitin product is the Glu40 variant of ubiquitin .

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A. The hydrolytic cleavage of UBE2N-Ub by MvcA into UBE2N and ubiquitin (Q40E variant). Reactions containing the indicated proteins were allowed to proceed for 30 min and samples resolved by SDS-PAGE were detected by CBB staining (left). Mass spectrometric analysis of the protein band corresponding to ubiquitin produced by MvcA from UBE2N-Ub (right). The ubiquitin product is the Glu40 variant of ubiquitin.


C . Dose - dependent cleavage of UBE2N - Ub by MvcA . A series of reactions containing UBE2N - Ub and MvcA at the indicated molar ratios were established and the reactions were allowed to proceed for 30 min before SDS - PAGE and CBB staining . Note that the activity was detectable in a reaction starting at the molar ratio of between UBE2N - Ub and MvcA is 2048 : 1 .

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C. Dose-dependent cleavage of UBE2N-Ub by MvcA. A series of reactions containing UBE2N-Ub and MvcA at the indicated molar ratios were established and the reactions were allowed to proceed for 30 min before SDS-PAGE and CBB staining. Note that the activity was detectable in a reaction starting at the molar ratio of between UBE2N-Ub and MvcA is 2048:1.


D . Time - dependent cleavage of UBE2N - Ub by MvcA . UBE2N - Ub was incubated with MvcA at the molar ratio of 128 . Samples taken at the indicated time points were resolved by SDS - PAGE and detected by CBB staining .

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D. Time-dependent cleavage of UBE2N-Ub by MvcA. UBE2N-Ub was incubated with MvcA at the molar ratio of 128. Samples taken at the indicated time points were resolved by SDS-PAGE and detected by CBB staining.


E . UBE2N produced from UBE2N - Ub via MvcA catalyzed deubiquitination is active in catalyzing the formation of polyubiquitin chains . UBE2N - Ub was preincubated with MvcA or its inactive mutant for 30 min . Then a cocktail containing E1 , UBE2V2 , 3xHA - ubiquitin , the E3 enzyme TRAF6 and ATP was added into reactions . Ubiquitination was allowed to proceed for 30 min . Proteins in samples resolved by SDS - PAGE were transferred onto nitrocellulose membranes and blotted with the HA specific antibody . Note the robust formation of polyubiquitin chains in the reaction receiving MvcA ( middle lane ) .

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E. UBE2N produced from UBE2N-Ub via MvcA catalyzed deubiquitination is active in catalyzing the formation of polyubiquitin chains. UBE2N-Ub was preincubated with MvcA or its inactive mutant for 30 min. Then a cocktail containing E1, UBE2V2, 3xHA-ubiquitin, the E3 enzyme TRAF6 and ATP was added into reactions. Ubiquitination was allowed to proceed for 30 min. Proteins in samples resolved by SDS-PAGE were transferred onto nitrocellulose membranes and blotted with the HA specific antibody. Note the robust formation of polyubiquitin chains in the reaction receiving MvcA (middle lane).


H . Mutational analysis of residues important for substrate recognition by MvcA predicted by structural analysis . For deubiquitination reactions , the indicated amounts of MvcA or its mutants were incubated with 2 . 0 μg UBE2N for 5 min in 25 μl reactions . For ubiquitin deamidase activity , the indicated amounts of MvcA or its mutants were incubated with 10 μg of ubiquitin for 60 min in 25 μl reactions . The ratio of cleavage was analyzed by SDS - PAGE and then densitometry . Each bar represents the ratio of cleavage or deamidation obtained from three independent experiments . Bars and error bars : means and standard error of the mean ( SEM ) .

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H. Mutational analysis of residues important for substrate recognition by MvcA predicted by structural analysis. For deubiquitination reactions, the indicated amounts of MvcA or its mutants were incubated with 2.0 μg UBE2N for 5 min in 25 μl reactions. For ubiquitin deamidase activity, the indicated amounts of MvcA or its mutants were incubated with 10 μg of ubiquitin for 60 min in 25 μl reactions. The ratio of cleavage was analyzed by SDS-PAGE and then densitometry. Each bar represents the ratio of cleavage or deamidation obtained from three independent experiments. Bars and error bars: means and standard error of the mean (SEM).


I - J . The roles of ubiquitin contact residues in the activity of MvcA . I . The indicated amounts of MvcA or its mutants were incubated with 2 μg UBE2N - Ub for 5 min in 25 μl reactions . The ratio of cleavage was analyzed by SDS - PAGE and then densitometry . Each bar represents the ratio of cleavage obtained from three independent experiments . Bars and error bars : means and standard error of the mean ( SEM ) . J . The activity of the MvcA mutant harboring three mutations . 0 . 05 μg , 0 . 25 μg and 1 . 25 μg of MvcA or the mutant was incubated with 2 μg UBE2N - Ub in 25 μl reactions . After 30 min incubation , the products resolved by SDS - PAGE were detected by CBB staining . Note that the mutant did not detectable cleave UBE2N - Ub even in the reaction received the highest amount of protein .

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I-J. The roles of ubiquitin contact residues in the activity of MvcA. I. The indicated amounts of MvcA or its mutants were incubated with 2 μg UBE2N-Ub for 5 min in 25 μl reactions. The ratio of cleavage was analyzed by SDS-PAGE and then densitometry. Each bar represents the ratio of cleavage obtained from three independent experiments. Bars and error bars: means and standard error of the mean (SEM). J. The activity of the MvcA mutant harboring three mutations. 0.05 μg, 0.25 μg and 1.25 μg of MvcA or the mutant was incubated with 2 μg UBE2N-Ub in 25 μl reactions. After 30 min incubation, the products resolved by SDS-PAGE were detected by CBB staining. Note that the mutant did not detectable cleave UBE2N-Ub even in the reaction received the highest amount of protein.


A . Overexpression of MavC but not the inactive mutant MavCC74A in the ∆ mavC ∆ mvcA mutant background causes IκBα accumulation in infected cells . U937 cells infected with the indicated bacterial strains at an MOI of 5 were probed for IκBα . Tubulin was probed as a loading control .

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A. Overexpression of MavC but not the inactive mutant MavCC74A in the ∆mavC∆mvcA mutant background causes IκBα accumulation in infected cells. U937 cells infected with the indicated bacterial strains at an MOI of 5 were probed for IκBα. Tubulin was probed as a loading control.


B . Prolonged UBE2N modification by MavC restricts bacterial vacuole maturation . HEK293 cells transfected to express the FcγII receptor and 4xFlag - MavC or the C74A mutant were challenged at an MOI of 1 with opsonized L . pneumophila that expresses GFP for 12 h . The vacuole was scored and categorized by the number of bacteria it contained . Data ( mean + / - SE ) shown were from three independent experiments , each with at least 300 vacuoles counted . * , p < 0 . 05 ; * * , p < 0 . 01 ; n . s . , not significant .

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B. Prolonged UBE2N modification by MavC restricts bacterial vacuole maturation. HEK293 cells transfected to express the FcγII receptor and 4xFlag-MavC or the C74A mutant were challenged at an MOI of 1 with opsonized L. pneumophila that expresses GFP for 12 h. The vacuole was scored and categorized by the number of bacteria it contained. Data (mean +/- SE) shown were from three independent experiments, each with at least 300 vacuoles counted. *, p<0.05; **, p<0.01; n.s., not significant.


C . Overexpression of MavC but not MavCC74A affects intracellular growth of the ∆ mavC ∆ mvcA mutant of L . pneumophila . Raw264 . 7 cells were infected with the indicated bacterial strains and intracellular bacteria were determined at the indicated time points . Each strain was examined in triplicate and the values shown were mean + / - SE . Similar results were obtained in two independent experiments . * , p < 0 . 1 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 ( t - test by comparing p ( MavC ) with p ( MavCC74A ) ) .

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C. Overexpression of MavC but not MavCC74A affects intracellular growth of the ∆mavC∆mvcA mutant of L. pneumophila. Raw264.7 cells were infected with the indicated bacterial strains and intracellular bacteria were determined at the indicated time points. Each strain was examined in triplicate and the values shown were mean +/- SE. Similar results were obtained in two independent experiments. *, p<0.1; **, p<0.01; ***, p<0.001(t-test by comparing p(MavC) with p(MavCC74A)).


D . MvcA is required for reversal of UBE2N modification during L . pneumophila infection . U937 cells infected with wild type or the ∆ mvcA mutant were withdrawn at the indicated time points . Samples resolved by SDS - PAGE were probed for UBE2N , translocated MavC and MvcA . Tubulin was probed as a loading control . Note that in samples infected with the ∆ mvcA strain , the level of UBE2N - Ub decreased at slower rates .

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D. MvcA is required for reversal of UBE2N modification during L. pneumophila infection. U937 cells infected with wild type or the ∆mvcA mutant were withdrawn at the indicated time points. Samples resolved by SDS-PAGE were probed for UBE2N, translocated MavC and MvcA. Tubulin was probed as a loading control. Note that in samples infected with the ∆mvcA strain, the level of UBE2N-Ub decreased at slower rates.


E . MvcA becomes detectable in intracellular bacteria 5 h after uptake . U937 macrophages infected with wild type L . pneumophila at an MOI of 10 were lysed at the indicated time and recovered bacteria were probed by immunoblotting for MavC and MvcA , respectively . The bacterial isocitrate dehydrogenase ( ICDH ) was probed sequentially as a loading control .

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E. MvcA becomes detectable in intracellular bacteria 5 h after uptake. U937 macrophages infected with wild type L. pneumophila at an MOI of 10 were lysed at the indicated time and recovered bacteria were probed by immunoblotting for MavC and MvcA, respectively. The bacterial isocitrate dehydrogenase (ICDH) was probed sequentially as a loading control.


F . MvcA restores the nuclear translocation of p65 inhibited by MavC during L . pneumophila infection . Raw264 . 7 cells were infected with the indicated L . pneumophila strains for 2 h at an MOI of 1 . Fixed cells were immunostained with antibodies specific for p65 and the bacterium , respectively . Host nuclei were labeled with Hoechst . Representative images of cells in each sample category were shown ( upper panels ) ; nuclear localization of p65 was determined by scoring at least 300 infected cells ( lower panel ) . Results shown were from three independent experiments done in triplicate and the values shown were mean + / - SE ; t - test .

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F. MvcA restores the nuclear translocation of p65 inhibited by MavC during L. pneumophila infection. Raw264.7 cells were infected with the indicated L. pneumophila strains for 2 h at an MOI of 1. Fixed cells were immunostained with antibodies specific for p65 and the bacterium, respectively. Host nuclei were labeled with Hoechst. Representative images of cells in each sample category were shown (upper panels); nuclear localization of p65 was determined by scoring at least 300 infected cells (lower panel). Results shown were from three independent experiments done in triplicate and the values shown were mean +/- SE; t-test.


G . Overexpression of MvcA but not its inactive mutant MvcAC83A promotes IκBα degradation during L . pneumophila infection . U937 cells were infected with the indicated bacterial strains at an MOI of 5 . Lysates of cells infected for the indicated time were probed for IκBα . Tubulin was probed as a loading control . IκBα levels for cells infected with the strains ∆ mvA ( pMvcA ) and ∆ mvA ( pMvcAC83A ) at the indicated time points were evaluated by measuring the band intensity from three independent experiments ( lower panel ) , the values shown were mean + / - SE ; t - test .

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G. Overexpression of MvcA but not its inactive mutant MvcAC83A promotes IκBα degradation during L. pneumophila infection. U937 cells were infected with the indicated bacterial strains at an MOI of 5. Lysates of cells infected for the indicated time were probed for IκBα. Tubulin was probed as a loading control. IκBα levels for cells infected with the strains ∆mvA(pMvcA) and ∆mvA(pMvcAC83A) at the indicated time points were evaluated by measuring the band intensity from three independent experiments (lower panel), the values shown were mean +/- SE; t-test.


B . Growth of DiCre - 3D7 transgenic lines following LDH1 , HAD4 , and Lipin depletion with 100 nM rapamycin and 2 . 5 mM glucosamine was assessed relative to uninduced parasite cultures . The untransfected DiCre - 3D7 parasite line was used as a negative control . Data are presented as the mean ± standard error of the mean ( SEM ) from three independent replicates .

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B. Growth of DiCre-3D7 transgenic lines following LDH1, HAD4, and Lipin depletion with 100 nM rapamycin and 2.5 mM glucosamine was assessed relative to uninduced parasite cultures. The untransfected DiCre-3D7 parasite line was used as a negative control. Data are presented as the mean ± standard error of the mean (SEM) from three independent replicates.


C . Untargeted LC - MS analysis of LDH1 depletion ( one cycle of rapamycin and glucosamine treatment ) indicated minimal but selective metabolic changes . m / z feature intensities are plotted as the log2 ratio of treated / untreated and the Benjamini - Hochberg corrected P values across six biological replicates plotted as - log10 ( P ) . Below , LDH1 substrate and product ( pyruvate and lactate respectively ) abundance plotted as the ratio of treated / untreated from biological replicates ( mean ± SEM ) and the parental DiCre line presented as the negative control .

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C. Untargeted LC-MS analysis of LDH1 depletion (one cycle of rapamycin and glucosamine treatment) indicated minimal but selective metabolic changes. m/z feature intensities are plotted as the log2 ratio of treated/untreated and the Benjamini-Hochberg corrected P values across six biological replicates plotted as -log10(P). Below, LDH1 substrate and product (pyruvate and lactate respectively) abundance plotted as the ratio of treated/untreated from biological replicates (mean±SEM) and the parental DiCre line presented as the negative control.


D . Loss of Lipin ( PF3D7 _ 0303200 ) leads to accumulation of various lipid species ( three cycles of rapamycin and glucosamine treatment ) . Data are presented as the mean log2 fold change Lipin depleted ( treated ) enriched trophozoite - stage iRBCs to untreated controls from three to six biological replicates ( ± SD ) . The schematic depicts the proposed lipid phosphatase activity of Lipin .

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D. Loss of Lipin (PF3D7_0303200) leads to accumulation of various lipid species (three cycles of rapamycin and glucosamine treatment). Data are presented as the mean log2 fold change Lipin depleted (treated) enriched trophozoite-stage iRBCs to untreated controls from three to six biological replicates (± SD). The schematic depicts the proposed lipid phosphatase activity of Lipin.


E . Loss of HAD4 leads to increases in intracellular levels of several nucleotides and intermediates in lower glycolysis . Data are represented as the mean log2 fold change of enriched trophozoite - stage iRBCs treated for three cycles compared to untreated controls from three / four biological replicates . Three nucleotide - nucleoside pairs are depicted in the top inset panels , HAD4 disruption leads to accumulation of the nucleotide - monophosphate and depletion of the nucleoside . The schematic depicts lower glycolysis and triose - phosphate interconversion , with the corresponding metabolite levels following HAD4 disruption depicted in the lower right inset .

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E. Loss of HAD4 leads to increases in intracellular levels of several nucleotides and intermediates in lower glycolysis. Data are represented as the mean log2 fold change of enriched trophozoite-stage iRBCs treated for three cycles compared to untreated controls from three/four biological replicates. Three nucleotide-nucleoside pairs are depicted in the top inset panels, HAD4 disruption leads to accumulation of the nucleotide-monophosphate and depletion of the nucleoside. The schematic depicts lower glycolysis and triose-phosphate interconversion, with the corresponding metabolite levels following HAD4 disruption depicted in the lower right inset.


B . Relative parasitemia was assessed for the untransfected DiCre 3D7 parental line and transfectant parasite lines with AMR1 under inducible disruption . Rapamycin / glucosamine treatment began on cycle zero and parasitemia determined by flow cytometry and compared to identical lines that were left untreated . Data represents the mean relative parasitemia ± SEM from three biological replicates ( y - axis ) across seven replication cycles ( x - axis ) .

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B. Relative parasitemia was assessed for the untransfected DiCre 3D7 parental line and transfectant parasite lines with AMR1 under inducible disruption. Rapamycin/glucosamine treatment began on cycle zero and parasitemia determined by flow cytometry and compared to identical lines that were left untreated. Data represents the mean relative parasitemia ± SEM from three biological replicates (y-axis) across seven replication cycles (x-axis).


C . Metabolite profile following three cycles of rapamycin and glucosamine treatment . The abundance of isoprenoid biosynthestic intermediates ( 1 - deoxy - xylulose - 5 - P ( DOXP ) , methyl - erithritol - 4 - P ( MEP ) , methyl - erithritol - cyclo - 2 , 4 - PP ( MEcPP ) , and isopentyl - PP ) , and hemoglobin - like peptides are presented as the mean log2 fold change from four biological replicates ( ± SD ) .

C. Metabolite profile following three cycles of rapamycin and glucosamine treatment. The abundance of isoprenoid biosynthestic intermediates (1-deoxy-xylulose-5-P (DOXP), methyl-erithritol-4-P (MEP), methyl-erithritol-cyclo-2,4-PP (MEcPP), and isopentyl-PP), and hemoglobin-like peptides are presented as the mean log2 fold change from four biological replicates (±SD).


C . HA - tagged SHMT - M detection via immunofluorescence microscopy . DAPI was used to visualise the nucleus , and MitoTracker CMXros used to illuminate the mitochondrion . Anti - HA signal partially colocalises with the mitochondrion .

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C. HA-tagged SHMT-M detection via immunofluorescence microscopy. DAPI was used to visualise the nucleus, and MitoTracker CMXros used to illuminate the mitochondrion. Anti-HA signal partially colocalises with the mitochondrion.


D . Pyrimidine biosynthetic intermediates accumulate when SHMT - M is disrupted compared to the DiCre parental line ( C ) following rapamycin / glucosamine treatment ( mean ± SEM ) . Pyrimidine biosynthetic disruption islinked to dihydroorotate dehydrogenase ( DHODH ) inhibition and blockage of the mitochondrial electron transport chain ( mETC ) . Ubiquinone ( Q ) is the necessary electron carrier for the mETC .

O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O B-MEASURED_VAR 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 B-MEASURED_VAR O O O O O O O O O O O O

D. Pyrimidine biosynthetic intermediates accumulate when SHMT-M is disrupted compared to the DiCre parental line (C) following rapamycin/glucosamine treatment (mean ±SEM). Pyrimidine biosynthetic disruption islinked to dihydroorotate dehydrogenase (DHODH) inhibition and blockage of the mitochondrial electron transport chain (mETC). Ubiquinone (Q) is the necessary electron carrier for the mETC.


E . Asexual growth when SHMT - M is depleted . Parasitemia was determined across seven replication cycles ( x - axis ) via flow cytometry and presented as the mean ± SEM from three independent experiments . Decylubiquinone ( Dub ) was added at 10 µM to determine if the growth defect observed was due to impaired ubiquinone synthesis or recycling .

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E. Asexual growth when SHMT-M is depleted. Parasitemia was determined across seven replication cycles (x-axis) via flow cytometry and presented as the mean ± SEM from three independent experiments. Decylubiquinone (Dub) was added at 10 µM to determine if the growth defect observed was due to impaired ubiquinone synthesis or recycling.


B . Kaplan - Meier curve represents the survival of BALB / c mice over time . Four BALB / c mice in each experimental group were infected i . v with 104 L . monocytogenes wild - type ( EGD - e ) or ΔinlK mutant .

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B. Kaplan-Meier curve represents the survival of BALB/c mice over time. Four BALB/c mice in each experimental group were infected i.v with 104 L. monocytogenes wild-type (EGD-e) or ΔinlK mutant.


C . The L . monocytogenes EGD - e wild - type strain ( WT ) , the ΔinlK mutant ( ΔinlK ) and the complemented strain ( ΔinlK + pPL2 inlK ) 104 CFU were inoculated i . v into BALB / c mice . Animals were euthanized 24 h , 48 h , 72 h or 96 h after infection and organs were recovered , homogenized , and homogenates serially plated on BHI . The number of bacteria able to colonize liver ( left panel ) and spleen ( right panel ) is expressed as log10 CFU . Four animals per bacterial strain , per time points and per experiment were used . Statistical analyses were performed on the results of 3 independent experiments using the Student t test . P values of < 0 . 05 were considered statistically different and are labeled here as * .

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C. The L. monocytogenes EGD-e wild-type strain (WT), the ΔinlK mutant (ΔinlK) and the complemented strain (ΔinlK+pPL2 inlK) 104 CFU were inoculated i.v into BALB/c mice. Animals were euthanized 24 h, 48 h, 72 h or 96 h after infection and organs were recovered, homogenized, and homogenates serially plated on BHI. The number of bacteria able to colonize liver (left panel) and spleen (right panel) is expressed as log10 CFU. Four animals per bacterial strain, per time points and per experiment were used. Statistical analyses were performed on the results of 3 independent experiments using the Student t test. P values of <0.05 were considered statistically different and are labeled here as *.


A . InlK amino acid sequence . The signal sequence is underlined and the different regions of leucine rich repeats ( LRRs ) are outlined . The consensus pentapeptide LPXTG at the C - terminal end is boxed .

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A. InlK amino acid sequence. The signal sequence is underlined and the different regions of leucine rich repeats (LRRs) are outlined. The consensus pentapeptide LPXTG at the C-terminal end is boxed.


B . Detection by immunofluorescence microscopy of InlK over - expressing in L . monocytogenes EGD - e ( WT ) , ΔinlK , WT + pADc - inlK , ΔinlK + pPRT - inlK and the ΔsrtA mutant over - expressing inlK ( ΔsrtA + pPRT - inlK ) grown in BHI medium using the rabbit polyclonal anti - InlK antibody . InlK was detected at the surface of InlK over - expressing bacteria ( WT + pADc - inlK and ΔinlK + pPRT - inlK ) , whereas it was undetectable at the surface WT bacteria or at the surface of the ΔstrA mutant over - expressing inlK .

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B. Detection by immunofluorescence microscopy of InlK over-expressing in L. monocytogenes EGD-e (WT), ΔinlK, WT+pADc-inlK, ΔinlK+pPRT-inlK and the ΔsrtA mutant over-expressing inlK (ΔsrtA+pPRT-inlK) grown in BHI medium using the rabbit polyclonal anti-InlK antibody. InlK was detected at the surface of InlK over-expressing bacteria (WT+pADc-inlK and ΔinlK+pPRT-inlK), whereas it was undetectable at the surface WT bacteria or at the surface of the ΔstrA mutant over-expressing inlK.


C . Detection of InlK by Western blot on total lysates of L . monocytogenes EGD - e ( WT ) , ΔinlK and ΔinlK + pPRT - inlK grown in BHI using the rabbit polyclonal anti - InlK antibody . Decreased concentrations of recombinant purified InlK were used as a positive control .

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C. Detection of InlK by Western blot on total lysates of L. monocytogenes EGD-e (WT), ΔinlK and ΔinlK+pPRT-inlK grown in BHI using the rabbit polyclonal anti-InlK antibody. Decreased concentrations of recombinant purified InlK were used as a positive control.


D . Detection of secreted InlK in the supernatant of ΔsrtA mutants over - expressing InlK . Western blotting was carried out on trichloroacetic acid precipitates of ΔsrtA and ΔsrtA + pPRT - inlK culture ( OD600 = 1 ) supernatants using the rabbit polyclonal anti - InlK antibody .

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D. Detection of secreted InlK in the supernatant of ΔsrtA mutants over-expressing InlK. Western blotting was carried out on trichloroacetic acid precipitates of ΔsrtA and ΔsrtA+pPRT-inlK culture (OD600 = 1) supernatants using the rabbit polyclonal anti-InlK antibody.


E . Detection of purified recombinant InlK protein with rabbit polyclonal anti - live Listeria antibody , rabbit polyclonal anti - killed Listeria antibody , rabbit polyclonal anti - InlK and a rabbit pre - immune serum . InlK was detected only with the rabbit polyclonal anti - live Listeria antibody indicating that it is expressed during the in vivo infectious process . BSA was used as control protein . Two different amounts of proteins were tested ( 500 ng and 200 ng ) to access signal specificity .

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E. Detection of purified recombinant InlK protein with rabbit polyclonal anti-live Listeria antibody, rabbit polyclonal anti-killed Listeria antibody, rabbit polyclonal anti-InlK and a rabbit pre-immune serum. InlK was detected only with the rabbit polyclonal anti-live Listeria antibody indicating that it is expressed during the in vivo infectious process. BSA was used as control protein. Two different amounts of proteins were tested (500 ng and 200 ng) to access signal specificity.


A . Bacterial pull - down of purified GST - MVP with the L . monocytogenes strains WT + pPRT - empty , ΔinlK + pPRT - empty , ΔinlK + pPRT - inlK and WT + pPRT - inlJ . GST - MVP bound to InlK over - expressing bacteria ( ΔinlK + pPRT - inlK ) but not to other bacteria . GST - ScarA was used as control of the specificity of the MVP precipitation by InlK over - expressing bacteria .

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A. Bacterial pull-down of purified GST-MVP with the L. monocytogenes strains WT+pPRT-empty, ΔinlK+pPRT-empty, ΔinlK+pPRT-inlK and WT+pPRT-inlJ. GST-MVP bound to InlK over-expressing bacteria (ΔinlK+pPRT-inlK) but not to other bacteria. GST-ScarA was used as control of the specificity of the MVP precipitation by InlK over-expressing bacteria.


B . Bacterial pull - down of MVP - GFP from transfected HeLa cell lysates with the L . monocytogenes strains WT + pPRT - empty , ΔinlK + pPRT - empty , ΔinlK + pPRT - inlK and WT + pPRT - inlJ . MVP - GFP bound to InlK over - expressing bacteria ( ΔinlK + pPRT - inlK ) but not to other bacteria .

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B. Bacterial pull-down of MVP-GFP from transfected HeLa cell lysates with the L. monocytogenes strains WT+pPRT-empty, ΔinlK+pPRT-empty, ΔinlK+pPRT-inlK and WT+pPRT-inlJ. MVP-GFP bound to InlK over-expressing bacteria (ΔinlK+pPRT-inlK) but not to other bacteria.


C . Co - immunoprecipitation ( Co - IP ) of purified InlK ( 20 µg ) with endogenous MVP of HT29 cells . The right panel shows anti - MVP co - IP performed on HT29 cell lysates . The left panel shows the control anti - MVP co - IP performed on lysis buffer .

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C. Co-immunoprecipitation (Co-IP) of purified InlK (20 µg) with endogenous MVP of HT29 cells. The right panel shows anti-MVP co-IP performed on HT29 cell lysates. The left panel shows the control anti-MVP co-IP performed on lysis buffer.


D . Detection of MVP recruitment at the surface of InlK over - expressing bacteria . HeLa cells were transfected with MVP - GFP ( green ) , infected with L . monocytogenes EGD - e wild - type ( WT ) , ΔinlK or ΔinlK + pPRT - inlK for 4 h , fixed for fluorescence light microscopy , and stained with anti - Listeria antibodies ( red ) . Inset regions are magnified . The scale bar represents 1 µm .

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D. Detection of MVP recruitment at the surface of InlK over-expressing bacteria. HeLa cells were transfected with MVP-GFP (green), infected with L. monocytogenes EGD-e wild-type (WT), ΔinlK or ΔinlK+pPRT-inlK for 4 h, fixed for fluorescence light microscopy, and stained with anti-Listeria antibodies (red). Inset regions are magnified. The scale bar represents 1 µm.


A . Detection of MVP recruitment at the surface of intracellular InlK over - expressing bacteria . HeLa cells were transfected with MVP - GFP ( red ) , infected with InlK expressing Listeria ( ΔinlK + pPRT - inlK ) for 4 h , fixed for fluorescence light microscopy . Intra - ( only green ) and extracellular ( cyan = green + blue ) bacteria were differentially stained with anti - Listeria antibody ( cf Material and Methods ) . Inset regions are magnified . Arrows indicate another intracellular bacterium which recruit MVP - GFP . The scale bar represents 1 µm . The right panel represents the quantification of the intracellular bacteria that recruit MVP ( mean % ± SEM % ) shown in the left panel . Statistical analyses were performed on the results of 3 independent experiments using the Student ' s t test . No significant difference was found between the 4 time points .

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A. Detection of MVP recruitment at the surface of intracellular InlK over-expressing bacteria. HeLa cells were transfected with MVP-GFP (red), infected with InlK expressing Listeria (ΔinlK+pPRT-inlK) for 4 h, fixed for fluorescence light microscopy. Intra- (only green) and extracellular (cyan = green+blue) bacteria were differentially stained with anti- Listeria antibody (cf Material and Methods). Inset regions are magnified. Arrows indicate another intracellular bacterium which recruit MVP-GFP. The scale bar represents 1 µm. The right panel represents the quantification of the intracellular bacteria that recruit MVP (mean%±SEM%) shown in the left panel. Statistical analyses were performed on the results of 3 independent experiments using the Student's t test. No significant difference was found between the 4 time points.


B . Detection of MVP recruitment at the surface of intracytosolic InlK over - expressing bacteria . HeLa cells were transfected with MVP - tomato ( red ) and YFP - CBD ( green ) , infected with InlK over - expressing Listeria ( ΔinlK + pPRT - inlK ) for 4 h , fixed for fluorescence light microscopy and stained with phalloidin ( blue ) . MVP positive bacteria were also labeled with YFP - CBD revealing that MVP was recruited by intracytosolic bacteria after the lysis of the internalization vacuole . Inset regions are magnified . The scale bar represents 1 µm .

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B. Detection of MVP recruitment at the surface of intracytosolic InlK over-expressing bacteria. HeLa cells were transfected with MVP-tomato (red) and YFP-CBD (green), infected with InlK over-expressing Listeria (ΔinlK+pPRT-inlK) for 4 h, fixed for fluorescence light microscopy and stained with phalloidin (blue). MVP positive bacteria were also labeled with YFP-CBD revealing that MVP was recruited by intracytosolic bacteria after the lysis of the internalization vacuole. Inset regions are magnified. The scale bar represents 1 µm.


C . Kinetics of MVP and actin recruitment at the surface of InlK over - expressing bacteria . HeLa cells were transfected with MVP - tomato ( red ) and actin - GFP ( green ) , infected with InlK over - expressing Listeria ( ΔinlK + pPRT - inlK ) for 4 h , and prepared for real - time video microscopy . Image series were collected every 15 min for 2 h . The left part shows an MVP positive bacterium that never recruits actin . The right part shows MVP replacement by actin around the bacterium . No colocalization of MVP - Tomato and actin - GFP was detected . Time is indicated along the Y axis . The entire image sequence can be viewed as Video S1 .

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C. Kinetics of MVP and actin recruitment at the surface of InlK over-expressing bacteria. HeLa cells were transfected with MVP-tomato (red) and actin-GFP (green), infected with InlK over-expressing Listeria (ΔinlK+pPRT-inlK) for 4 h, and prepared for real-time video microscopy. Image series were collected every 15 min for 2 h. The left part shows an MVP positive bacterium that never recruits actin. The right part shows MVP replacement by actin around the bacterium. No colocalization of MVP-Tomato and actin-GFP was detected. Time is indicated along the Y axis. The entire image sequence can be viewed as Video S1.


A . Impaired recruitment of p62 to MVP positive Listeria . HeLa cells were transfected with MVP - GFP ( green ) , infected with InlK over - expressing Listeria ( ΔinlK + pPRT - inlK ) for 4 h , fixed for fluorescence light microscopy , and stained with phalloidin ( blue ) and anti - p62 antibody ( red ) . Inset regions are magnified . Arrows indicate independent bacteria The scale bar represents 1 µm . The vast majority of MVP - positive bacteria were completely devoid of anti - p62 labeling ( 95 . 1 ± 2 . 0 % ; mean ± SEM from n = 3 experiments ) but 4 . 9 ± 2 . 0 % ( mean ± SEM from n = 3 experiments ) were stained at one pole with MVP and at the other pole with p62 .

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A. Impaired recruitment of p62 to MVP positive Listeria. HeLa cells were transfected with MVP-GFP (green), infected with InlK over-expressing Listeria (ΔinlK+pPRT-inlK) for 4 h, fixed for fluorescence light microscopy, and stained with phalloidin (blue) and anti-p62 antibody (red). Inset regions are magnified. Arrows indicate independent bacteria The scale bar represents 1 µm. The vast majority of MVP-positive bacteria were completely devoid of anti-p62 labeling (95.1±2.0%; mean ± SEM from n = 3 experiments) but 4.9±2.0% (mean ± SEM from n = 3 experiments) were stained at one pole with MVP and at the other pole with p62.


B . Impaired recruitment of GFP - LC3 on MVP positive Listeria . HeLa cells were transfected with MVP - tomato ( red ) and GFP - LC3 ( green ) , infected with InlK over - expressing Listeria ( ΔinlK + pPRT - inlK ) for 4 h , fixed for fluorescence light microscopy , and stained with phalloidin ( blue ) . Inset regions are magnified . The scale bar represents 1 µm . MVP and / or actin positive bacteria were never recognized by GFP - LC3 . Arrows point to bacteria at different steps of the infection process : 1 ) InlK over - expressing bacterium is totally covered by MVP ; 2 ) bacterium is partially labeled with MVP ( at the poles ) and actin ( at the center ) ; 3 ) bacterium is completely covered by actin ; 4 ) bacterium is enclosed in an GFP - LC3 positive autophagosome .

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B. Impaired recruitment of GFP-LC3 on MVP positive Listeria. HeLa cells were transfected with MVP-tomato (red) and GFP-LC3 (green), infected with InlK over-expressing Listeria (ΔinlK+pPRT-inlK) for 4 h, fixed for fluorescence light microscopy, and stained with phalloidin (blue). Inset regions are magnified. The scale bar represents 1 µm. MVP and/or actin positive bacteria were never recognized by GFP-LC3. Arrows point to bacteria at different steps of the infection process: 1) InlK over-expressing bacterium is totally covered by MVP; 2) bacterium is partially labeled with MVP (at the poles) and actin (at the center); 3) bacterium is completely covered by actin; 4) bacterium is enclosed in an GFP-LC3 positive autophagosome.


C . Kinetics of autophagy escape for MVP positive Listeria . Jeg3 cells were transfected with MVP - tomato ( red ) and GFP - LC3 ( green ) , infected with InlK over - expressing Listeria ( ΔinlK + pPRT - inlK ) for 4 h , and prepared for real - time video microscopy . Image series were collected every 5 min for 2 h . Time is indicated along the Y axis . The left panel shows that the GFP - LC3 membranous aggregate detaches from the MVP positive Listeria . The entire image sequence can be viewed as Video S2 . The right panel shows that the GFP - LC3 membranous aggregate on MVP positive bacteria does not lead to an autophagosome formation , whereas those bacteria efficiently divided . The entire image sequence can be viewed as Video S3 .

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C. Kinetics of autophagy escape for MVP positive Listeria. Jeg3 cells were transfected with MVP-tomato (red) and GFP-LC3 (green), infected with InlK over-expressing Listeria (ΔinlK+pPRT-inlK) for 4 h, and prepared for real-time video microscopy. Image series were collected every 5 min for 2 h. Time is indicated along the Y axis. The left panel shows that the GFP-LC3 membranous aggregate detaches from the MVP positive Listeria. The entire image sequence can be viewed as Video S2. The right panel shows that the GFP-LC3 membranous aggregate on MVP positive bacteria does not lead to an autophagosome formation, whereas those bacteria efficiently divided. The entire image sequence can be viewed as Video S3.


D . Impaired recruitment of GFP - LC3 and ubiquitin to MVP positive ΔactA Listeria . HeLa cells were transfected with MVP - tomato ( red ) and GFP - LC3 ( green ) , infected with InlK over - expressing ΔactA ( ΔactA + pADc - inlK ) for 4 h , fixed for fluorescence light microscopy , and stained with anti - ubiquitin antibody ( blue ) and DAPI ( white ) . Inset regions are magnified . The scale bar represents 1 µm .

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D. Impaired recruitment of GFP-LC3 and ubiquitin to MVP positive ΔactA Listeria. HeLa cells were transfected with MVP-tomato (red) and GFP-LC3 (green), infected with InlK over-expressing ΔactA (ΔactA+pADc-inlK) for 4 h, fixed for fluorescence light microscopy, and stained with anti-ubiquitin antibody (blue) and DAPI (white). Inset regions are magnified. The scale bar represents 1 µm.


E . LC3 levels in infected RAW 267 . 4 macrophages . Left panel : RAW 267 . 4 macrophages were infected with L . monocytogenes EGD ( WT ) , ΔactA or ΔactA + InlK for 6 h . Cell total lysates were immunobloted for LC3 and actin . Western blot is representative from 3 independent experiments . Right panel : Quantification of the relative LC3 - II level ( mean ± SEM ) shown in the left panel . Statistical analyses were performed on the results of 3 independent experiments using the Student ' s t test . P values of < 0 . 05 were considered statistically different .

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E. LC3 levels in infected RAW 267.4 macrophages. Left panel: RAW 267.4 macrophages were infected with L. monocytogenes EGD (WT), ΔactA or ΔactA+InlK for 6 h. Cell total lysates were immunobloted for LC3 and actin. Western blot is representative from 3 independent experiments. Right panel: Quantification of the relative LC3-II level (mean ± SEM) shown in the left panel. Statistical analyses were performed on the results of 3 independent experiments using the Student's t test. P values of <0.05 were considered statistically different.


A . InlK and ActA expression in Listeria strains used for survival assays . Total lysates of L . monocytogenes EGD - ( pADc - GFP ) , EGD - ( pADc - inlK ) , ΔactA - ( pADc - GFP ) and ΔactA - ( pADc - inlK ) grown in BHI were immunoblotted using anti - ActA and anti - InlK antibodies .

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A. InlK and ActA expression in Listeria strains used for survival assays. Total lysates of L. monocytogenes EGD-(pADc-GFP), EGD-(pADc-inlK), ΔactA-(pADc-GFP) and ΔactA-(pADc-inlK) grown in BHI were immunoblotted using anti-ActA and anti-InlK antibodies.


B . Intracellular survival of EGD - ( pADc - GFP ) , EGD - ( pADc - inlK ) , ΔactA - ( pADc - GFP ) and ΔactA - ( pADc - inlK ) in RAW 267 . 4 macrophages . Statistical analyses were performed on the results of 3 independent experiments using the Student t test . P values of < 0 . 05 were considered statistically different and are labeled here as * .

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B. Intracellular survival of EGD-(pADc-GFP), EGD-(pADc-inlK), ΔactA-(pADc-GFP) and ΔactA-(pADc-inlK) in RAW 267.4 macrophages. Statistical analyses were performed on the results of 3 independent experiments using the Student t test. P values of <0.05 were considered statistically different and are labeled here as *.


C . Intracellular survival of ΔactA - ( pADc - GFP ) and ΔactA - ( pADc - inlK ) in MVP - GFP transfected Jeg3 cells . Statistical analyses were performed on the results of 3 independent experiments using the Student ' s t test . P values of < 0 . 05 were considered statistically different and are labeled here as * .

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C. Intracellular survival of ΔactA-(pADc-GFP) and ΔactA-(pADc-inlK) in MVP-GFP transfected Jeg3 cells. Statistical analyses were performed on the results of 3 independent experiments using the Student's t test. P values of <0.05 were considered statistically different and are labeled here as *.


D . Intracellular survival of ΔactA - ( pADc - GFP ) and ΔactA - ( pADc - inlK ) in MVP - GFP transfected HeLa cells . Statistical analyses were performed on the results of 3 independent experiments using the Student ' s t test . P values of < 0 . 05 were considered statistically different and are labeled here as * .

D. Intracellular survival of ΔactA-(pADc-GFP) and ΔactA-(pADc-inlK) in MVP-GFP transfected HeLa cells. Statistical analyses were performed on the results of 3 independent experiments using the Student's t test. P values of <0.05 were considered statistically different and are labeled here as *.


E . MVP levels in RAW 267 . 4 macrophages treated with MVP - siRNA . Western blot is representative from 3 independent experiments .

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E. MVP levels in RAW 267.4 macrophages treated with MVP-siRNA. Western blot is representative from 3 independent experiments.


F . Intracellular survival of ΔactA - ( pADc - GFP ) and ΔactA - ( pADc - inlK ) in MVP knock - down RAW 267 . 4 macrophages . Statistical analyses were performed on the results of 3 independent experiments using the Student t test . P values of < 0 . 05 were considered statistically different and are labeled here as * .

F. Intracellular survival of ΔactA-(pADc-GFP) and ΔactA-(pADc-inlK) in MVP knock-down RAW 267.4 macrophages. Statistical analyses were performed on the results of 3 independent experiments using the Student t test. P values of <0.05 were considered statistically different and are labeled here as *.


( b ) Density gradient fractions of NRK cells starved for 8 h were analysed by transmission electron microscopy ( upper panel ) and immunoblotted for LC3 or LAMP1 ( lower panel ) . Proteins in fraction 1 were identified by mass spectrometry analysis . Three replicates were carried out from three independent purifications . Scale bar , 2 μm .

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(b) Density gradient fractions of NRK cells starved for 8 h were analysed by transmission electron microscopy (upper panel) and immunoblotted for LC3 or LAMP1 (lower panel). Proteins in fraction 1 were identified by mass spectrometry analysis. Three replicates were carried out from three independent purifications. Scale bar, 2 μm.


( e ) NRK cells grown on the SAMCell were transfected with FAM - RNAi by reverse transfection and the transfection efficiency was monitored by microscopy . Left panel , FAM - RNAi - positive cells ; right panel , differential interference contrast ( DIC ) image of the cells in the left panel .

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(e) NRK cells grown on the SAMCell were transfected with FAM-RNAi by reverse transfection and the transfection efficiency was monitored by microscopy. Left panel, FAM-RNAi-positive cells; right panel, differential interference contrast (DIC) image of the cells in the left panel.


( a ) NRK cells stably expressing CFP - LC3 ; LAMP1 - YFP were transfected with nonspecific ( NS ) - or Cltc ( clathrin heavy chain ) - RNAi and starved for 12 h . Scale bar , 5 μm . ( b ) Cells from a were assessed for enlarged autolysosomes after starvation and quantified . n = 100 cells from three independent experiments . Error bars indicate the s . d .

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(a) NRK cells stably expressing CFP-LC3; LAMP1-YFP were transfected with nonspecific (NS)- or Cltc (clathrin heavy chain)-RNAi and starved for 12 h. Scale bar, 5 μm. (b) Cells from a were assessed for enlarged autolysosomes after starvation and quantified. n = 100 cells from three independent experiments. Error bars indicate the s.d.


( c ) Representative TEM micrographs of nonspecific - or Cltc - RNAi - transfected NRK cells after 12 h of serum starvation . Scale bar , 5 μm .

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(c) Representative TEM micrographs of nonspecific- or Cltc-RNAi-transfected NRK cells after 12 h of serum starvation. Scale bar, 5 μm.


( d ) NRK cell lines stably expressing LAMP1 - Cherry ; clathrin - GFP were starved for 0 or 4 h and observed by confocal microscopy . As a control , clathrin - GFP - expressing cells were starved for 0 or 4 h and then stained with MitoTracker red . Scale bar , 5 μm . The insets in a , c and d represent enlarged regions of interest . ( e ) Cells from d were analysed for Pearson ' s co - localization coefficent ( R ( r ) ) . n = 30 cells from three independent experiments . Error bars indicate the s . d .

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(d) NRK cell lines stably expressing LAMP1-Cherry; clathrin-GFP were starved for 0 or 4 h and observed by confocal microscopy. As a control, clathrin-GFP-expressing cells were starved for 0 or 4 h and then stained with MitoTracker red. Scale bar, 5 μm. The insets in a, c and d represent enlarged regions of interest. (e) Cells from d were analysed for Pearson's co-localization coefficent (R(r)). n = 30 cells from three independent experiments. Error bars indicate the s.d.


( f ) NRK cells stably expressing LAMP1 - Cherry ; clathrin - GFP were starved for 4 h and time - lapse images were acquired by spinning - disc microscopy . Scale bar , 500 nm . The white arrows indicate a clathrin punctum moving along a LAMP1 - positive structure .

(f) NRK cells stably expressing LAMP1-Cherry; clathrin-GFP were starved for 4 h and time-lapse images were acquired by spinning-disc microscopy. Scale bar, 500 nm. The white arrows indicate a clathrin punctum moving along a LAMP1-positive structure.


( g ) NRK cells were starved for 0 , 4 or 6 h and then subjected to cell fractionation . Fractions were analysed by western blotting with antibodies against LAMP2 and clathrin heavy chain . 1 is the top fraction . The outline indicates the Lamp2 - positive fraction .

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(g) NRK cells were starved for 0, 4 or 6 h and then subjected to cell fractionation. Fractions were analysed by western blotting with antibodies against LAMP2 and clathrin heavy chain. 1 is the top fraction. The outline indicates the Lamp2-positive fraction.


( h ) Cell lines stably expressing clathrin - GFP were starved for 4 h , stained with antibodies against GFP and LAMP1 , analysed by 3D - SIM . Scale bar , 2 . 5 μm . The right panels show enlarged regions of interest from the left panel : T , top view ; L , lateral view ; B , bottom view .

(h) Cell lines stably expressing clathrin-GFP were starved for 4 h, stained with antibodies against GFP and LAMP1, analysed by 3D-SIM. Scale bar, 2.5 μm. The right panels show enlarged regions of interest from the left panel: T, top view; L, lateral view; B, bottom view.


( i ) Left panel , representative TEM micrographs of purified autolysosomes from starved clathrin - GFP - expressing cells . Scale bar , 250 nm . Right panel , SIM image of purified autolysosomes stained with GFP and LAMP1 from the same experiment . Scale bar , 5 μm .

(i) Left panel, representative TEM micrographs of purified autolysosomes from starved clathrin-GFP-expressing cells. Scale bar, 250 nm. Right panel, SIM image of purified autolysosomes stained with GFP and LAMP1 from the same experiment. Scale bar, 5 μm.


( j ) NRK or stable clathrin - GFP - expressing NRK cell lines were analysed by pre - embedding - immuno - electron microscopy using antibodies against GFP . Scale bar , 100 nm . ( k ) Quantification of the average number of gold particles per image from control or clathrin - GFP cells . n = 25 images from three independent experiments . The error bars indicate the s . d . Uncropped images of blots are shown in Supplementary Fig . S6 .

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(j) NRK or stable clathrin-GFP-expressing NRK cell lines were analysed by pre-embedding-immuno-electron microscopy using antibodies against GFP. Scale bar, 100 nm. (k) Quantification of the average number of gold particles per image from control or clathrin-GFP cells. n = 25 images from three independent experiments. The error bars indicate the s.d. Uncropped images of blots are shown in Supplementary Fig. S6.


( a ) NRK cell lines stably expressing CFP - LC3 ; LAMP1 - YFP were transfected with nonspecific ( NS ) - or PIP5K1B - RNAi and starved for 12 h . Scale bars , 5 μm . ( b ) Cells from a were assessed for enlarged autolysosomes in a blind fashion after starvation and quantified . n = 100 cells from three independent experiments . Error bars indicate the s . d .

(a) NRK cell lines stably expressing CFP-LC3; LAMP1-YFP were transfected with nonspecific (NS)- or PIP5K1B-RNAi and starved for 12 h. Scale bars, 5 μm. (b) Cells from a were assessed for enlarged autolysosomes in a blind fashion after starvation and quantified. n = 100 cells from three independent experiments. Error bars indicate the s.d.


( c ) Representative TEM micrographs of nonspecific - or PIP5K1B - RNAi - transfected NRK cells after 12 h of serum starvation . Scale bar , 5 μm .

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(c) Representative TEM micrographs of nonspecific- or PIP5K1B-RNAi-transfected NRK cells after 12 h of serum starvation. Scale bar, 5 μm.


( d ) NRK cells were starved for 4 h , stained with antibodies against PIP5K1B and LAMP1 , and then analysed by 3D - SIM . Scale bar , 2 . 5 μm . The right panels show enlarged regions of interest from the left panel : T , top view ; L , lateral view ; B , bottom view .

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(d) NRK cells were starved for 4 h, stained with antibodies against PIP5K1B and LAMP1, and then analysed by 3D-SIM. Scale bar, 2.5 μm. The right panels show enlarged regions of interest from the left panel: T, top view; L, lateral view; B, bottom view.


( e ) Fractions from Fig . 2f were analysed by western blotting with an antibody against PIP5K1B . The outline indicates the LAMP2 - positive fraction .

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(e) Fractions from Fig. 2f were analysed by western blotting with an antibody against PIP5K1B. The outline indicates the LAMP2-positive fraction.


( f ) NRK cells were starved for 4 h , autolysosomes were purified by density fractionation and the quality of purified autolysosomes was monitored by TEM ( left panel ) . Right panel , purified autolysosomes stained with antibodies against PI ( 4 ) P and PtdIns ( 4 , 5 ) P2 . Scale bar , 500 nm .

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(f) NRK cells were starved for 4 h, autolysosomes were purified by density fractionation and the quality of purified autolysosomes was monitored by TEM (left panel). Right panel, purified autolysosomes stained with antibodies against PI(4)P and PtdIns(4,5)P2. Scale bar, 500 nm.


( g ) NRK cells were starved for 0 or 4 h and stained with antibody against PI ( 4 ) P . Scale bar , 5 μm .

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(g) NRK cells were starved for 0 or 4 h and stained with antibody against PI(4)P. Scale bar, 5 μm.


( h ) NRK cells lines stably expressing OSBP - PH - GFP ; LAMP1 - Cherry were starved for 0 , 4 , or 8 h and observed by confocal microscopy . Scale bars , 5 μm .

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(h) NRK cells lines stably expressing OSBP-PH-GFP;LAMP1-Cherry were starved for 0, 4, or 8 h and observed by confocal microscopy. Scale bars, 5 μm.


( i ) NRK cell lines stably expressing OSBP - PH - GFP ; LAMP1 - Cherry were transfected with PIP5K1B - RNAi . At 60 h after transfection , cells were starved for 12 h and analysed by confocal microscopy . Scale bar , 5 μm .

(i) NRK cell lines stably expressing OSBP-PH-GFP;LAMP1-Cherry were transfected with PIP5K1B-RNAi. At 60 h after transfection, cells were starved for 12 h and analysed by confocal microscopy. Scale bar, 5 μm.


( j ) NRK cell lines stably expressing LAMP1 - Cherry ; clathrin - GFP were transfected with nonspecific - or PIP5K1B - RNAi and starved for 12 h , and then observed by confocal microscopy . Scale bars , 5 μm . The insets in a , h - j represent enlarged regions of interest .

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(j) NRK cell lines stably expressing LAMP1-Cherry;clathrin-GFP were transfected with nonspecific- or PIP5K1B-RNAi and starved for 12 h, and then observed by confocal microscopy. Scale bars, 5 μm. The insets in a, h-j represent enlarged regions of interest.


( k ) Cells from g were analysed for Pearson ' s co - localization coefficent ( R ( r ) ) by Olympus Fluoview . n = 30 cells from three independent experiments . Error bars indicate the s . d . Uncropped images of blots are shown in Supplementary Fig . S6 .

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(k) Cells from g were analysed for Pearson's co-localization coefficent (R(r)) by Olympus Fluoview. n = 30 cells from three independent experiments. Error bars indicate the s.d. Uncropped images of blots are shown in Supplementary Fig. S6.


( a ) NRK cells stably expressing CFP - LC3 ; LAMP1 - YFP were transfected with nonspecific ( NS ) - or PIP5K1A - RNAi and starved for 10 h . The insets represent enlarged regions of interest . Scale bar , 5 μm . ( b ) Cells from a were assessed for the presence of elongated reformation tubules in a blind fashion after starvation and quantified . n = 100 cells from three independent experiments . Error bars indicate the s . d . ( c ) Cells from a were assessed for the length of reformation tubules in a blind fashion after starvation and quantified . 50 cells were counted . The average tubule length is indicated above each column . ( d ) Diagram of reformation tubules . R , radius of main body ; L , length . ( e ) Representative image of reformation tubules in nonspecific - or PIP5K1A - RNAi - transfected cells . The top and bottom panels show two randomly selected images of the respective samples .

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(a) NRK cells stably expressing CFP-LC3;LAMP1-YFP were transfected with nonspecific (NS)- or PIP5K1A-RNAi and starved for 10 h. The insets represent enlarged regions of interest. Scale bar, 5 μm. (b) Cells from a were assessed for the presence of elongated reformation tubules in a blind fashion after starvation and quantified. n = 100 cells from three independent experiments. Error bars indicate the s.d. (c) Cells from a were assessed for the length of reformation tubules in a blind fashion after starvation and quantified. 50 cells were counted. The average tubule length is indicated above each column. (d) Diagram of reformation tubules. R, radius of main body; L, length. (e) Representative image of reformation tubules in nonspecific- or PIP5K1A-RNAi-transfected cells. The top and bottom panels show two randomly selected images of the respective samples.


( f ) Cells from a were assessed for the L / R ratio of reformation tubules in a blind fashion after starvation and quantified . 25 cells were counted . The average L / R is indicated above each column .

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(f) Cells from a were assessed for the L/R ratio of reformation tubules in a blind fashion after starvation and quantified. 25 cells were counted. The average L/R is indicated above each column.


( g ) Purified reformation tubules were monitored by TEM . Scale bar , 250 nm .

(g) Purified reformation tubules were monitored by TEM. Scale bar, 250 nm.


( h ) Purified reformation tubules stained with antibodies against PI ( 4 ) P , PtdIns ( 4 , 5 ) P2 and PIP5K1A . Scale bar , 250 nm .

(h) Purified reformation tubules stained with antibodies against PI(4)P, PtdIns(4,5)P2 and PIP5K1A. Scale bar, 250 nm.


( i ) Fractions from Fig . 2f were analysed by western blotting with an antibody against PIP5K1A . The outline indicates the LAMP2 - positive fraction .

(i) Fractions from Fig. 2f were analysed by western blotting with an antibody against PIP5K1A. The outline indicates the LAMP2-positive fraction.


( j ) NRK cells stably expressing CFP - LC3 ; LAMP1 - YFP were starved for 8 h , and the budding of proto - lysosomes was monitored by spinning - disc microscopy . Scale bar , 100 nm .

(j) NRK cells stably expressing CFP-LC3;LAMP1-YFP were starved for 8 h, and the budding of proto-lysosomes was monitored by spinning-disc microscopy. Scale bar, 100 nm.


( k ) NRK cell lines stably expressing LAMP1 - Cherry ; clathrin - GFP were starved for 8 h and observed by confocal microscopy . The right panels show enlarged regions of interest . Scale bar , 5 μm .

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(k) NRK cell lines stably expressing LAMP1-Cherry;clathrin-GFP were starved for 8 h and observed by confocal microscopy. The right panels show enlarged regions of interest. Scale bar, 5 μm.


( l ) NRK cell lines stably expressing LAMP1 - Cherry ; clathrin - GFP were transfected with nonspecific - or PIP5K1A - RNAi and starved for 8 h . Scale bar , 5 μm . The middle panels show enlarged regions of interest from the upper panels . The distance between clathrin dots on each reformation tubule in the upper panels is shown in diagrammatic form in the lower panels . Black line : reformation tubules ; red dot : clathrin . ( m ) The distance between two clathrin dots on reformation tubules ( RTs ) in cells from l was accessed in a blind fashion . Twenty - five cells were counted . The average distance between two clathrin dots is indicated above each column . Uncropped images of blots are shown in Supplementary Fig . S6 .

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(l) NRK cell lines stably expressing LAMP1-Cherry;clathrin-GFP were transfected with nonspecific- or PIP5K1A-RNAi and starved for 8 h. Scale bar, 5 μm. The middle panels show enlarged regions of interest from the upper panels. The distance between clathrin dots on each reformation tubule in the upper panels is shown in diagrammatic form in the lower panels. Black line: reformation tubules; red dot: clathrin. (m) The distance between two clathrin dots on reformation tubules (RTs) in cells from l was accessed in a blind fashion. Twenty-five cells were counted. The average distance between two clathrin dots is indicated above each column. Uncropped images of blots are shown in Supplementary Fig. S6.


( a ) NRK cells stably expressing CFP - LC3 ; LAMP1 - YFP were transfected with nonspecific ( NS ) - or AP2 - RNAi and starved for 12 h . Scale bars , 5 μm . ( b ) Cells from a were assessed for enlarged autolysosomes in a blind fashion after starvation and quantified . n = 100 cells from three independent experiments . Error bars indicate the s . d .

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(a) NRK cells stably expressing CFP-LC3; LAMP1-YFP were transfected with nonspecific (NS)- or AP2-RNAi and starved for 12 h. Scale bars, 5 μm. (b) Cells from a were assessed for enlarged autolysosomes in a blind fashion after starvation and quantified. n = 100 cells from three independent experiments. Error bars indicate the s.d.


( c ) Representative TEM micrographs of nonspecific - or AP2 - RNAi - transfected NRK cells after 12 h of serum starvation . Scale bars , 5 μm .

(c) Representative TEM micrographs of nonspecific- or AP2-RNAi-transfected NRK cells after 12 h of serum starvation. Scale bars, 5 μm.


( d ) NRK cells were starved for 4 h , stained with antibodies against AP2 and LAMP1 , and then analysed by 3D - SIM . Scale bar , 5 μm . The right panels show enlarged regions of interest from the left panel : T , top view ; L , lateral view ; B , bottom view .

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(d) NRK cells were starved for 4 h, stained with antibodies against AP2 and LAMP1, and then analysed by 3D-SIM. Scale bar, 5 μm. The right panels show enlarged regions of interest from the left panel: T, top view; L, lateral view; B, bottom view.


( e ) Fractions from Fig . 2f were analysed by western blotting with an antibody against AP2α . The outline indicates the LAMP2 - positive fraction .

(e) Fractions from Fig. 2f were analysed by western blotting with an antibody against AP2α. The outline indicates the LAMP2-positive fraction.


( g ) Purified autolysosomes were treated with trypsin , incubated with rat brain cytosol , washed and then analysed by western blot with antibodies against AP2α and LAMP2 .

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(g) Purified autolysosomes were treated with trypsin, incubated with rat brain cytosol, washed and then analysed by western blot with antibodies against AP2α and LAMP2.


( h ) Stripped autolysosomes were pretreated with GFP or PtdIns ( 4 , 5 ) P2 antibody and then incubated with rat brain cytosol . Autolysosomes were washed and then analysed by western blotting with antibodies against AP2α and LAMP2 .

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(h) Stripped autolysosomes were pretreated with GFP or PtdIns(4,5)P2 antibody and then incubated with rat brain cytosol. Autolysosomes were washed and then analysed by western blotting with antibodies against AP2α and LAMP2.


( i ) NRK cell lines stably expressing LAMP1 - Cherry ; clathrin - GFP were transfected with nonspecific - or AP2 - RNAi and starved for 12 h . Scale bar , 5 μm . The insets in a , c and i represent enlarged regions of interest . ( j ) Cells from i were analysed for the Pearson ' s co - localization coefficent ( R ( r ) ) by Olympus Fluoview . n = 30 cells from three independent experiments ) . Error bars indicate the s . d . Uncropped images of blots are shown in Supplementary Fig . S6 .

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(i) NRK cell lines stably expressing LAMP1-Cherry;clathrin-GFP were transfected with nonspecific- or AP2-RNAi and starved for 12 h. Scale bar, 5 μm. The insets in a, c and i represent enlarged regions of interest. (j) Cells from i were analysed for the Pearson's co-localization coefficent (R(r)) by Olympus Fluoview. n = 30 cells from three independent experiments). Error bars indicate the s.d. Uncropped images of blots are shown in Supplementary Fig. S6.


( a ) NRK cells stably expressing CFP - LC3 ; LAMP1 - YFP were transfected with nonspecific ( NS ) - or AP4 - RNAi and starved for 12 h . Scale bars , 5 μm . ( b ) Cells from a were assessed for enlarged autolysosomes in a blind fashion after starvation and quantified . n = 100 cells from three independent experiments . Error bars indicate the s . d .

(a) NRK cells stably expressing CFP-LC3; LAMP1-YFP were transfected with nonspecific (NS)- or AP4-RNAi and starved for 12 h. Scale bars, 5 μm. (b) Cells from a were assessed for enlarged autolysosomes in a blind fashion after starvation and quantified. n = 100 cells from three independent experiments. Error bars indicate the s.d.


( c ) Representative TEM micrographs of nonspecific - or AP4 - RNAi - transfected NRK cells after 12 h of serum starvation . Scale bars , 5 μm .

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(c) Representative TEM micrographs of nonspecific- or AP4-RNAi-transfected NRK cells after 12 h of serum starvation. Scale bars, 5 μm.


( d ) NRK cells were starved for 0 , 4 , 8 or 12 h , and isolated lysosomes / autolysosome were analysed by western blotting using antibodies against AP4 or LAMP2 .

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(d) NRK cells were starved for 0, 4, 8 or 12 h, and isolated lysosomes/autolysosome were analysed by western blotting using antibodies against AP4 or LAMP2.


( e ) Purified reformation tubules were stained with antibodies against AP4 and LAMP1 . Scale bar , 2 μm . The bottom row shows enlarged regions of interest from the top panel .

(e) Purified reformation tubules were stained with antibodies against AP4 and LAMP1. Scale bar, 2 μm. The bottom row shows enlarged regions of interest from the top panel.


( f ) NRK cells were transfected with GFP and LAMP1 - GFP . At 20 h after transfection , cells were starved for 0 or 8 h , and then collected and analysed by immunoprecipitation ( IP ) and western blotting ( WB ) of proteins as indicated , using stably expressed LAMP1 - GFP as bait . TCL , total cell lysate .

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(f) NRK cells were transfected with GFP and LAMP1-GFP. At 20 h after transfection, cells were starved for 0 or 8 h, and then collected and analysed by immunoprecipitation (IP) and western blotting (WB) of proteins as indicated, using stably expressed LAMP1-GFP as bait. TCL, total cell lysate.


The insets in a , c and g represent enlarged regions of interest . ( g ) NRK cell lines stably expressing LAMP1 - Cherry ; clathrin - GFP were transfected with nonspecific - or AP4 - RNAi and starved for 12 h . Scale bars , 5 μm . ( h ) Cells from g were analysed for the Pearson ' s co - localization coefficent ( R ( r ) ) by Olympus Fluoview . n = 30 cells from three independent experiments . Error bars indicate the s . d . Uncropped images of blots are shown in Supplementary Fig . S6 .

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The insets in a, c and g represent enlarged regions of interest. (g) NRK cell lines stably expressing LAMP1-Cherry; clathrin-GFP were transfected with nonspecific- or AP4-RNAi and starved for 12 h. Scale bars, 5 μm. (h) Cells from g were analysed for the Pearson's co-localization coefficent (R(r)) by Olympus Fluoview. n = 30 cells from three independent experiments. Error bars indicate the s.d. Uncropped images of blots are shown in Supplementary Fig. S6.


( a ) Purified autolysosomes were treated with trypsin . The trypsin - treated ( stripped ) autolysosomes were incubated with rat brain cytosol and reaction mixture , and then analysed by western blotting . HC , heavy chain .

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(a) Purified autolysosomes were treated with trypsin. The trypsin-treated (stripped) autolysosomes were incubated with rat brain cytosol and reaction mixture, and then analysed by western blotting. HC, heavy chain.


( b ) Scanning electron micrographs of purified autolysosomes , rat brain cytosol , trypsin - treated purified autolysosomes and trypsin - treated purified autolysosomes after the membrane budding assay . Scale bar , 250 nm . The arrows indicate buds .

(b) Scanning electron micrographs of purified autolysosomes, rat brain cytosol, trypsin-treated purified autolysosomes and trypsin-treated purified autolysosomes after the membrane budding assay. Scale bar, 250 nm. The arrows indicate buds.


( c ) Trypsin - treated autolysosomes were incubated with rat brain cytosol and reaction mixture in the presence of antibodies against GFP , clathrin , AP2 , AP3 , AP4 and PtdIns ( 4 , 5 ) P2 , and then analysed by western blotting .

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(c) Trypsin-treated autolysosomes were incubated with rat brain cytosol and reaction mixture in the presence of antibodies against GFP, clathrin, AP2, AP3, AP4 and PtdIns(4,5)P2, and then analysed by western blotting.


( d ) Reactions from c were visualized by scanning electron microscopy . Scale bar , 250 nm . Arrow indicates bud . ( e ) Samples from d were quantified for the number of buds on each autolysosome . Error bars represent s . d . n = 50 autolysosomes from three independent experiments . Uncropped images of blots are shown in Supplementary Fig . S6 .

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(d) Reactions from c were visualized by scanning electron microscopy. Scale bar, 250 nm. Arrow indicates bud. (e) Samples from d were quantified for the number of buds on each autolysosome. Error bars represent s.d. n = 50 autolysosomes from three independent experiments. Uncropped images of blots are shown in Supplementary Fig. S6.


( a ) NRK cells were transfected with nonspecific ( NS ) - or Cltc ( clathrin heavy chain ) - RNAi and starved for 0 , 4 , 8 or 12 h . Scale bar , 5 mm . Cells were stained with antibodies against LC3 . ( b ) Cells from a were assessed for LC3 puncta in a blind fashion after starvation and quantified . n = 100 cells from three independent experiments . Error bars indicate the s . d .

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(a) NRK cells were transfected with nonspecific (NS)- or Cltc (clathrin heavy chain)-RNAi and starved for 0, 4, 8 or 12 h. Scale bar, 5 mm. Cells were stained with antibodies against LC3. (b) Cells from a were assessed for LC3 puncta in a blind fashion after starvation and quantified. n = 100 cells from three independent experiments. Error bars indicate the s.d.


A Representative immunofluorescence microscopy staining of GFP of small intestinal tissue of LGR5 ‐ GFP knock ‐ in ( LGR5 ‐ GFPki ) mice . Arrowheads and numbers indicate ISPC positions in the crypts . Scale bar : 20 μm . B Quantification of GFP staining intensity of intestinal stem and progenitor cells ( ISPCs ) at indicated positions in basal crypts of LGR5 ‐ GFPkimice ( n = 80 crypts from four mice ) . Each dot presents one cell . Red lines : mean values ± SEM . Unpaired two ‐ tailed Student ' s t ‐ test .

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A Representative immunofluorescence microscopy staining of GFP of small intestinal tissue of LGR5‐GFP knock‐in (LGR5‐GFPki) mice. Arrowheads and numbers indicate ISPC positions in the crypts. Scale bar: 20 μm.B Quantification of GFP staining intensity of intestinal stem and progenitor cells (ISPCs) at indicated positions in basal crypts of LGR5‐GFPkimice (n = 80 crypts from four mice). Each dot presents one cell. Red lines: mean values ± SEM. Unpaired two‐tailed Student's t‐test.


C Representative immunofluorescence microscopy staining of Msi1 of small intestinal tissue of young mTerc + / + mice . Dashed line outlines a crypt . Arrowheads and numbers indicate ISPC positions in the crypts . Scale bar : 20 μm . D Quantification of Msi1 staining intensity of ISPCs at indicated positions in basal crypts of young mTerc + / + mice ( n = 150 crypts from three mice ) . Each dot presents one cell . Red lines : mean values ± SEM . Unpaired two ‐ tailed Student ' s t ‐ test .

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C Representative immunofluorescence microscopy staining of Msi1 of small intestinal tissue of young mTerc+/+mice. Dashed line outlines a crypt. Arrowheads and numbers indicate ISPC positions in the crypts. Scale bar: 20 μm.D Quantification of Msi1 staining intensity of ISPCs at indicated positions in basal crypts of young mTerc+/+mice (n = 150 crypts from three mice). Each dot presents one cell. Red lines: mean values ± SEM. Unpaired two‐tailed Student's t‐test.


E Representative FACS plots showing gating of LGR5hi and LGR5lo populations within the LGR5 + ( GFP ‐ positive ) cell gating .

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E Representative FACS plots showing gating of LGR5hi and LGR5lo populations within the LGR5+ (GFP‐positive) cell gating.


F Representative FACS plots showing gating of LGR5hi ‐ high , LGR5hi ‐ low , LGR5lo ‐ high , and LGR5lo ‐ low populations within the LGR5hi gating .

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F Representative FACS plots showing gating of LGR5hi‐high, LGR5hi‐low, LGR5lo‐high, and LGR5lo‐low populations within the LGR5hi gating.


G - I mRNA expression of indicated genes relative to GAPDH in LGR5hi ‐ high , LGR5hi ‐ low , LGR5lo ‐ high , and LGR5lo ‐ low populations ( n = 3 mice ) . Mean values ± SEM are given . Unpaired two ‐ tailed Student ' s t ‐ test .

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G-I mRNA expression of indicated genes relative to GAPDH in LGR5hi‐high, LGR5hi‐low, LGR5lo‐high, and LGR5lo‐low populations (n = 3 mice). Mean values ± SEM are given. Unpaired two‐tailed Student's t‐test.


A mRNA expression of indicated components of the canonical Wnt / β ‐ catenin pathway in freshly isolated small intestinal crypts of 12 ‐ to 16 ‐ month ‐ old G3 mTerc − / − mice and age ‐ matched mTerc + / + mice ( n = 5 mice per group ) .

A mRNA expression of indicated components of the canonical Wnt/β‐catenin pathway in freshly isolated small intestinal crypts of 12‐ to 16‐month‐old G3 mTerc−/−mice and age‐matched mTerc+/+mice (n = 5 mice per group).


B Axin2 mRNA expression in LGR5 + cells of 12 ‐ to 16 ‐ month ‐ old G3 mTerc − / − mice and mTerc + / + mice ( n = 3 mice per group ) .

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B Axin2 mRNA expression in LGR5+ cells of 12‐ to 16‐month‐old G3 mTerc−/−mice and mTerc+/+mice (n = 3 mice per group).


C mRNA expression of indicated components of the canonical Wnt / β ‐ catenin pathway and of the p53 target gene p21 in cultured crypts of 2 ‐ month ‐ old G3 mTerc − / − mice and mTerc + / + mice ( n = 3 mice per group ) .

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C mRNA expression of indicated components of the canonical Wnt/β‐catenin pathway and of the p53 target gene p21 in cultured crypts of 2‐month‐old G3 mTerc−/−mice and mTerc+/+mice (n = 3 mice per group).


A - J Flow cytometry analysis of freshly isolated crypt cells from the small intestine of young ( 2 - 3 month ) and old ( 12 - 16 month ) LGR5 ‐ GFPki , mTerc + / + mice and LGR5 ‐ GFPki , G3 mTerc − / − mice ( n = 4 mice per group ) . ( A , B , D , E , H , I ) Representative FACS plots depicting the analysis of LGR5 + cells . Note the reduction in LGR5 + cells in G3 mTerc − / − mice with the remaining cells showing almost exclusively weak expression of GFP ( LGR5lo ) and that within the fraction of LGR5hi cells , the cells with high LGR5 ‐ reporter activity ( LGR5hi ‐ high ) are preferentially depleted in response to IR . ( C , F ) Quantification of ( C ) the number of LGR5 + cells and ( F ) the number of LGR5hi and LGR5lo cells . ( G ) Quantification of the percentage of LGR5hi cells and LGR5lo cells within the fraction of LGR5 + cells . ( J ) Quantification of the survival rate of LGR5hi ‐ high cells and LGR5hi ‐ low cells within the fraction of LGR5hi cells comparing old G3 mTerc − / − mice to mTerc + / + mice .

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A-J Flow cytometry analysis of freshly isolated crypt cells from the small intestine of young (2-3 month) and old (12-16 month) LGR5‐GFPki, mTerc+/+mice and LGR5‐GFPki, G3 mTerc−/−mice (n = 4 mice per group). (A, B, D, E, H, I) Representative FACS plots depicting the analysis of LGR5+ cells. Note the reduction in LGR5+ cells in G3 mTerc−/−mice with the remaining cells showing almost exclusively weak expression of GFP (LGR5lo) and that within the fraction of LGR5hi cells, the cells with high LGR5‐reporter activity (LGR5hi‐high) are preferentially depleted in response to IR. (C, F) Quantification of (C) the number of LGR5+ cells and (F) the number of LGR5hi and LGR5lo cells. (G) Quantification of the percentage of LGR5hi cells and LGR5lo cells within the fraction of LGR5+ cells. (J) Quantification of the survival rate of LGR5hi‐high cells and LGR5hi‐low cells within the fraction of LGR5hi cells comparing old G3 mTerc−/−mice to mTerc+/+mice.


K - O Immunofluorescence microscopy staining of GFP in basal crypts of 9 ‐ month ‐ old LGR5 ‐ GFPki , G3 mTerc − / − mice and age ‐ matched LGR5 ‐ GFPki , mTerc + / + mice ( n = 2 mice per group ) . ( K , L ) Representative pictures are given . Dashed lines outline the crypts . Arrowheads and numbers indicate ISPC positions in the crypts . Scale bar : 20 μm . ( M ) Quantification of absolute number of GFP + ISPCs at indicated positions at the crypt base per 50 crypts . ( N ) The histogram depicts the depletion rate ( % ) of LGR5 + cells in LGR5 ‐ GFPki , G3 mTerc − / − mice compared to age ‐ matched LGR5 ‐ GFPki , mTerc + / + mice at indicated positions in the basal crypts . ( O ) Quantification of GFP staining intensity of ISPCs at indicated positions in positively stained basal crypts . Note that the staining intensity of position 4 cells is equal in mTerc + / + mice and G3 mTerc − / − mice and is significantly lower than position 1 and 2 cells of mTerc + / + mice .

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K-O Immunofluorescence microscopy staining of GFP in basal crypts of 9‐month‐old LGR5‐GFPki, G3 mTerc−/−mice and age‐matched LGR5‐GFPki, mTerc+/+mice (n = 2 mice per group). (K, L) Representative pictures are given. Dashed lines outline the crypts. Arrowheads and numbers indicate ISPC positions in the crypts. Scale bar: 20 μm. (M) Quantification of absolute number of GFP+ISPCs at indicated positions at the crypt base per 50 crypts. (N) The histogram depicts the depletion rate (%) of LGR5+ cells in LGR5‐GFPki, G3 mTerc−/−mice compared to age‐matched LGR5‐GFPki, mTerc+/+mice at indicated positions in the basal crypts. (O) Quantification of GFP staining intensity of ISPCs at indicated positions in positively stained basal crypts. Note that the staining intensity of position 4 cells is equal in mTerc+/+mice and G3 mTerc−/−mice and is significantly lower than position 1 and 2 cells of mTerc+/+mice.


A - I Staining of small intestinal tissue sections of 12 ‐ to 16 ‐ month ‐ old G3 mTerc − / − mice and age ‐ matched mTerc + / + mice ( n = 4 - 5 mice per group ) . Representative pictures of ( A , B ) H & amp ; amp ; E staining , ( C , F , I ) Quantification of the absolute numbers of ISPCs at the indicated positions in the crypt base per 50 crypts as determined by counting of ( C ) spindle ‐ shaped cells in between and adjacent to the Paneth cells . Mean values ± SEM are given . Unpaired two ‐ tailed Student ' s t ‐ test .

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A-I Staining of small intestinal tissue sections of 12‐ to 16‐month‐old G3 mTerc−/− mice and age‐matched mTerc+/+ mice (n = 4-5 mice per group). Representative pictures of (A, B) H&amp;E staining, (C, F, I) Quantification of the absolute numbers of ISPCs at the indicated positions in the crypt base per 50 crypts as determined by counting of (C) spindle‐shaped cells in between and adjacent to the Paneth cells. Mean values ± SEM are given. Unpaired two‐tailed Student's t‐test.


A - I Staining of small intestinal tissue sections of 12 ‐ to 16 ‐ month ‐ old G3 mTerc − / − mice and age ‐ matched mTerc + / + mice ( n = 4 - 5 mice per group ) . Representative pictures of ( D , E ) PCNA staining . Arrowheads and numbers indicate the position of ISPCs in the basal crypts . Scale bars : 20 μm . ( C , F , I ) Quantification of the absolute numbers of ISPCs at the indicated positions in the crypt base per 50 crypts as determined by counting of cells staining positive for ( F ) PCNA . Mean values ± SEM are given . Unpaired two ‐ tailed Student ' s t ‐ test .

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A-I Staining of small intestinal tissue sections of 12‐ to 16‐month‐old G3 mTerc−/−mice and age‐matched mTerc+/+mice (n = 4-5 mice per group). Representative pictures of (D, E) PCNA staining. Arrowheads and numbers indicate the position of ISPCs in the basal crypts. Scale bars: 20 μm. (C, F, I) Quantification of the absolute numbers of ISPCs at the indicated positions in the crypt base per 50 crypts as determined by counting of cells staining positive for (F) PCNA . Mean values ± SEM are given. Unpaired two‐tailed Student's t‐test.


A - I Staining of small intestinal tissue sections of 12 ‐ to 16 ‐ month ‐ old G3 mTerc − / − mice and age ‐ matched mTerc + / + mice ( n = 4 - 5 mice per group ) . Representative pictures of ( ( G , H ) Msi1 staining are given . Arrowheads and numbers indicate the position of ISPCs in the basal crypts . Scale bars : 20 μm . ( C , F , I ) Quantification of the absolute numbers of ISPCs at the indicated positions in the crypt base per 50 crypts as determined by counting of ( I ) Msi1 . Mean values ± SEM are given . Unpaired two ‐ tailed Student ' s t ‐ test .

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A-I Staining of small intestinal tissue sections of 12‐ to 16‐month‐old G3 mTerc−/− mice and age‐matched mTerc+/+ mice (n = 4-5 mice per group). Representative pictures of ((G, H) Msi1 staining are given. Arrowheads and numbers indicate the position of ISPCs in the basal crypts. Scale bars: 20 μm. (C, F, I) Quantification of the absolute numbers of ISPCs at the indicated positions in the crypt base per 50 crypts as determined by counting of (I) Msi1. Mean values ± SEM are given. Unpaired two‐tailed Student's t‐test.