( a - c ) Analysis on day 2 after infection with PR8 virus of wild - type mice ( WT ) or Ripk2 − / − mice injected intraperitoneally with control rabbit serum and Ripk2 − / − mice injected intraperitoneally with anti - IL - 18 neutralizing antiserum ( αIL - 18Ab ) 3 h before infection , for IL - 18 , IFN - γ and CCL2 amounts in whole lung homogenates ( a ; t . p . , total protein ) , and for neutrophil numbers by immunohistochemistry ( b ) and by flow cytometry ( c ) . Original magnification , × 20 .

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(a-c) Analysis on day 2 after infection with PR8 virus of wild-type mice (WT) or Ripk2−/− mice injected intraperitoneally with control rabbit serum and Ripk2−/− mice injected intraperitoneally with anti-IL-18 neutralizing antiserum (αIL-18Ab) 3 h before infection, for IL-18, IFN-γ and CCL2 amounts in whole lung homogenates (a; t.p., total protein), and for neutrophil numbers by immunohistochemistry (b) and by flow cytometry (c). Original magnification, ×20.


( d ) Analysis of survival of indicated mice .

(d) Analysis of survival of indicated mice.


( e ) Neutrophil numbers in WT , Ripk2 − / − and Ripk2 − / − Il18 − / − mice on day 2 after PR8 infection .

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(e) Neutrophil numbers in WT, Ripk2−/− and Ripk2−/−Il18−/− mice on day 2 after PR8 infection.


( f ) Survival of Ripk2 − / − Il18 − / − , Ripk2 − / − and WT mice . * P 0 . 05 , * * P 0 . 01 , * * * P 0 . 001 ( one - way ANOVA with Tukey ' s post - hoc test ( a , c , e ) and log - rank test ( d , f ) ) . Data are representative of two independent experiments ( mean ± s . e . m . ; n = 6 - 8 mice per experiment ) .

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(f) Survival of Ripk2−/−Il18−/−, Ripk2−/− and WT mice. *P 0.05, **P 0.01, ***P 0.001 (one-way ANOVA with Tukey's post-hoc test (a,c,e) and log-rank test (d,f)). Data are representative of two independent experiments (mean ± s.e.m.; n = 6-8 mice per experiment).


( a , b ) RIPK2 bone marrow chimeras were analyzed at day 2 after PR8 infection for contribution to elevated IL - 18 production by using IL - 18 immunohistochemistry ( a ) and IL - 18 ELISA ( b ) of whole lung homogenates . WT , wild type ; t . p . , total protein . In a , blue arrows denote IL - 18 + macrophages , and red arrows denote IL - 18 + bronchial epithelium . Original magnification , × 200 .

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(a,b) RIPK2 bone marrow chimeras were analyzed at day 2 after PR8 infection for contribution to elevated IL-18 production by using IL-18 immunohistochemistry (a) and IL-18 ELISA (b) of whole lung homogenates. WT, wild type; t.p., total protein. In a, blue arrows denote IL-18+ macrophages, and red arrows denote IL-18+ bronchial epithelium. Original magnification, ×200.


( c ) Flow cytometry analysis of NK cell counts in Ripk2 − / − and wild - type mice .

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(c) Flow cytometry analysis of NK cell counts in Ripk2−/− and wild-type mice.


( d ) Analysis of wild - type and Ripk2 − / − NK cells for IFN - γ production on a per cell basis .

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(d) Analysis of wild-type and Ripk2−/− NK cells for IFN-γ production on a per cell basis.


( e ) Accumulation of T and B cells in lungs .

(e) Accumulation of T and B cells in lungs.


( f ) Analysis of CD8 + T cells for IFN - γ and CD44 expression in wild - type and Ripk2 − / − mice . * P 0 . 05 , * * P 0 . 01 ( one - way ANOVA with Tukey ' s post - hoc test ( b ) and unpaired two - sided Student ' s t - test ( d , f ) ) . Data are representative of two experiments ( a , b , f ) or three experiments ( c - e ) ( mean ± s . e . m . ; n = 6 - 8 mice per experiment ) .

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(f) Analysis of CD8+ T cells for IFN-γ and CD44 expression in wild-type and Ripk2−/− mice. *P 0.05, **P 0.01 (one-way ANOVA with Tukey's post-hoc test (b) and unpaired two-sided Student's t-test (d,f)). Data are representative of two experiments (a,b,f) or three experiments (c-e) (mean ± s.e.m.; n = 6-8 mice per experiment).


( a ) Immunoblots showing active caspase - 1 ( casp - 1 ) p20 compared to GAPDH loading controls in wild - type ( WT ) , Ripk2 − / − and Nod2 − / − BMDCs after PR8 infection .

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(a) Immunoblots showing active caspase-1 (casp-1) p20 compared to GAPDH loading controls in wild-type (WT), Ripk2−/− and Nod2−/− BMDCs after PR8 infection.


( b ) IL - 18 release in PR8 - infected WT , Ripk2 − / − and Nod2 − / − BMDCs .

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(b) IL-18 release in PR8-infected WT, Ripk2−/− and Nod2−/− BMDCs.


( c , d ) Immunoblot analysis showing active casp - 1 p20 ( c ) and IL - 18 production ( d ) in BMDCs infected for 4 h with Listeria monocytogenes at multiplicity of infection of 10 .

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(c,d) Immunoblot analysis showing active casp-1 p20 (c) and IL-18 production (d) in BMDCs infected for 4 h with Listeria monocytogenes at multiplicity of infection of 10.


( e , f ) IL - 18 and casp - 1 activation in Ripk2 − / − BMDCs treated with the NLRP3 inflammasome - specific inhibitor glyburide ( glyb . ) during PR8 infection .

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(e,f) IL-18 and casp-1 activation in Ripk2−/− BMDCs treated with the NLRP3 inflammasome-specific inhibitor glyburide (glyb.) during PR8 infection.


( g ) Immunoblot analysis of casp - 1 p20 during Listeria infection of WT and Aim2 − / − BMDCs . * P 0 . 01 , * * P 0 . 001 ( one - way ANOVA with Tukey ' s post - hoc test ) . Data are representative of six experiments ( a , b ) or three experiments ( c - g ) ( mean ± s . d . ; n = 2 - 3 replicates per experiment ) .

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(g) Immunoblot analysis of casp-1 p20 during Listeria infection of WT and Aim2−/− BMDCs. *P 0.01, **P 0.001 (one-way ANOVA with Tukey's post-hoc test). Data are representative of six experiments (a,b) or three experiments (c-g) (mean ± s.d.; n = 2-3 replicates per experiment).


( a ) Immunoblot analysis of wild - type ( WT ) , Ripk2 − / − and Nod2 − / − BMDCs for LC3 - II ( marker for autophagy ) after 18 h of PR8 infection .

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(a) Immunoblot analysis of wild-type (WT), Ripk2−/− and Nod2−/− BMDCs for LC3-II (marker for autophagy) after 18 h of PR8 infection.


( b , c ) Numbers of LC3 - II + puncta ( b ) , counted in at least 100 cells from five random fields from confocal images ( c ) of BMDCs after PR8 infection and / or chloroquine treatment . Original magnification , × 400 .

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(b,c) Numbers of LC3-II+ puncta (b), counted in at least 100 cells from five random fields from confocal images (c) of BMDCs after PR8 infection and/or chloroquine treatment. Original magnification, ×400.


( d , e ) Immunoblots of LC3 - II and caspase - 1 ( casp - 1 ; e ) and IL - 18 production ( d ) in wild - type and Ripk2 − / − BMDCs after treatment with rapamycin ( Rap ) .

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(d,e) Immunoblots of LC3-II and caspase-1 (casp-1; e) and IL-18 production (d) in wild-type and Ripk2−/− BMDCs after treatment with rapamycin (Rap).


( f ) Immunoblot analysis for LC3 - II in lung lysates of PR8 - infected wild - type and Ripk2 − / − mice ( 1 - 5 indicate individual mice ) .

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(f) Immunoblot analysis for LC3-II in lung lysates of PR8-infected wild-type and Ripk2−/− mice (1-5 indicate individual mice).


( g , h ) Lungneutrophil infiltration ( g ) in IAV - infected Ripk2 − / − mice treated with rapamycin , Ripk2 − / − mice and WT mice ( t . p . , total protein ) . * P 0 . 05 , * * P 0 . 01 , * * * P 0 . 001 ( Mann - Whitney test ( b ) ; one - way ANOVA with Tukey ' s post - hoc test ( d , g , h ) ) .

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(g,h) Lungneutrophil infiltration (g) in IAV-infected Ripk2−/− mice treated with rapamycin, Ripk2−/− mice and WT mice (t.p., total protein). *P 0.05, **P 0.01, ***P 0.001 (Mann-Whitney test (b); one-way ANOVA with Tukey's post-hoc test (d,g,h)).


( g , h ) IL - 18 and IFN - γ amounts ( h ) in IAV - infected Ripk2 − / − mice treated with rapamycin , Ripk2 − / − mice and WT mice ( t . p . , total protein ) . * P 0 . 05 , * * P 0 . 01 , * * * P 0 . 001 ( Mann - Whitney test ( b ) ; one - way ANOVA with Tukey ' s post - hoc test ( d , g , h ) ) .

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(g,h) IL-18 and IFN-γ amounts (h) in IAV-infected Ripk2−/− mice treated with rapamycin, Ripk2−/− mice and WT mice (t.p., total protein). *P 0.05, **P 0.01, ***P 0.001 (Mann-Whitney test (b); one-way ANOVA with Tukey's post-hoc test (d,g,h)).


( a - c ) Flow cytometry analysis of wild - type ( WT ) and Ripk2 − / − BMDCs stained with the mitochondrial superoxide - specific stain MitoSOX during IAV or Listeria infection ( a , b ) and with the general mitochondrial stain MitoTracker Green during IAV infection ( c ) .

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(a-c) Flow cytometry analysis of wild-type (WT) and Ripk2−/− BMDCs stained with the mitochondrial superoxide-specific stain MitoSOX during IAV or Listeria infection (a,b) and with the general mitochondrial stain MitoTracker Green during IAV infection (c).


( d , e ) Analysis of IL - 18 production and immunoblotting to analyze caspase - 1 ( casp - 1 ) p20 activation in IAV - infected BMDCs that had been pretreated with the ROS inhibitor NAC .

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(d,e) Analysis of IL-18 production and immunoblotting to analyze caspase-1 (casp-1) p20 activation in IAV-infected BMDCs that had been pretreated with the ROS inhibitor NAC.


( f ) Immunoblot of casp - 1 p20 in cell lysates of IAV - infected BMDCs pretreated with the mitochondrial superoxide inhibitor mitoTEMPO .

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(f) Immunoblot of casp-1 p20 in cell lysates of IAV-infected BMDCs pretreated with the mitochondrial superoxide inhibitor mitoTEMPO.


( g ) Confocal microscopy analysis of BMDCs stained for mitochondria ( red ) and LC3 ( green ) for colocalization as an indicator of mitophagy . Original magnification , × 400 ; inset ( red boxed region ) was digitally enlarged × 10 . * P 0 . 01 ( one - way ANOVA with Tukey ' s post - hoc test ( d ) and unpaired two - sided Student ' s t - test ( g ) ) . Data are representative of nine experiments ( a ) , three experiments ( b , d - g ) or six experiments ( c ) ( mean ± s . d . ; n = 2 - 3 replicates per experiment ) .

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(g) Confocal microscopy analysis of BMDCs stained for mitochondria (red) and LC3 (green) for colocalization as an indicator of mitophagy. Original magnification, ×400; inset (red boxed region) was digitally enlarged ×10. *P 0.01 (one-way ANOVA with Tukey's post-hoc test (d) and unpaired two-sided Student's t-test (g)). Data are representative of nine experiments (a), three experiments (b,d-g) or six experiments (c) (mean ± s.d.; n = 2-3 replicates per experiment).


( a , b ) IL - 18 amounts ( a ) , and caspase - 1 ( casp - 1 ) p20 and LC3 - II immunoblots ( b ) in PR8 - infected wild - type ( WT ) BMDCs treated with the p38 and RIPK2 kinase inhibitor SB203580 .

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(a,b) IL-18 amounts (a), and caspase-1 (casp-1) p20 and LC3-II immunoblots (b) in PR8-infected wild-type (WT) BMDCs treated with the p38 and RIPK2 kinase inhibitor SB203580.


( c ) Immunoblots for phosphorylation of ULK1 at Ser555 in WT and Ripk2 − / − BMDCs after PR8 infection .

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(c) Immunoblots for phosphorylation of ULK1 at Ser555 in WT and Ripk2−/− BMDCs after PR8 infection.


( d ) Immunoblots for casp - 1 p20 in untreated or NAC - treated WT and Ulk1 − / − BMDCs after PR8 infection .

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(d) Immunoblots for casp-1 p20 in untreated or NAC-treated WT and Ulk1−/− BMDCs after PR8 infection.


( e ) Flow cytometry analysis of MitoSOX staining in PR8 - infected WT and Ulk1 − / − BMDCs .

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(e) Flow cytometry analysis of MitoSOX staining in PR8-infected WT and Ulk1−/− BMDCs.


( f ) MitoTracker Green staining of PR8 - infected WT and Ulk1 − / − BMDCs .

(f) MitoTracker Green staining of PR8-infected WT and Ulk1−/− BMDCs.


Effects of basic and bulky hydrophobic amino acids on LT - induced pyroptosis and caspase - 1 activation in RAWRA cells . Cells were pre - treated with 10 mM indicated amino acids prior to LT stimulation . Cell viability was measured by using the ATP assay and data shown are mean values ± SD from three replicates ( A ) . Mock , DMEM medium .

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Effects of basic and bulky hydrophobic amino acids on LT-induced pyroptosis and caspase-1 activation in RAWRA cells. Cells were pre-treated with 10 mM indicated amino acids prior to LT stimulation. Cell viability was measured by using the ATP assay and data shown are mean values ± SD from three replicates (A). Mock, DMEM medium.


Cell supernatants were subjected to anti - caspase - 1 immunoblotting and the lysates were blotted with the anti - tubulin antibody ( B ) . p10 , mature caspase - 1 ; p45 , caspase - 1 precursor .

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Cell supernatants were subjected to anti-caspase-1 immunoblotting and the lysates were blotted with the anti-tubulin antibody (B). p10, mature caspase-1; p45, caspase-1 precursor.


C , D Effects of basic and bulky hydrophobic amino acids on LT - induced RFP - ASC specks formation in RAWRA cells . Fluorescence images were taken on a confocal microscopy ( C ) . The percentages of cells showing RFP - ASC specks ( mean values ± SD from three replicates ) are in ( D ) .

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C, D Effects of basic and bulky hydrophobic amino acids on LT-induced RFP-ASC specks formation in RAWRA cells. Fluorescence images were taken on a confocal microscopy (C). The percentages of cells showing RFP-ASC specks (mean values ± SD from three replicates) are in (D).


E Effects of basic and bulky hydrophobic amino acids on LT - induced caspase - 1 activation in Tlr4 - / - iBMDMs . Cells were treated as that in ( A ) . Cell supernatants were subjected to anti - caspase - 1 immunoblotting and the lysates were blotted with the anti - tubulin or anti - MEK3 antibody .

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E Effects of basic and bulky hydrophobic amino acids on LT-induced caspase-1 activation in Tlr4-/- iBMDMs. Cells were treated as that in (A). Cell supernatants were subjected to anti-caspase-1 immunoblotting and the lysates were blotted with the anti-tubulin or anti-MEK3 antibody.


A RFP - ASC specks formation assay of the effect of Ubr2 knockdown on LT - induced inflammasome activation . RAWRA cells were transfected with a control siRNA or Ubr2 - targeting siRNA mixtures for 60 h followed by LT treatment . The numbers in the merged panel are the percentages of the cells showing RFP - ASC specks .

A RFP-ASC specks formation assay of the effect of Ubr2 knockdown on LT-induced inflammasome activation. RAWRA cells were transfected with a control siRNA or Ubr2-targeting siRNA mixtures for 60 h followed by LT treatment. The numbers in the merged panel are the percentages of the cells showing RFP-ASC specks.


B Effect of Ubr2 stable knockdown on caspase - 1 activation in RAWRA cells . Cells stably expressing a control or Ubr2 - targeting shRNA were incubated with WT LT ( + ) or its E687C mutant ( - ) for 3 h .

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B Effect of Ubr2 stable knockdown on caspase-1 activation in RAWRA cells. Cells stably expressing a control or Ubr2-targeting shRNA were incubated with WT LT (+) or its E687C mutant (-) for 3 h.


C Effects of Ubr2 knockdown ( siRNA # 05 ) on LT - induced NLRP1B inflammasome activation reconstituted in 293T cells . Cells were treated with WT LT ( + ) or its E687C mutant ( - ) .

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C Effects of Ubr2 knockdown (siRNA #05) on LT-induced NLRP1B inflammasome activation reconstituted in 293T cells. Cells were treated with WT LT (+) or its E687C mutant (-).


D , E Effect of Ubr2 knockout on NLRP1B or NAIP2 - NLRC4 inflammasome activation . WT and Ubr2 - / - iBMDMs were treated with LT or BsaK for 3 h . Cell viability was measured by using the ATP assay and data shown are mean values ± SD from three replicates ( D ) .

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D, E Effect of Ubr2 knockout on NLRP1B or NAIP2-NLRC4 inflammasome activation. WT and Ubr2-/- iBMDMs were treated with LT or BsaK for 3 h. Cell viability was measured by using the ATP assay and data shown are mean values ± SD from three replicates (D).


A Immunoblotting of caspase - 1 activation in Ubrs - knockdown BMDMs derived from the 129 / Sv mice . Cells were transfected with the indicated siRNA for 60 h , and then treated with WT LT ( + ) or its E687C mutant ( - ) for 3 h .

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A Immunoblotting of caspase-1 activation in Ubrs-knockdown BMDMs derived from the 129/Sv mice. Cells were transfected with the indicated siRNA for 60 h, and then treated with WT LT (+) or its E687C mutant (-) for 3 h.


Immunoblotting of caspase - 1 activation in E2s - knockdown BMDMs derived from the 129 / Sv mice .

Immunoblotting of caspase-1 activation in E2s-knockdown BMDMs derived from the 129/Sv mice.


Additional Ube2o siRNAs were used to confirm the inhibitory effect of Ube2o knockdown on LT - induced caspase - 1 activation ( C ) . Ube2o knockdown efficiency was measured by qPCR and are shown as mean values ± SD from three replicates .

Additional Ube2o siRNAs were used to confirm the inhibitory effect of Ube2o knockdown on LT-induced caspase-1 activation (C). Ube2o knockdown efficiency was measured by qPCR and are shown as mean values ± SD from three replicates.


RFP - ASC specks formation assay of the effect of Ube2o knockdown ( siRNA Ube2o - 01 ) on LT - induced inflammasome activation in RAWRA cells . The percentages of cells showing RFP - ASC specks ( mean values ± SD from three replicates ) are in ( D ) .

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RFP-ASC specks formation assay of the effect of Ube2o knockdown (siRNA Ube2o-01) on LT-induced inflammasome activation in RAWRA cells. The percentages of cells showing RFP-ASC specks (mean values ± SD from three replicates) are in (D).


F Co - immunoprecipitation interaction of UBE2O and UBR2 . Myc - UBE2O and Flag - UBR2 were co - expressed in 293T cells and cell lysates were subjected to anti - Flag immunoprecipitation followed by immunoblotting analyses as shown .

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F Co-immunoprecipitation interaction of UBE2O and UBR2. Myc-UBE2O and Flag-UBR2 were co-expressed in 293T cells and cell lysates were subjected to anti-Flag immunoprecipitation followed by immunoblotting analyses as shown.


B Effect of LT treatment on NLRP1B protein level . 293T cells were transfected with Flag - NLRP1B expression plasmid for 24 h , and cells were then treated with WT LT ( + ) or its E687C mutant ( - ) for 4 h . Shown is the anti - Flag immunoblot of the total cell lysates .

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B Effect of LT treatment on NLRP1B protein level. 293T cells were transfected with Flag-NLRP1B expression plasmid for 24 h, and cells were then treated with WT LT (+) or its E687C mutant (-) for 4 h. Shown is the anti-Flag immunoblot of the total cell lysates.


C RFP - ASC specks formation in 293T cells expressing an indicated NLRP1B variant . Cells were treated with LT for 3 h . Scale bar , 20 μm . 1 - 983 and 984 - C ( residues 984 to the C terminus ) are the FIIND - mediated autocleaved fragments of NLRP1B .

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C RFP-ASC specks formation in 293T cells expressing an indicated NLRP1B variant. Cells were treated with LT for 3 h. Scale bar, 20 μm. 1-983 and 984-C (residues 984 to the C terminus) are the FIIND-mediated autocleaved fragments of NLRP1B.


Effect of MG132 on LT - induced NLRP1B degradation . 293T cells expressing NLRP1B ( 1 - 983 ) - HA were treated with LT and MG132 for 8 h . Shown are the anti - HA and anti - tubulin immunoblots of the total cell lysates .

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Effect of MG132 on LT-induced NLRP1B degradation. 293T cells expressing NLRP1B (1-983)-HA were treated with LT and MG132 for 8 h. Shown are the anti-HA and anti-tubulin immunoblots of the total cell lysates.


E Effect of MG132 and Leu on LT - induced NLRP1B degradation . 293T cells expressing NLRP1B ( 1 - 983 ) - HA were treated with LT and MG132 for 8 h . 10 mM Leu was added to cells 30 min prior to LT treatment . Shown are the anti - HA and anti - tubulin immunoblots of the total cell lysates .

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E Effect of MG132 and Leu on LT-induced NLRP1B degradation. 293T cells expressing NLRP1B (1-983)-HA were treated with LT and MG132 for 8 h. 10 mM Leu was added to cells 30 min prior to LT treatment. Shown are the anti-HA and anti-tubulin immunoblots of the total cell lysates.


F LT - induced ubiquitination of NLRP1B . 293T cells expressing NLRP1B ( 1 - 983 ) - HA were treated with LT and / or MG132 for 6 h . Cells were then harvested for anti - HA immunoprecipitation and subjected to immunoblotting analyses using indicated antibodies .

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F LT-induced ubiquitination of NLRP1B. 293T cells expressing NLRP1B (1-983)-HA were treated with LT and/or MG132 for 6 h. Cells were then harvested for anti-HA immunoprecipitation and subjected to immunoblotting analyses using indicated antibodies.


B - F Jejunal spheroids were cultured in differentiation medium containing 0 - 1 , 000 nM dmPGE2 or Iloprost . ( B ) Representative bright - field images of spheroids treated with dmPGE2 . Scale bars , 200 µm .

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 B-CONTROLLED_VAR O O O O O O O

B-F Jejunal spheroids were cultured in differentiation medium containing 0-1,000 nM dmPGE2 or Iloprost. (B) Representative bright-field images of spheroids treated with dmPGE2. Scale bars, 200 µm.


B - F Jejunal spheroids were cultured in differentiation medium containing 0 - 1 , 000 nM dmPGE2 or Iloprost . ( C ) Quantification of average spheroid area ± s . e . m . relative to spheroids treated with 0 nM dmPGE2 ( average area was 3761 µm2 for 0 nM group ; n = 4 images from two independent experiments ) .

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

B-F Jejunal spheroids were cultured in differentiation medium containing 0-1,000 nM dmPGE2 or Iloprost. (C) Quantification of average spheroid area ± s.e.m. relative to spheroids treated with 0 nM dmPGE2 (average area was 3761 µm2 for 0 nM group; n = 4 images from two independent experiments).


B - F Jejunal spheroids were cultured in differentiation medium containing 0 - 1 , 000 nM dmPGE2 or Iloprost . ( D ) Representative histological sections of spheroids treated with 1 µM dmPGE2 or an equivalent volume of DMSO and stained for hematoxylin . Scale bars , 100 µm .

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

B-F Jejunal spheroids were cultured in differentiation medium containing 0-1,000 nM dmPGE2 or Iloprost. (D) Representative histological sections of spheroids treated with 1 µM dmPGE2 or an equivalent volume of DMSO and stained for hematoxylin. Scale bars, 100 µm.


B - F Jejunal spheroids were cultured in differentiation medium containing 0 - 1 , 000 nM dmPGE2 or Iloprost . ( E ) Quantification of the average expression ± s . e . m . of Cldn4mRNA relative to the 0 nM treatment group ( n = 3 independent experiments ) .

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

B-F Jejunal spheroids were cultured in differentiation medium containing 0-1,000 nM dmPGE2 or Iloprost. (E) Quantification of the average expression ± s.e.m. of Cldn4mRNA relative to the 0 nM treatment group (n = 3 independent experiments).


B - F Jejunal spheroids were cultured in differentiation medium containing 0 - 1 , 000 nM dmPGE2 or Iloprost . ( F ) Quantification of average spheroid area ± s . e . m . relative to spheroids treated with 0 nM Iloprost ( n = 4 images from two independent experiments ) .

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

B-F Jejunal spheroids were cultured in differentiation medium containing 0-1,000 nM dmPGE2 or Iloprost. (F) Quantification of average spheroid area ± s.e.m. relative to spheroids treated with 0 nM Iloprost (n = 4 images from two independent experiments).


A Quantification of the average expression ± s . e . m . of Ptger1 , Ptger2 , Ptger3 , Ptger4 , and Ptgir mRNAs in whole - thickness lung , ileum or colon tissues or in jejunal spheroids cultured in stem cell ( Stem ) , WAE ( dmPGE2 ) or enterocyte ( EP4i ) medium ( n = 3 independent experiments ) . Data are presented as fold change compared to lung tissue . n . d . , not detected .

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

A Quantification of the average expression ± s.e.m. of Ptger1, Ptger2, Ptger3, Ptger4, and Ptgir mRNAs in whole-thickness lung, ileum or colon tissues or in jejunal spheroids cultured in stem cell (Stem), WAE (dmPGE2) or enterocyte (EP4i) medium (n = 3 independent experiments). Data are presented as fold change compared to lung tissue. n.d., not detected.


B - D Jejunal spheroids were cultured in differentiation medium with DMSO only or with 1 µM dmPGE2 and pharmacological inhibitors of EP1 ( EP1i , SC 51322 ) , EP2 ( EP2i , PF 04418948 ) , EP3 ( EP3i , L - 798 , 106 ) or EP4 ( EP4i , L - 161 , 982 ) at a concentration of 10 µM or an equivalent volume of DMSO vehicle . * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 compared to the DMSO only group by one - way ANOVA and Dunnett ' s post - test . ( B ) Representative bright field images . Scale bars , 200 µm .

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 B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-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 O O O O O O O O O O O O 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-D Jejunal spheroids were cultured in differentiation medium with DMSO only or with 1 µM dmPGE2 and pharmacological inhibitors of EP1 (EP1i, SC 51322), EP2 (EP2i, PF 04418948), EP3 (EP3i, L-798,106) or EP4 (EP4i, L-161,982) at a concentration of 10 µM or an equivalent volume of DMSO vehicle. **p<0.01, ***p<0.001, ****p<0.0001 compared to the DMSO only group by one-way ANOVA and Dunnett's post-test. (B) Representative bright field images. Scale bars, 200 µm.


B - D Jejunal spheroids were cultured in differentiation medium with DMSO only or with 1 µM dmPGE2 and pharmacological inhibitors of EP1 ( EP1i , SC 51322 ) , EP2 ( EP2i , PF 04418948 ) , EP3 ( EP3i , L - 798 , 106 ) or EP4 ( EP4i , L - 161 , 982 ) at a concentration of 10 µM or an equivalent volume of DMSO vehicle . * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 compared to the DMSO only group by one - way ANOVA and Dunnett ' s post - test . ( C ) Quantification of average spheroid area ± s . e . m . relative to spheroids treated with DMSO alone ( n = 4 images from two independent experiments ) .

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 B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-D Jejunal spheroids were cultured in differentiation medium with DMSO only or with 1 µM dmPGE2 and pharmacological inhibitors of EP1 (EP1i, SC 51322), EP2 (EP2i, PF 04418948), EP3 (EP3i, L-798,106) or EP4 (EP4i, L-161,982) at a concentration of 10 µM or an equivalent volume of DMSO vehicle. **p<0.01, ***p<0.001, ****p<0.0001 compared to the DMSO only group by one-way ANOVA and Dunnett's post-test. (C) Quantification of average spheroid area ± s.e.m. relative to spheroids treated with DMSO alone (n = 4 images from two independent experiments).


B - D Jejunal spheroids were cultured in differentiation medium with DMSO only or with 1 µM dmPGE2 and pharmacological inhibitors of EP1 ( EP1i , SC 51322 ) , EP2 ( EP2i , PF 04418948 ) , EP3 ( EP3i , L - 798 , 106 ) or EP4 ( EP4i , L - 161 , 982 ) at a concentration of 10 µM or an equivalent volume of DMSO vehicle . * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 compared to the DMSO only group by one - way ANOVA and Dunnett ' s post - test . ( D ) Quantification of the average expression ± s . e . m . of Cldn4 mRNA relative to stem cellspheroids ( n = 3 independent experiments ) .

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 B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-D Jejunal spheroids were cultured in differentiation medium with DMSO only or with 1 µM dmPGE2 and pharmacological inhibitors of EP1 (EP1i, SC 51322), EP2 (EP2i, PF 04418948), EP3 (EP3i, L-798,106) or EP4 (EP4i, L-161,982) at a concentration of 10 µM or an equivalent volume of DMSO vehicle. **p<0.01, ***p<0.001, ****p<0.0001 compared to the DMSO only group by one-way ANOVA and Dunnett's post-test. (D) Quantification of the average expression ± s.e.m. of Cldn4 mRNA relative to stem cellspheroids (n = 3 independent experiments).


E , F Tat - Cre mediated recombination of Ptger4flox / flox ( fl / fl ) jejunal spheroids to generate Ptger4flox / Δ ( fl / Δ ) and Ptger4Δ / Δ ( Δ / Δ ) spheroid lines . ( E ) Schematic and representative PCR genotyping results . M , marker lane . bp , base pairs . ( F ) Quantification of the average expression ± s . e . m . of Ptger4 mRNA in spheroids cultured in stem cell media relative to fl / fl genotype ( n = 3 independent experiments ) . * * p < 0 . 01 , * * * * p < 0 . 0001 by 2 - way ANOVA and Dunnett ' s multiple comparisons post - test with the EP4i - treated fl / fl group set as the 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 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, F Tat-Cre mediated recombination of Ptger4flox/flox (fl/fl) jejunal spheroids to generate Ptger4flox/Δ (fl/Δ) and Ptger4Δ/Δ (Δ/Δ) spheroid lines. (E) Schematic and representative PCR genotyping results. M, marker lane. bp, base pairs. (F) Quantification of the average expression ± s.e.m. of Ptger4 mRNA in spheroids cultured in stem cell media relative to fl/fl genotype (n = 3 independent experiments). **p<0.01, ****p<0.0001 by 2-way ANOVA and Dunnett's multiple comparisons post-test with the EP4i-treated fl/fl group set as the control.


G - I Ptger4flox / flox , Ptger4flox / Δ and Ptger4Δ / Δ spheroids were cultured in differentiation medium with 1 µM dmPGE2 or 10 µM EP4i . * p < 0 . 05 , * * p < 0 . 01 , * * * * p < 0 . 0001 by 1 - way ANOVA and Tukey ' s post - test . ( G ) Representative bright - field images . Scale bars , 200 µm . ( H ) Quantification of average spheroid area ± s . e . m . ( n = 4 images per group from two independent experiments ) relative to EP4i - treated Ptger4flox / flox spheroids .

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

G-I Ptger4flox/flox, Ptger4flox/Δ and Ptger4Δ/Δ spheroids were cultured in differentiation medium with 1 µM dmPGE2 or 10 µM EP4i. *p<0.05, **p<0.01, ****p<0.0001 by 1-way ANOVA and Tukey's post-test. (G) Representative bright-field images. Scale bars, 200 µm. (H) Quantification of average spheroid area ± s.e.m. (n = 4 images per group from two independent experiments) relative to EP4i-treated Ptger4flox/flox spheroids.


G - I Ptger4flox / flox , Ptger4flox / Δ and Ptger4Δ / Δ spheroids were cultured in differentiation medium with 1 µM dmPGE2 or 10 µM EP4i . * p < 0 . 05 , * * p < 0 . 01 , * * * * p < 0 . 0001 by 1 - way ANOVA and Tukey ' s post - test . ( I ) average expression ± s . e . m . of Cldn4 mRNA ( n = 3 independent experiments ) relative to EP4i - treated Ptger4flox / flox spheroids .

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

G-I Ptger4flox/flox, Ptger4flox/Δ and Ptger4Δ/Δ spheroids were cultured in differentiation medium with 1 µM dmPGE2 or 10 µM EP4i. *p<0.05, **p<0.01, ****p<0.0001 by 1-way ANOVA and Tukey's post-test. (I) average expression ± s.e.m. of Cldn4 mRNA (n = 3 independent experiments) relative to EP4i-treated Ptger4flox/flox spheroids.


J - L Humanileal spheroids were cultured in differentiation medium with 1 µM dmPGE2 or 10 µM EP4i . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 by 1 - way ANOVA and Tukey ' s post - test . ( J ) Representative bright - field images . Scale bars , 200 µm . ( K ) Quantification of average spheroid area ± s . e . m . ( n = 4 images per group from 3 independent donor lines examined over 3 passages each ) relative to stem spheroids .

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 O O O O O O O O O

J-L Humanileal spheroids were cultured in differentiation medium with 1 µM dmPGE2 or 10 µM EP4i. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 by 1-way ANOVA and Tukey's post-test. (J) Representative bright-field images. Scale bars, 200 µm. (K) Quantification of average spheroid area ± s.e.m. (n = 4 images per group from 3 independent donor lines examined over 3 passages each) relative to stem spheroids.


J - L Humanileal spheroids were cultured in differentiation medium with 1 µM dmPGE2 or 10 µM EP4i . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 by 1 - way ANOVA and Tukey ' s post - test . ( L ) the average expression ± s . e . m . of CLDN4 mRNA ( n = 3 independent donor lines ) relative to stem spheroids .

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

J-L Humanileal spheroids were cultured in differentiation medium with 1 µM dmPGE2 or 10 µM EP4i. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 by 1-way ANOVA and Tukey's post-test. (L) the average expression ± s.e.m. of CLDN4 mRNA (n = 3 independent donor lines) relative to stem spheroids.


B Heatmap showing unsupervised hierarchical clustering of genes that were significantly ( FDR - adjusted p < 0 . 05 ) and differentially expressed between the three groups ( n = 4 samples per treatment from two independent experiments ) . Each column is one sample and each row is one gene . Red and blue indicate genes with enriched or de - enriched expression in a particular group , respectively . The dendrogram for the gene clustering is colored to highlight major gene clusters 1 - 6 . The number of probes in each cluster is in parentheses .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Heatmap showing unsupervised hierarchical clustering of genes that were significantly (FDR-adjusted p<0.05) and differentially expressed between the three groups (n = 4 samples per treatment from two independent experiments). Each column is one sample and each row is one gene. Red and blue indicate genes with enriched or de-enriched expression in a particular group, respectively. The dendrogram for the gene clustering is colored to highlight major gene clusters 1-6. The number of probes in each cluster is in parentheses.


E , F Spheroids were cultured as indicated for 24 hours followed by a 1 - hr pulse with EdU to mark the cells undergoing DNA synthesis . ( E ) Representative images of EdU staining ( red ) . Nuclei are visualized with bisbenzimide ( blue ) . Scale bars , 20 µm . ( F ) Quantification of EdU - positive nuclei as a percent of the total nuclei ( n = minimum of 15 spheroids counted per sample from 2 independent experiments ) . * * * * p < 0 . 0001 by 1 - way ANOVA and Tukey ' s post - 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 B-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

E, F Spheroids were cultured as indicated for 24 hours followed by a 1-hr pulse with EdU to mark the cells undergoing DNA synthesis. (E) Representative images of EdU staining (red). Nuclei are visualized with bisbenzimide (blue). Scale bars, 20 µm. (F) Quantification of EdU-positive nuclei as a percent of the total nuclei (n = minimum of 15 spheroids counted per sample from 2 independent experiments). ****p<0.0001 by 1-way ANOVA and Tukey's post-test.


G Graph of the fold change in background - subtracted luminescence ± s . e . m . ( relative to 0 hr measurement ) of Cdc25A - CBRLuc spheroids ( n = 3 independent experiments with 4 technical replicates ) . * * * * p < 0 . 0001 for dmPGE2 - and EP4i - treated spheroids compared to stem cells by repeated measures 2 - way ANOVA ( variable = treatment ) . p < 0 . 001 at the 16 hr , 20 hr and 24 hr time points by Dunnett ' s post - test comparing dmPGE2 - treated and EP4i - treated spheroids to the stem cell 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 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 B-CONTROLLED_VAR O O O O O O O O O O O O O

G Graph of the fold change in background-subtracted luminescence ± s.e.m. (relative to 0 hr measurement) of Cdc25A-CBRLuc spheroids (n = 3 independent experiments with 4 technical replicates). ****p<0.0001 for dmPGE2- and EP4i-treated spheroids compared to stem cells by repeated measures 2-way ANOVA (variable = treatment). p<0.001 at the 16 hr, 20 hr and 24 hr time points by Dunnett's post-test comparing dmPGE2-treated and EP4i-treated spheroids to the stem cell control.


H Quantification of the average expression ± s . e . m . of Lgr5 and Mki67 mRNAs in jejunal spheroids cultured in stem cell ( Stem ) or in differentiation medium with the indicated supplements relative to the stem cell group ( n = 3 independent experiments ) . * * p < 0 . 01 , * * * * p < 0 . 0001 by one - way ANOVA and Dunnett ' s post - test . n . d . , not detected .

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

H Quantification of the average expression ± s.e.m. of Lgr5 and Mki67 mRNAs in jejunal spheroids cultured in stem cell (Stem) or in differentiation medium with the indicated supplements relative to the stem cell group (n = 3 independent experiments). **p<0.01, ****p<0.0001 by one-way ANOVA and Dunnett's post-test. n.d., not detected.


D Representative images of spheroids stained for Cldn4 ( red ) . Nuclei are visualized with bisbenzimide ( blue ) ( n = 2 independent experiments ) . 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

D Representative images of spheroids stained for Cldn4 (red). Nuclei are visualized with bisbenzimide (blue) (n = 2 independent experiments). Scale bars, 20 µm.


E , F Jejunal spheroids were cultured as in Figure 2B - D . Quantification of the average expression ± s . e . m . of Dpcr1 ( E ) and Cd55b mRNAs relative to DMSO group ( n = 3 independent experiments ) . * p < 0 . 05 , * * * * p < 0 . 0001 by one - way ANOVA and Dunnett ' s post - 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

E, F Jejunal spheroids were cultured as in Figure 2B-D. Quantification of the average expression ± s.e.m. of Dpcr1 (E) and Cd55b mRNAs relative to DMSO group (n = 3 independent experiments). *p<0.05, ****p<0.0001 by one-way ANOVA and Dunnett's post-test.


G - J Representative transmission electron microscopy ( TEM ) images of spheroids or an in vivo WAE cell from a biopsy - injured mousecolon . ( G ) The basal plasma membranes are outlined in orange solid lines , lateral plasma membranes are indicated with orange arrowheads , and nuclei are outlined with wide yellow dashed lines . Insets show a magnified view of the apical cell surface .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-J Representative transmission electron microscopy (TEM) images of spheroids or an in vivo WAE cell from a biopsy-injured mousecolon. (G) The basal plasma membranes are outlined in orange solid lines, lateral plasma membranes are indicated with orange arrowheads, and nuclei are outlined with wide yellow dashed lines. Insets show a magnified view of the apical cell surface.


G - J Representative transmission electron microscopy ( TEM ) images of spheroids or an in vivo WAE cell from a biopsy - injured mousecolon . Quantification of cytoplasmic : nuclear ratio ( H ) and microvillar length ( I ) from the TEM images ( n = minimum of 5 images per group ) . * p < 0 . 05 , * * p < 0 . 01 , * * * * p < 0 . 0001 by 1 - way ANOVA and Tukey ' s post - 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

G-J Representative transmission electron microscopy (TEM) images of spheroids or an in vivo WAE cell from a biopsy-injured mousecolon. Quantification of cytoplasmic:nuclear ratio (H) and microvillar length (I) from the TEM images (n = minimum of 5 images per group). *p<0.05, **p<0.01, ****p<0.0001 by 1-way ANOVA and Tukey's post-test.


G - J Representative transmission electron microscopy ( TEM ) images of spheroids or an in vivo WAE cell from a biopsy - injured mousecolon . ( J ) Higher power image of the cytoplasm . Single mitochondria are outlined with a narrow blue dashed line , vacuole structures are indicated with red asterisks . Scale bars , 1 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

G-J Representative transmission electron microscopy (TEM) images of spheroids or an in vivo WAE cell from a biopsy-injured mousecolon. (J) Higher power image of the cytoplasm. Single mitochondria are outlined with a narrow blue dashed line, vacuole structures are indicated with red asterisks. Scale bars, 1 µm.


K Representative image of a spheroid stained for β - catenin ( green ) and F - actin ( red ) . Nuclei are visualized with bisbenzimide ( blue ) . A similarly stained section of a small intestinal ulcer is shown for comparison . Arrowheads indicate the apical cell membrane . Scale bars , 50 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

K Representative image of a spheroid stained for β-catenin (green) and F-actin (red). Nuclei are visualized with bisbenzimide (blue). A similarly stained section of a small intestinal ulcer is shown for comparison. Arrowheads indicate the apical cell membrane. Scale bars, 50 µm.


A Representative image of a spheroid stained for β - catenin ( green ) and F - actin ( red ) . Nuclei are visualized with bisbenzimide ( blue ) . A similarly stained section of a mouse villus is shown for comparison . Arrowheads indicate the apical cell membrane . Scale bars , 50 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

A Representative image of a spheroid stained for β-catenin (green) and F-actin (red). Nuclei are visualized with bisbenzimide (blue). A similarly stained section of a mouse villus is shown for comparison. Arrowheads indicate the apical cell membrane. Scale bars, 50 µm.


B , C Jejunal spheroids were cultured as indicated . ( B ) Quantification of the average expression ± s . e . m . of Fabp1 , Ace2 , and Maoa mRNAs ( n = 3 independent experiments ) . * p < 0 . 05 , * * p < 0 . 01 by one - way ANOVA and Tukey ' s post - 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

B, C Jejunal spheroids were cultured as indicated. (B) Quantification of the average expression ± s.e.m. of Fabp1, Ace2, and Maoa mRNAs (n = 3 independent experiments). *p<0.05, **p<0.01 by one-way ANOVA and Tukey's post-test.


B , C Jejunal spheroids were cultured as indicated . ( C ) Representative images of spheroids stained for Ace2 ( green ) and β - catenin ( red ) . Nuclei are visualized with bisbenzimide ( blue ) . 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

B, C Jejunal spheroids were cultured as indicated. (C) Representative images of spheroids stained for Ace2 (green) and β-catenin (red). Nuclei are visualized with bisbenzimide (blue). Scale bars, 20 µm.


F , G Representative TEM images of EP4i - treated spheroids . ( F ) The basal plasma membranes are outlined in orange solid lines , lateral plasma membranes are indicated with orange arrowheads , and nuclei are outlined with wide yellow dashed lines . Insets show a magnified view of the apical cell surface . ( G ) Higher power image of the cytoplasm . Single mitochondria are outlined with a narrow blue dashed line , vacuole structures are indicated with red asterisks . Scale bars , 1 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

F, G Representative TEM images of EP4i-treated spheroids. (F) The basal plasma membranes are outlined in orange solid lines, lateral plasma membranes are indicated with orange arrowheads, and nuclei are outlined with wide yellow dashed lines. Insets show a magnified view of the apical cell surface. (G) Higher power image of the cytoplasm. Single mitochondria are outlined with a narrow blue dashed line, vacuole structures are indicated with red asterisks. Scale bars, 1 µm.


H Graph of spheroid oxygen consumption rate ( OCR ) to extracellular acidification rate ( ECAR ) ratio expressed as mean ± s . e . m . ( n = 3 independent experiments ) . * * p < 0 . 01by 1 - way ANOVA and Tukey ' s post - 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

H Graph of spheroid oxygen consumption rate (OCR) to extracellular acidification rate (ECAR) ratio expressed as mean ± s.e.m. (n = 3 independent experiments). **p<0.01by 1-way ANOVA and Tukey's post-test.


A - D Quantification of the average expression ± s . e . m . of Dpcr1 and Cd55b mRNAs ( A , C , E ) or Fabp1 and Ace2 mRNAs ( B , D , F ) in jejunal spheroids cultured in differentiation medium containing EP4i or dmPGE2 . ( A , B ) Gene expression analyzed 2 , 6 , 12 or 24 hours after the start of treatment ( n = 3 independent experiments ) . * * * p < 0 . 001 , * * * * p < 0 . 0001 comparing the two treatment groups and † p < 0 . 05 , † † † p < 0 . 001 , † † † † p < 0 . 0001 compared to the 2 hr time point of the same medium by 2 - way ANOVA and Sidak ' s multiple comparisons test . ( C , D ) Gene expression was analyzed after culturing spheroids in EP4i ( E ) or dmPGE2 ( P ) for the first 12 hours followed by wash out and re - feeding with EP4i or dmPGE2 for the second 12 hours as shown ( n = 3 independent experiments ) . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 and * * * * p < 0 . 0001 by one - way ANOVA and Dunnett ' s post - 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 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 O O 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 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

A-D Quantification of the average expression ± s.e.m. of Dpcr1 and Cd55b mRNAs (A, C, E) or Fabp1 and Ace2 mRNAs (B, D, F) in jejunal spheroids cultured in differentiation medium containing EP4i or dmPGE2. (A, B) Gene expression analyzed 2, 6, 12 or 24 hours after the start of treatment (n = 3 independent experiments). ***p<0.001, ****p<0.0001 comparing the two treatment groups and †p<0.05, †††p<0.001, ††††p<0.0001 compared to the 2 hr time point of the same medium by 2-way ANOVA and Sidak's multiple comparisons test. (C, D) Gene expression was analyzed after culturing spheroids in EP4i (E) or dmPGE2 (P) for the first 12 hours followed by wash out and re-feeding with EP4i or dmPGE2 for the second 12 hours as shown (n = 3 independent experiments). *p<0.05, **p<0.01, ***p<0.001 and ****p<0.0001 by one-way ANOVA and Dunnett's post-test.


E , F Gene expression was analyzed in Ptger4flox / flox , Ptger4flox / Δ and Ptger4Δ / Δspheroids and expressed as fold change relative to EP4i - treated Ptger4flox / flox ( n = 3 independent experiments ) . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 by two - way ANOVA and Tukey ' s post - test comparing the genotypes within a treatment group . † † † p < 0 . 001 , † † † † p < 0 . 0001 by two - way ANOVA and Sidak ' s post - test comparing the effects of the treatments within a genotype .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, F Gene expression was analyzed in Ptger4flox/flox, Ptger4flox/Δ and Ptger4Δ/Δspheroids and expressed as fold change relative to EP4i-treated Ptger4flox/flox (n = 3 independent experiments). *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 by two-way ANOVA and Tukey's post-test comparing the genotypes within a treatment group. †††p<0.001, ††††p<0.0001 by two-way ANOVA and Sidak's post-test comparing the effects of the treatments within a genotype.


A , B Jejunal spheroids were cultured in differentiation medium containing 10 µM of Forskolin or an equivalent volume of DMSO . ( A ) Representative bright - field images . Scale bars , 200 µm .

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

A, B Jejunal spheroids were cultured in differentiation medium containing 10 µM of Forskolin or an equivalent volume of DMSO. (A) Representative bright-field images. Scale bars, 200 µm.


A , B Jejunal spheroids were cultured in differentiation medium containing 10 µM of Forskolin or an equivalent volume of DMSO . ( B ) Quantification of the average expression ± s . e . m . of Cldn4 mRNA relative to the DMSO treatment group ( n = 3 independent experiments ) . * p < 0 . 05 compared to DMSO group by unpaired t test .

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

A, B Jejunal spheroids were cultured in differentiation medium containing 10 µM of Forskolin or an equivalent volume of DMSO. (B) Quantification of the average expression ± s.e.m. of Cldn4 mRNA relative to the DMSO treatment group (n = 3 independent experiments). *p<0.05 compared to DMSO group by unpaired t test.


C , D Representative Western blots for β - catenin detected in nuclear ( Nuc ) and cytoplasmic ( Cyt ) protein lysates . Lamin A / C and Actin were used as loading controls for the nuclear and cytoplasmic fractions , respectively ( n = 3 independent experiments ) . ( C ) Spheroids were cultured in stem , enterocyte ( EP4i ) or WAE ( dmPGE2 ) media . ( D ) Spheroids were cultured in enterocyte medium with DMSO or 10 µM CHIR 99021 .

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

C, D Representative Western blots for β-catenin detected in nuclear (Nuc) and cytoplasmic (Cyt) protein lysates. Lamin A/C and Actin were used as loading controls for the nuclear and cytoplasmic fractions, respectively (n = 3 independent experiments). (C) Spheroids were cultured in stem, enterocyte (EP4i) or WAE (dmPGE2) media. (D) Spheroids were cultured in enterocyte medium with DMSO or 10 µM CHIR 99021.


E , F Spheroids were cultured in enterocyte medium with the indicated concentrations of CHIR 99021 . Quantification of the average expression ± s . e . m . of Cldn4 ( E ) and Fabp1 ( F ) mRNAs shown as fold change relative to 0 µM group ( n = 3 independent experiments ) . * * p < 0 . 01 , * * * * p < 0 . 0001 as determined by one - way ANOVA and Dunnett ' s post - test .

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

E, F Spheroids were cultured in enterocyte medium with the indicated concentrations of CHIR 99021. Quantification of the average expression ± s.e.m. of Cldn4 (E) and Fabp1 (F) mRNAs shown as fold change relative to 0 µM group (n = 3 independent experiments). **p<0.01, ****p<0.0001 as determined by one-way ANOVA and Dunnett's post-test.


G Representative images of Ptger4Δ / Δspheroids cultured in differentiation medium with DMSO or 10 µM CHIR 99021 and stained for β - catenin ( green ) and F - actin ( red ) . Nuclei are visualized with bisbenzimide ( blue ) . Scale bars , 20 µm .

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

G Representative images of Ptger4Δ/Δspheroids cultured in differentiation medium with DMSO or 10 µM CHIR 99021 and stained for β-catenin (green) and F-actin (red). Nuclei are visualized with bisbenzimide (blue). Scale bars, 20 µm.


H Graph of TOPflash ( TOP ) to FOPflash ( FOP ) luciferase reporter ratios ± s . e . m . ( n = 3 independent experiments ) in transfected 293FT cells treated with conditioned mediums collected from L - WRN cells and diluted as indicated . L - WRN CM had been produced in the presence or absence of the porcupine inhibitor C59 ( 10 - fold dilutions , 10 µM to 1 pM ) . * p < 0 . 05 , * * * p < 0 . 001 , * * * * p < 0 . 0001 by 1 - way ANOVA and Dunnett ' s post - test compared to 0 % L - WRN CM group .

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

H Graph of TOPflash (TOP) to FOPflash (FOP) luciferase reporter ratios ± s.e.m. (n = 3 independent experiments) in transfected 293FT cells treated with conditioned mediums collected from L-WRN cells and diluted as indicated. L-WRN CM had been produced in the presence or absence of the porcupine inhibitor C59 (10-fold dilutions, 10 µM to 1 pM). *p<0.05, ***p<0.001, ****p<0.0001 by 1-way ANOVA and Dunnett's post-test compared to 0% L-WRN CM group.


I , J Spheroids were cultured in enterocyte media , WAE media or WAE media containing 100 pM C59 . Quantification of the average expression ± s . e . m . of Cldn4 ( I ) and Fabp1 ( J ) mRNAs shown as fold change relative to EP4i group ( n = 3 independent experiments ) . * * * p < 0 . 001 , * * * * p < 0 . 0001 as determined by one - way ANOVA and Tukey ' s post - test .

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

I, J Spheroids were cultured in enterocyte media, WAE media or WAE media containing 100 pM C59. Quantification of the average expression ± s.e.m. of Cldn4 (I) and Fabp1 (J) mRNAs shown as fold change relative to EP4i group (n = 3 independent experiments). ***p<0.001, ****p<0.0001 as determined by one-way ANOVA and Tukey's post-test.


K Quantification of the average expression ± s . e . m . of Axin2 mRNA in spheroids cultured in stem cell or in differentiation medium with the indicated supplements relative to stem ( n = 3 independent experiments ) . * * * * p < 0 . 0001 compared to stem cell group as determined by one - way ANOVA and Dunnett ' s post - 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

K Quantification of the average expression ± s.e.m. of Axin2 mRNA in spheroids cultured in stem cell or in differentiation medium with the indicated supplements relative to stem (n = 3 independent experiments). ****p<0.0001 compared to stem cell group as determined by one-way ANOVA and Dunnett's post-test.


A Representative whole - mount images of wounds from Ptger4flox / flox ( Ptger4fl / fl ) and Villin - Cre Ptger4flox / flox ( VilCrePtger4fl / fl ) mice four days post - biopsy injury . Scale bars , 750 µm . Wounds are outlined with a black dashed line . Asterisk indicates fibrin clot . B , C Quantification of percent healing ( 1 - [ Day 4 wound area / original wound area ] x 100 ) ( B ) and fibrin clot areas ( C ) ± s . e . m . ( n = 7 - 8 mice per genotype with 3 - 4 wounds each , three independent experiments ) . * p = 0 . 0270 , * * p = 0 . 0057 by two - tailed unpaired 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

A Representative whole-mount images of wounds from Ptger4flox/flox (Ptger4fl/fl) and Villin-Cre Ptger4flox/flox (VilCrePtger4fl/fl) mice four days post-biopsy injury. Scale bars, 750 µm. Wounds are outlined with a black dashed line. Asterisk indicates fibrin clot.B, C Quantification of percent healing (1 - [Day 4 wound area/original wound area] x 100) (B) and fibrin clot areas (C) ± s.e.m. (n = 7-8 mice per genotype with 3-4 wounds each, three independent experiments). *p=0.0270, **p=0.0057 by two-tailed unpaired t-test.


D , E Representative images of serial wound tissue sections from the indicated genotypes . ( D ) Fluorescent images of sections stained for Claudin 4 ( red ) . Nuclei are visualized with bisbenzimide ( blue ) . Epithelial crypts adjacent to the wound bed are indicated by dashed yellow lines . Layer of adherent WAE cells indicated by white arrowheads . Non - adherent residual WAE cells indicated by orange arrows .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Representative images of serial wound tissue sections from the indicated genotypes. (D) Fluorescent images of sections stained for Claudin 4 (red). Nuclei are visualized with bisbenzimide (blue). Epithelial crypts adjacent to the wound bed are indicated by dashed yellow lines. Layer of adherent WAE cells indicated by white arrowheads. Non-adherent residual WAE cells indicated by orange arrows.


D , E Representative images of serial wound tissue sections from the indicated genotypes . ( E ) Grayscale images of hematoxylin and eosin - stained sections . Insets of the boxed regions show a higher magnification of the region typically containing WAE cells ( red arrowheads , dashed line indicates border with wound bed ) . Orange arrow indicates region of non - adherent residual WAE cells . Scale bars , 100 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

D, E Representative images of serial wound tissue sections from the indicated genotypes. (E) Grayscale images of hematoxylin and eosin-stained sections. Insets of the boxed regions show a higher magnification of the region typically containing WAE cells (red arrowheads, dashed line indicates border with wound bed). Orange arrow indicates region of non-adherent residual WAE cells. Scale bars, 100 µm.


( A ) BACE1 internalization kinetics after 20 minutes ( WT : 19 . 79 ± 1 . 89 % , KO : 13 . 28 ± 1 . 57 % , p WT vs KO = 0 . 017 ) and 40 minutes of HA antibody chase ( WT : 15 . 99 ± 2 . 04 , KO : 9 . 29 ± 1 . 33 , pWT vs KO = 0 . 010 ) is significantly decreased in AP - 2µ KO neurons ( 27 WT and 29 KO neurons ) . Data information : All graphs show mean ± SEM , statistical analysis was performed by unpaired two - tailed Student ' s t ‐ test n . s . - non - significant . * indicates p ≤ 0 . 05 , * * indicates p ≤ 0 . 01 , * * * indicates p ≤ 0 . 001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) BACE1 internalization kinetics after 20 minutes (WT: 19.79±1.89%, KO: 13.28±1.57%, p WT vs KO=0.017) and 40 minutes of HA antibody chase (WT: 15.99±2.04, KO: 9.29±1.33, pWT vs KO=0.010) is significantly decreased in AP-2µ KO neurons (27 WT and 29 KO neurons). Data information: All graphs show mean ± SEM, statistical analysis was performed by unpaired two-tailed Student's t‐test n.s.-non-significant. * indicates p≤0.05, ** indicates p≤0.01, *** indicates p≤0.001.


( B ) Representative examples of internalized BACE1 after 5 min of HA antibody pulse in HA - BACE1 - eGFP - overexpressing WT and KO neurons . Scale bar : 5µm .

O O O O O O O O O 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) Representative examples of internalized BACE1 after 5 min of HA antibody pulse in HA-BACE1-eGFP-overexpressing WT and KO neurons. Scale bar: 5µm.


( C , D ) Internalized BACE1 levels after 20 min of HA antibody pulse in HA - BACE1 - eGFP - overexpressing WT and KO neurons , calculated as the HA ( internalized ) / eGFP ( total ) signal intensity ratio ( WT : 0 . 50 ± 0 . 052 , KO : 0 . 23 ± 0 . 036 , p = 0 . 000 , 30 WT and 36 KO neurons , N = 4 biological replicates ) . Scale bar : 5 µm . Data information : Experimental designs are depicted in the top panels where analyzed BACE1 fraction is marked with the red square . All graphs show mean ± SEM , statistical analysis was performed by unpaired two - tailed Student ' s t ‐ test n . s . - non - significant . * indicates p ≤ 0 . 05 , * * indicates p ≤ 0 . 01 , * * * indicates p ≤ 0 . 001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, D) Internalized BACE1 levels after 20 min of HA antibody pulse in HA-BACE1-eGFP-overexpressing WT and KO neurons, calculated as the HA (internalized)/eGFP (total) signal intensity ratio (WT: 0.50±0.052, KO: 0.23±0.036, p=0.000, 30 WT and 36 KO neurons, N=4 biological replicates). Scale bar: 5 µm. Data information: Experimental designs are depicted in the top panels where analyzed BACE1 fraction is marked with the red square. All graphs show mean ± SEM, statistical analysis was performed by unpaired two-tailed Student's t‐test n.s.-non-significant. * indicates p≤0.05, ** indicates p≤0.01, *** indicates p≤0.001.


( E , F ) Recycled BACE1 levels after 20 min of HA antibody pulse in HA - BACE1 - eGFP - overexpressing WT and KO neurons , calculated as the HA ( recycled ) / eGFP ( total ) signal intensity ratio ( WT : 0 . 29 ± 0 . 031 , KO : 0 . 53 ± 0 . 040 , p < 000 , 30 WT and 32 KO neurons , N = 4 biological replicates ) . Scale bar : 5 µm . Data information : Experimental designs are depicted in the top panels where analyzed BACE1 fraction is marked with the red square . All graphs show mean ± SEM , statistical analysis was performed by unpaired two - tailed Student ' s t ‐ test n . s . - non - significant . * indicates p ≤ 0 . 05 , * * indicates p ≤ 0 . 01 , * * * indicates p ≤ 0 . 001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, F) Recycled BACE1 levels after 20 min of HA antibody pulse in HA-BACE1-eGFP-overexpressing WT and KO neurons, calculated as the HA(recycled)/eGFP(total) signal intensity ratio (WT: 0.29±0.031, KO: 0.53±0.040, p<000, 30 WT and 32 KO neurons, N=4 biological replicates). Scale bar: 5 µm. Data information: Experimental designs are depicted in the top panels where analyzed BACE1 fraction is marked with the red square. All graphs show mean ± SEM, statistical analysis was performed by unpaired two-tailed Student's t‐test n.s.-non-significant. * indicates p≤0.05, ** indicates p≤0.01, *** indicates p≤0.001.


Recycled levels of BACE1 after 20 min of HA antibody pulse in HA - BACE1 - mCherry - transfected WT and KO neurons , co - expressing either eGFP - RAB4 or eGFP - RAB4 - S22N pWTRAB4 vs pWTRAB4S22N > 0 . 999 , 34 - 38 neurons , N = 4 biological replicates ) . Scale bar : 5 µm . Data information : Experimental designs are depicted in the top panels , where analyzed BACE1 fraction is marked with the red square .

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

Recycled levels of BACE1 after 20 min of HA antibody pulse in HA-BACE1-mCherry-transfected WT and KO neurons, co-expressing either eGFP-RAB4 or eGFP-RAB4-S22N pWTRAB4 vs pWTRAB4S22N>0.999, 34-38 neurons, N=4 biological replicates). Scale bar: 5 µm. Data information: Experimental designs are depicted in the top panels , where analyzed BACE1 fraction is marked with the red square.


Recycled levels of BACE1 after 20 min of HA antibody pulse in HA - BACE1 - mCherry - transfected WT and KO neurons , co - expressing either eGFP - RAB4 or eGFP - RAB4 - S22N , calculated as the HA ( recycled ) / mCherry ( total ) signal intensity ratio ( WTRAB4 : 0 . 14 ± 0 . 02 , KORAB4 : 0 . 20 ± 0 . 02 , WTRAB4 - S22N : 0 . 14 ± 0 . 02 , KORAB4S22N : 0 . 14 ± 0 . 01 , pWTRAB4 vs pKORAB4 = 0 . 033 , pKORAB4 vs pKORAB4S22N = 0 . 035 , pWTRABS22N vs pKORABS22N = 0 . 999 , pWTRAB4 vs pWTRAB4S22N > 0 . 999 , 34 - 38 neurons , N = 4 biological replicates ) . Scale bar : 5 µm . Data information : All graphs show mean ± SEM , statistical analysis was performed by two - way ANOVA in n . s . - non - significant . * indicates p ≤ 0 . 05 , * * indicates p ≤ 0 . 01 , * * * indicates p ≤ 0 . 001 .

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

Recycled levels of BACE1 after 20 min of HA antibody pulse in HA-BACE1-mCherry-transfected WT and KO neurons, co-expressing either eGFP-RAB4 or eGFP-RAB4-S22N, calculated as the HA(recycled)/mCherry(total) signal intensity ratio (WTRAB4: 0.14±0.02, KORAB4: 0.20±0.02, WTRAB4-S22N: 0.14±0.02, KORAB4S22N:0.14±0.01, pWTRAB4 vs pKORAB4=0.033, pKORAB4 vs pKORAB4S22N=0.035, pWTRABS22N vs pKORABS22N=0.999, pWTRAB4 vs pWTRAB4S22N>0.999, 34-38 neurons, N=4 biological replicates). Scale bar: 5 µm. Data information: All graphs show mean ± SEM, statistical analysis was performed by two-way ANOVA in n.s.-non-significant. * indicates p≤0.05, ** indicates p≤0.01, *** indicates p≤0.001.


Recycled levels of BACE1 after 20 min of HA antibody chase in HA - BACE1 - mCherry - transfected WT and KO neurons , co - expressing either eGFP - RAB11 or eGFP - RAB11 - S22N , calculated as the HA ( recycled ) / mCherry ( total ) signal intensity ratio ( WTRAB11 : 0 . 11 ± 0 . 01 , KORAB11 : 0 . 29 ± 0 . 02 , WTRAB11A - S25N : 0 . 14 ± 0 . 02 , KORAB11A - S25N : 0 . 19 ± 0 . 02 , pWTRAB11 vs pKORAB11 < 0 . 0001 , pWTRAB11 vs pWTRAB11A - S25N = 0 . 761 , pKORAB11 vs pKORAB11A - S25N = 0 . 001 , pWTRAB11A - S25N vs pKORAB11A - S25N = 0 . 175 , 28 - 37 neurons per condition , N = 3 biological replicates ) . Data information : All graphs show mean ± SEM , statistical analysis was performed by two - way ANOVA n . s . - non - significant . * indicates p ≤ 0 . 05 , * * indicates p ≤ 0 . 01 , * * * indicates p ≤ 0 . 001 .

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

Recycled levels of BACE1 after 20 min of HA antibody chase in HA-BACE1-mCherry-transfected WT and KO neurons, co-expressing either eGFP-RAB11 or eGFP-RAB11-S22N, calculated as the HA(recycled)/mCherry(total) signal intensity ratio (WTRAB11: 0.11±0.01, KORAB11: 0.29±0.02, WTRAB11A-S25N: 0.14±0.02, KORAB11A-S25N: 0.19±0.02, pWTRAB11 vs pKORAB11<0.0001, pWTRAB11 vs pWTRAB11A-S25N=0.761, pKORAB11 vs pKORAB11A-S25N=0.001, pWTRAB11A-S25N vs pKORAB11A-S25N=0.175, 28-37 neurons per condition, N=3 biological replicates). Data information: All graphs show mean ± SEM, statistical analysis was performed by two-way ANOVA n.s.-non-significant. * indicates p≤0.05, ** indicates p≤0.01, *** indicates p≤0.001.


Internalized levels of BACE1 after 20 minutes of HA antibody uptake in HA - BACE1 - mCherry transfected WT and KO neurons , co - expressing either eGFP - RAB4 or eGFP - RAB4 - S22N pWTRAB4 vs pWTRAB4S22N = 0 . 450 , 27 - 37 neurons per condition , N = 3 biological replicates ) . Scale bar : 5 µm . Data information : Experimental designs are depicted in the top panels where analyzed BACE1 fraction is marked with the red square .

Internalized levels of BACE1 after 20 minutes of HA antibody uptake in HA-BACE1-mCherry transfected WT and KO neurons, co-expressing either eGFP-RAB4 or eGFP-RAB4-S22N pWTRAB4 vs pWTRAB4S22N=0.450, 27-37 neurons per condition, N=3 biological replicates). Scale bar: 5 µm. Data information: Experimental designs are depicted in the top panels where analyzed BACE1 fraction is marked with the red square.


Internalized levels of BACE1 after 20 minutes of HA antibody uptake in HA - BACE1 - mCherry transfected WT and KO neurons , co - expressing either eGFP - RAB4 or eGFP - RAB4 - S22N , calculated as the HA ( internalized ) / mCherry ( total ) signal intensity ratio ( WTRAB4 : 0 . 54 ± 0 . 05 , KORAB4 : 0 . 33 ± 0 . 02 , WTRAB4 - S22N : 0 . 63 ± 0 . 06 , KORAB11A - S25N : 0 . 50 ± 0 . 04 , pWTRAB4 vs pKORAB4 = 0 . 004 , pKORAB4 vs pKORAB4S22N = 0 . 046 , pWTRABS22N vs pKORABS22N = 0 . 182 , pWTRAB4 vs pWTRAB4S22N = 0 . 450 , 27 - 37 neurons per condition , N = 3 biological replicates ) . Scale bar : 5 µm . Data information : All graphs show mean ± SEM , statistical analysis was performed by ANOVA n . s . - non - significant . * indicates p ≤ 0 . 05 , * * indicates p ≤ 0 . 01 , * * * indicates p ≤ 0 . 001 .

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

Internalized levels of BACE1 after 20 minutes of HA antibody uptake in HA-BACE1-mCherry transfected WT and KO neurons, co-expressing either eGFP-RAB4 or eGFP-RAB4-S22N, calculated as the HA(internalized)/mCherry(total) signal intensity ratio (WTRAB4: 0.54±0.05, KORAB4: 0.33±0.02, WTRAB4-S22N: 0.63±0.06, KORAB11A-S25N: 0.50±0.04, pWTRAB4 vs pKORAB4=0.004, pKORAB4 vs pKORAB4S22N=0.046, pWTRABS22N vs pKORABS22N=0.182, pWTRAB4 vs pWTRAB4S22N=0.450, 27-37 neurons per condition, N=3 biological replicates). Scale bar: 5 µm. Data information: All graphs show mean ± SEM, statistical analysis was performed by ANOVA n.s.-non-significant. * indicates p≤0.05, ** indicates p≤0.01, *** indicates p≤0.001.


( L , M ) BACE1 surface levels are increased in AP - 2µ KO neurons , when compared to the WT set to 100 % ( KO : 129 . 65 ± 5 . 30 % , p = 0 . 005 , N = 4 biological replicates ) . ( N ) Surface versus total BACE1 ratio is not altered in AP - 2µ KO neurons when normalized to the WT set to 100 % ( KO : 83 . 02 ± 10 . 93 % , p = 0 . 109 , N = 4 biological replicates ) . Data information : All graphs show mean ± SEM , statistical analysis was performed by one - sample Student ' s t ‐ test in ( M , N ) . n . s . - non - significant . * indicates p ≤ 0 . 05 , * * indicates p ≤ 0 . 01 , * * * indicates p ≤ 0 . 001 .

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

(L, M) BACE1 surface levels are increased in AP-2µ KO neurons, when compared to the WT set to 100% (KO: 129.65±5.30%, p=0.005, N=4 biological replicates). (N) Surface versus total BACE1 ratio is not altered in AP-2µ KO neurons when normalized to the WT set to 100% (KO: 83.02±10.93%, p=0.109, N=4 biological replicates). Data information: All graphs show mean ± SEM, statistical analysis was performed by one-sample Student's t‐test in (M,N). n.s.-non-significant. * indicates p≤0.05, ** indicates p≤0.01, *** indicates p≤0.001.


( O , P ) Accumulation of endogenous BACE1 in RAB5 - positive early endosomes , quantified using Pearson ' s colocalization coefficient ( WT : 0 . 41 ± 0 . 01 , KO : 0 . 46 ± 0 . 02 , pWT vs pKO = 0 . 021 , 58 WT and 54 KO neurons , N = 4 biological replicates ) . Scale bar : 10 µm . Arrows indicate BACE1 accumulation within RAB5 - positive endosomes . Data information All graphs show mean ± SEM , statistical analysis was performed by unpaired two - tailed Student ' s t ‐ test n . s . - non - significant . * indicates p ≤ 0 . 05 , * * indicates p ≤ 0 . 01 , * * * indicates p ≤ 0 . 001 .

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

(O, P) Accumulation of endogenous BACE1 in RAB5-positive early endosomes, quantified using Pearson's colocalization coefficient (WT: 0.41±0.01, KO: 0.46±0.02, pWT vs pKO=0.021, 58 WT and 54 KO neurons, N=4 biological replicates). Scale bar: 10 µm. Arrows indicate BACE1 accumulation within RAB5-positive endosomes. Data information All graphs show mean ± SEM, statistical analysis was performed by unpaired two-tailed Student's t‐test n.s.-non-significant. * indicates p≤0.05, ** indicates p≤0.01, *** indicates p≤0.001.


( A , B ) BACE1 protein levels in cortical lysates of WT and AP - 2µ KO mice . Protein levels in the KO were normalized to the WT set to 100 % ( KO : 447 . 64 ± 48 . 78 % , p < 0 . 000 , N = 7 biological replicates ) . Data information : All graphs show mean ± SEM , statistical analysis was performed by one - sample Student ' s t ‐ test n . s . - non - significant . * indicates p ≤ 0 . 05 , * * indicates p ≤ 0 . 01 , * * * indicates p ≤ 0 . 001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, B) BACE1 protein levels in cortical lysates of WT and AP-2µ KO mice. Protein levels in the KO were normalized to the WT set to 100% (KO: 447.64±48.78%, p<0.000, N=7 biological replicates). Data information: All graphs show mean ± SEM, statistical analysis was performed by one-sample Student's t‐test n.s.-non-significant. * indicates p≤0.05, ** indicates p≤0.01, *** indicates p≤0.001.


( C , D ) Immunofluorescence levels of BACE1 are significantly increased in the cortex of AP - 2µ KO mice when normalized to the WT set to 100 % ( KO : 162 . 714 ± 11 . 082 % , p = 0 . 005 , N = 4 , biological replicates ) . Scale bar : 100µm . Data information : All graphs show mean ± SEM , statistical analysis was performed by one - sample Student ' s t ‐ test n . s . - non - significant . * indicates p ≤ 0 . 05 , * * indicates p ≤ 0 . 01 , * * * indicates p ≤ 0 . 001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, D) Immunofluorescence levels of BACE1 are significantly increased in the cortex of AP-2µ KO mice when normalized to the WT set to 100% (KO: 162.714±11.082%, p=0.005, N=4, biological replicates). Scale bar: 100µm. Data information: All graphs show mean ± SEM, statistical analysis was performed by one-sample Student's t‐test n.s.-non-significant. * indicates p≤0.05, ** indicates p≤0.01, *** indicates p≤0.001.


( E ) Localization of BACE1 in cultured WT and AP - 2µ KO neurons . Scale bars : 20 µm , 5 µm ( inserts ) .

O O O O 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) Localization of BACE1 in cultured WT and AP-2µ KO neurons. Scale bars: 20 µm, 5 µm (inserts).


( F ) Line plot of BACE1 immunofluorescence along the

(F) Line plot of BACE1 immunofluorescence along the neurites


Percentage of BACE1 puncta colocalizing with the LC3b ( WT : 21 . 75 ± 2 . 88 , KO : 30 . 28 ± 2 . 97 , p = 0 . 043 , WT = 41 , KO = 45 neurons , N = 4 biological replicates ) . Scale bar : 2µm . Arrowheads indicate BACE1 accumulation within LC3b - positive organelles .

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

Percentage of BACE1 puncta colocalizing with the LC3b (WT: 21.75±2.88, KO: 30.28±2.97, p=0.043, WT=41, KO=45 neurons, N=4 biological replicates). Scale bar: 2µm. Arrowheads indicate BACE1 accumulation within LC3b-positive organelles.


Percentage of BACE1 puncta colocalizing with the LC3b ( WT : 21 . 75 ± 2 . 88 , KO : 30 . 28 ± 2 . 97 , p = 0 . 043 , WT = 41 , KO = 45 neurons , N = 4 biological replicates ) . Scale bar : 2µm . Arrowheads indicate BACE1 accumulation within LC3b - positive organelles . Data information : All graphs show mean ± SEM , statistical analysis was performed by unpaired two - tailed Student ' s t ‐ test n . s . - non - significant . * indicates p ≤ 0 . 05 , * * indicates p ≤ 0 . 01 , * * * indicates p ≤ 0 . 001 .

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

Percentage of BACE1 puncta colocalizing with the LC3b (WT: 21.75±2.88, KO: 30.28±2.97, p=0.043, WT=41, KO=45 neurons, N=4 biological replicates). Scale bar: 2µm. Arrowheads indicate BACE1 accumulation within LC3b-positive organelles. Data information: All graphs show mean ± SEM, statistical analysis was performed by unpaired two-tailed Student's t‐test n.s.-non-significant. * indicates p≤0.05, ** indicates p≤0.01, *** indicates p≤0.001.


( I , J ) Percentage of BACE1 puncta colocalizing with the eGFP - RAB7 ( WT : 19 . 54 ± 1 . 91 , KO : 37 . 09 ± 3 . 13 % , p < 0 . 000 , WT = 44 and KO = 43 neurons , N = 4 biological replicates ) . Scale bar : 2µm . Arrowheads indicate BACE1 accumulation within RAB7 - positive organelles . Data information : All graphs show mean ± SEM , statistical analysis was performed by unpaired two - tailed Student ' s t ‐ test n . s . - non - significant . * indicates p ≤ 0 . 05 , * * indicates p ≤ 0 . 01 , * * * indicates p ≤ 0 . 001 .

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

(I, J) Percentage of BACE1 puncta colocalizing with the eGFP-RAB7 (WT:19.54±1.91, KO: 37.09±3.13%, p<0.000, WT=44 and KO=43 neurons, N=4 biological replicates). Scale bar: 2µm. Arrowheads indicate BACE1 accumulation within RAB7-positive organelles. Data information: All graphs show mean ± SEM, statistical analysis was performed by unpaired two-tailed Student's t‐test n.s.-non-significant. * indicates p≤0.05, ** indicates p≤0.01, *** indicates p≤0.001.


( K , L ) Colocalization of BACE1 and RAB7A in cortical sections of WT and AP - 2μ ΚΟ brains , quantified using Pearson ' s colocalization coefficient ( WT : 0 . 18 ± 0 . 04 , KO : 0 . 35 ± 0 . 05 , p = 0 . 038 , N = 4 biological replicates ) . Scale bar : 10 µm . Data information : All graphs show mean ± SEM , statistical analysis was performed by unpaired two - tailed Student ' s t ‐ test n . s . - non - significant . * indicates p ≤ 0 . 05 , * * indicates p ≤ 0 . 01 , * * * indicates p ≤ 0 . 001 .

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

(K, L) Colocalization of BACE1 and RAB7A in cortical sections of WT and AP-2μ ΚΟ brains, quantified using Pearson's colocalization coefficient (WT: 0.18±0.04, KO: 0.35±0.05, p=0.038, N=4 biological replicates). Scale bar: 10 µm. Data information: All graphs show mean ± SEM, statistical analysis was performed by unpaired two-tailed Student's t‐test n.s.-non-significant. * indicates p≤0.05, ** indicates p≤0.01, *** indicates p≤0.001.


( M Representative fluorescence images of WT neurons transfected with HA - BACE1 - mCherry and immunostained for Cathepsin D . Channels were false color - coded to better illustrate the colocalization . Scale bars : 10µm . Arrowheads indicate BACE1 accumulation in lysosomes in the WT Dotted line outlines the neuron .

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

(M Representative fluorescence images of WT neurons transfected with HA-BACE1-mCherry and immunostained for Cathepsin D. Channels were false color-coded to better illustrate the colocalization. Scale bars: 10µm. Arrowheads indicate BACE1 accumulation in lysosomes in the WT Dotted line outlines the neuron.


N ) Representative fluorescence images of AP - 2µ KO neurons transfected with HA - BACE1 - mCherry and immunostained for Cathepsin D . Channels were false color - coded to better illustrate the colocalization . Scale bars : 10µm . Arrowheads indicate BACE1 absence in the KO . Dotted line outlines the neuron .

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

N) Representative fluorescence images of AP-2µ KO neurons transfected with HA-BACE1-mCherry and immunostained for Cathepsin D. Channels were false color-coded to better illustrate the colocalization. Scale bars: 10µm. Arrowheads indicate BACE1 absence in the KO. Dotted line outlines the neuron.


( O ) Lysosomal localization of BACE1 is decreased in KO neurons ( WT : 73 . 05 ± 3 . 80 , KO : 57 . 68 ± 4 . 28 , p = 0 . 008 , 45 WT and 44 KO neurons , N = 4 biological replicates ) . Data information : All graphs show mean ± SEM , statistical analysis was performed by unpaired two - tailed Student ' s t ‐ test n . s . - non - significant . * indicates p ≤ 0 . 05 , * * indicates p ≤ 0 . 01 , * * * indicates p ≤ 0 . 001 .

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

(O) Lysosomal localization of BACE1 is decreased in KO neurons (WT: 73.05±3.80, KO: 57.68±4.28, p=0.008, 45 WT and 44 KO neurons, N=4 biological replicates). Data information: All graphs show mean ± SEM, statistical analysis was performed by unpaired two-tailed Student's t‐test n.s.-non-significant. * indicates p≤0.05, ** indicates p≤0.01, *** indicates p≤0.001.


( Q , R ) Representative fluorescence images of WT and AP - 2μ KO neurons transfected with BACE1 - mKeima - Red and co - transfected with eBFP to reveal the neuronal morphology . Scale bars : 10µm , 2µm in zoomed images .

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

(Q, R) Representative fluorescence images of WT and AP-2μ KO neurons transfected with BACE1-mKeima-Red and co-transfected with eBFP to reveal the neuronal morphology. Scale bars: 10µm, 2µm in zoomed images.


( S ) The 550nm / 438nm signal intensity ratio of BACE1 - mKeima - Red quantified over the entire cell is significantly reduced in the KO condition compared to the WT ( WT : 0 . 59 ± 0 . 04 , KO : 0 . 48 ± 0 . 04 , p = 0 . 041 , 28 WT and 25 KO neurons , N = 3 biological replicates ) . Data information : All graphs show mean ± SEM , statistical analysis was performed by unpaired two - tailed Student ' s t ‐ test n . s . - non - significant . * indicates p ≤ 0 . 05 , * * indicates p ≤ 0 . 01 , * * * indicates p ≤ 0 . 001 .

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

(S) The 550nm/438nm signal intensity ratio of BACE1-mKeima-Red quantified over the entire cell is significantly reduced in the KO condition compared to the WT (WT: 0.59±0.04, KO: 0.48±0.04, p=0.041, 28 WT and 25 KO neurons, N=3 biological replicates). Data information: All graphs show mean ± SEM, statistical analysis was performed by unpaired two-tailed Student's t‐test n.s.-non-significant. * indicates p≤0.05, ** indicates p≤0.01, *** indicates p≤0.001.


( A , B ) Representative fluorescence images ( A ) and corresponding kymographs ( B ) from time - lapse videos of control neurons transfected with HA - BACE1 - eGFP and co - transfected with AP - 2μ - mCherry . Scale bars : ( A ) 2μm , 1 . 6µm inserts , ( B ) x = 2μm , y = 5s . Arrowheads in ( A ) correspond to BACE1 carriers indicated by the same arrowheads in ( B ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, B) Representative fluorescence images (A) and corresponding kymographs (B) from time-lapse videos of control neurons transfected with HA-BACE1-eGFP and co-transfected with AP-2μ-mCherry. Scale bars: (A) 2μm, 1.6µm inserts, (B) x=2μm, y=5s. Arrowheads in (A) correspond to BACE1 carriers indicated by the same arrowheads in (B).


( C ) Representative fluorescence images and corresponding kymographs from time - lapse videos of WT and AP - 2μ KO neurons transfected with HA - BACE1 - eGFP . Scale bar : 5µm top panels , x = 2μm , y = 5s bottom panels .

O O O O O O O O O O O O O O O O O O O O O O O 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) Representative fluorescence images and corresponding kymographs from time-lapse videos of WT and AP-2μ KO neurons transfected with HA-BACE1-eGFP. Scale bar: 5µm top panels, x=2μm, y=5s bottom panels.


( D ) Loss of AP - 2μ significantly decreases the BACE1 motility ( WT : 37 . 42 ± 3 . 79 % , KO : 23 . 07 ± 3 . 07 % , p = 0 . 003 , WT = 31 , KO = 32 neurons , N = 4 biological replicates ) . Data information : All graphs show mean ± SEM , statistical analysis was performed by unpaired two - tailed Student ' s t ‐ test n . s . - non - significant . * indicates p ≤ 0 . 05 , * * indicates p ≤ 0 . 01 , * * * indicates p ≤ 0 . 001 .

(D) Loss of AP-2μ significantly decreases the BACE1 motility (WT: 37.42±3.79%, KO: 23.07±3.07%, p=0.003, WT=31, KO=32 neurons, N=4 biological replicates). Data information: All graphs show mean ± SEM, statistical analysis was performed by unpaired two-tailed Student's t‐test n.s.-non-significant. * indicates p≤0.05, ** indicates p≤0.01, *** indicates p≤0.001.


( E ) Loss of AP - 2μ significantly decreases the anterograde and retrograde BACE1 velocity ( WTAntero : 0 . 37 ± 0 . 02 μm / s , KOAntero : 0 . 22 ± 0 . 03 μm / s , pWTAntero vs pKOAntero < 0 . 000 ; WTRetro : 0 . 35 ± 0 . 02 μm / s , KORetro : 0 . 25 ± . 03 μm / s , pWTRetro vs pKORetro = 0 . 006 , WT = 35 and KO = 32 neurons , N = 4 biological replicates ) . Data information : All graphs show mean ± SEM , statistical analysis was performed by unpaired two - tailed Student ' s t ‐ test n . s . - non - significant . * indicates p ≤ 0 . 05 , * * indicates p ≤ 0 . 01 , * * * indicates p ≤ 0 . 001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Loss of AP-2μ significantly decreases the anterograde and retrograde BACE1 velocity (WTAntero: 0.37±0.02 μm/s, KOAntero: 0.22±0.03 μm/s, pWTAntero vs pKOAntero<0.000; WTRetro: 0.35±0.02 μm/s, KORetro: 0.25±.03 μm/s, pWTRetro vs pKORetro =0.006, WT=35 and KO=32 neurons, N=4 biological replicates). Data information: All graphs show mean ± SEM, statistical analysis was performed by unpaired two-tailed Student's t‐test n.s.-non-significant. * indicates p≤0.05, ** indicates p≤0.01, *** indicates p≤0.001.


( F ) Representative fluorescence images and corresponding kymographs from time - lapse videos of WT and AP - 2μ KO neurons co - transfected with HA - BACE1 - GFP and AP - 2μ - mRFP . Scale bar : 5µm top panels , x = 2μm , y = 5s bottom panels .

(F) Representative fluorescence images and corresponding kymographs from time-lapse videos of WT and AP-2μ KO neurons co-transfected with HA-BACE1-GFP and AP-2μ-mRFP. Scale bar: 5µm top panels, x=2μm, y=5s bottom panels.


( G ) Bi - directional mobility of BACE1 carriers in WT and AP - 2μ ΚΟ neurons , co - expressing the AP - 2μ - mRFP ( WT : 37 . 60 ± 2 . 20 % , KO : 25 . 61 ± 2 . 67 % , WTAP - 2μ - mRFP : 45 . 04 ± 3 . 27 % , KOAP - 2μ - mRFP : 40 . 07 ± 2 . 37 % , pWT vs KO = 0 . 005 , pKO vs KO + AP - 2μ - mRFP < 0 . 000 , pWT vs WT + AP - 2μ - mRFP = 0 . 186 , pWT + AP - 2μ - mRFP vs KO + AP - 2μ - mRFP = 0 . 570 , 43 - 53 neurons per condition , N = 4 biological replicates ) . Data information : All graphs show mean ± SEM , statistical analysis was performed by two - way ANOVA in ( G ) . n . s . - non - significant . * indicates p ≤ 0 . 05 , * * indicates p ≤ 0 . 01 , * * * indicates p ≤ 0 . 001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Bi-directional mobility of BACE1 carriers in WT and AP-2μ ΚΟ neurons, co-expressing the AP-2μ-mRFP (WT: 37.60±2.20%, KO: 25.61±2.67%, WTAP-2μ-mRFP: 45.04±3.27%, KOAP-2μ-mRFP: 40.07±2.37%, pWT vs KO=0.005, pKO vs KO+AP-2μ-mRFP<0.000, pWT vs WT+AP-2μ-mRFP=0.186, pWT+AP-2μ-mRFP vs KO+AP-2μ-mRFP=0.570, 43-53 neurons per condition, N=4 biological replicates). Data information: All graphs show mean ± SEM, statistical analysis was performed by two-way ANOVA in (G). n.s.-non-significant. * indicates p≤0.05, ** indicates p≤0.01, *** indicates p≤0.001.


( H ) Representative kymographs of WT and AP - 2μ KO neurons co - expressing the HA - BACE1 - GFP and tagRFP - LC3B . Scale bars : x = 2μm , y = 5s .

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

(H) Representative kymographs of WT and AP-2μ KO neurons co-expressing the HA-BACE1-GFP and tagRFP-LC3B. Scale bars: x=2μm, y=5s.


( I ) Reduced bi - directional mobility of BACE1 - containing autophagosomes in AP - 2µ KO neurons . ( WT : 33 . 53 ± 4 . 19 % , KO : 19 . 45 ± 5 . 14 % , p = 0 . 000 , WT = 37 and KO = 35 neurons , N = 4 biological replicates ) . Data information : All graphs show mean ± SEM , statistical analysis was performed by unpaired two - tailed Student ' s t ‐ test n . s . - non - significant . * indicates p ≤ 0 . 05 , * * indicates p ≤ 0 . 01 , * * * indicates p ≤ 0 . 001 .

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(I) Reduced bi-directional mobility of BACE1-containing autophagosomes in AP-2µ KO neurons. (WT: 33.53±4.19%, KO: 19.45±5.14%, p=0.000, WT=37 and KO=35 neurons, N=4 biological replicates). Data information: All graphs show mean ± SEM, statistical analysis was performed by unpaired two-tailed Student's t‐test n.s.-non-significant. * indicates p≤0.05, ** indicates p≤0.01, *** indicates p≤0.001.


( J ) Representative kymographs of WT neurons co - expressing HA - BACE1 - GFP and AP2 - αΑ WT or AP2 - αΑ Μut . Scale bar : 5µm top panels , x = 2μm , y = 5s bottom panels .

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(J) Representative kymographs of WT neurons co-expressing HA-BACE1-GFP and AP2-αΑ WT or AP2-αΑ Μut. Scale bar: 5µm top panels, x=2μm, y=5s bottom panels.