( g , h ) Lungneutrophil infiltration ( g ) in IAV - infected Ripk2 − / − mice treated with rapamycin , Ripk2 − / − mice WT mice ( t . p . , total ) . * P 0 . 05 , * * P 0 . 01 , * * * P 0 . 001 ( Mann - Whitney ( b ) ; one - way ANOVA with Tukey ' s post - hoc ( 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 IFN - γ amounts ( h ) in IAV - infected Ripk2 − / − mice treated with rapamycin , Ripk2 − / − mice WT mice ( t . p . , total ) . * P 0 . 05 , * * P 0 . 01 , * * * P 0 . 001 ( Mann - Whitney ( b ) ; one - way ANOVA with Tukey ' s post - hoc ( 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 of wild - type ( WT ) Ripk2 − / − BMDCs stained with mitochondrial superoxide - specific stain MitoSOX during IAV or Listeria infection ( a , b ) with 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 ) of IL - 18 production immunoblotting to analyze caspase - 1 ( casp - 1 ) p20 activation in IAV - infected BMDCs that had been pretreated with 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 lysates of IAV - infected BMDCs pretreated with 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 of BMDCs stained for mitochondria ( red ) 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 ( d ) unpaired two - sided Student ' s t - ( 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 ) , caspase - 1 ( casp - 1 ) p20 LC3 - II immunoblots ( b ) in PR8 - infected wild - type ( WT ) BMDCs treated with p38 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 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 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 of MitoSOX in PR8 - infected WT Ulk1 − / − BMDCs .

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


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

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


Effects of basic bulky hydrophobic on LT - induced pyroptosis caspase - 1 activation in RAWRA . were pre - treated with 10 mM indicated prior to LT stimulation . viability was measured by using ATP 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.


supernatants were subjected to anti - caspase - 1 immunoblotting lysates were blotted with 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 bulky hydrophobic on LT - induced RFP - ASC specks formation in RAWRA . were taken on a confocal microscopy ( C ) . percentages of 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 bulky hydrophobic on LT - induced caspase - 1 activation in Tlr4 - / - iBMDMs . were treated as that in ( A ) . supernatants were subjected to anti - caspase - 1 immunoblotting lysates were blotted with 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 of effect of Ubr2 knockdown on LT - induced inflammasome activation . RAWRA were transfected with a control siRNA or Ubr2 - targeting siRNA mixtures for 60 h followed by LT treatment . numbers in merged panel are percentages of showing RFP - ASC specks .

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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 . 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 . 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 Ubr2 - / - iBMDMs were treated with LT or BsaK for 3 h . viability was measured by using ATP 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 129 / Sv mice . were transfected with indicated siRNA for 60 h , 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 129 / Sv mice .

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


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

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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 of effect of Ube2o knockdown ( siRNA Ube2o - 01 ) on LT - induced inflammasome activation in RAWRA . percentages of 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 UBR2 . Myc - UBE2O Flag - UBR2 were co - expressed in 293T 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 level . 293T were transfected with Flag - NLRP1B expression plasmid for 24 h , were then treated with WT LT ( + ) or its E687C mutant ( - ) for 4 h . Shown is anti - Flag immunoblot of total 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 expressing an indicated NLRP1B variant . were treated with LT for 3 h . Scale bar , 20 μm . 1 - 983 984 - C ( residues 984 to C terminus ) are 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 expressing NLRP1B ( 1 - 983 ) - HA were treated with LT MG132 for 8 h . Shown are anti - HA anti - tubulin immunoblots of total 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 Leu on LT - induced NLRP1B degradation . 293T expressing NLRP1B ( 1 - 983 ) - HA were treated with LT MG132 for 8 h . 10 mM Leu was added to 30 min prior to LT treatment . Shown are anti - HA anti - tubulin immunoblots of total 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 expressing NLRP1B ( 1 - 983 ) - HA were treated with LT / or MG132 for 6 h . were then harvested for anti - HA immunoprecipitation 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 of spheroids treated with dmPGE2 . Scale bars , 200 µm .

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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 ) of average spheroid ± s . e . m . relative to spheroids treated with 0 nM dmPGE2 ( average was 3761 µm2 for 0 nM group ; n = 4 from two independent experiments ) .

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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 stained for hematoxylin . Scale bars , 100 µm .

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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 ) of average expression ± s . e . m . of Cldn4mRNA relative to 0 nM treatment group ( n = 3 independent experiments ) .

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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 ) of average spheroid ± s . e . m . relative to spheroids treated with 0 nM Iloprost ( n = 4 from two independent experiments ) .

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

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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 pharmacological inhibitors of EP1 ( EP1i , SC 51322 ) , EP2 ( EP2i , PF 04418948 ) , EP3 ( EP3i , L - 798 , 106 ) or EP4 ( EP4i , L - 161 , 982 ) at a of 10 µM or an equivalent volume of DMSO vehicle . * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 compared to DMSO only group by one - way ANOVA Dunnett ' s post - . ( B ) Representative bright field . Scale bars , 200 µm .

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

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

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

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

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

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 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 Tukey ' s post - . ( G ) Representative bright - field . Scale bars , 200 µm . ( H ) of average spheroid ± s . e . m . ( n = 4 per group from two independent experiments ) relative to EP4i - treated Ptger4flox / flox spheroids .

O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O 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 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 Tukey ' s post - . ( I ) average expression ± s . e . m . of Cldn4 mRNA ( n = 3 independent experiments ) relative to EP4i - treated Ptger4flox / flox spheroids .

O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR 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 B-MEASURED_VAR O O O O O O O O O O O O O B-CONTROLLED_VAR 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 Tukey ' s post - . ( J ) Representative bright - field . Scale bars , 200 µm . ( K ) of average spheroid ± s . e . m . ( n = 4 per group from 3 independent donor lines examined over 3 passages each ) relative to spheroids .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Tukey ' s post - . ( L ) average expression ± s . e . m . of CLDN4 mRNA ( n = 3 independent donor lines ) relative to spheroids .

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

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

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 with EdU to mark undergoing synthesis . ( E ) Representative of EdU ( red ) . Nuclei are visualized with bisbenzimide ( blue ) . Scale bars , 20 µm . ( F ) of EdU - positive nuclei as a of total nuclei ( n = minimum of 15 spheroids counted per sample from 2 independent experiments ) . * * * * p < 0 . 0001 by 1 - way ANOVA Tukey ' s post - .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 - EP4i - treated spheroids compared to by repeated measures 2 - way ANOVA ( variable = treatment ) . p < 0 . 001 at 16 hr , 20 hr 24 hr time points by Dunnett ' s post - comparing dmPGE2 - treated EP4i - treated spheroids to 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 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

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

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

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 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 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

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 . of average expression ± s . e . m . of Dpcr1 ( E ) Cd55b mRNAs relative to DMSO group ( n = 3 independent experiments ) . * p < 0 . 05 , * * * * p < 0 . 0001 by one - way ANOVA Dunnett ' s post - .

O 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 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

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 ( ) of spheroids or an in vivo WAE from a biopsy - injured mousecolon . ( G ) basal plasma membranes are outlined in orange solid lines , lateral plasma membranes are indicated with orange arrowheads , nuclei are outlined with wide yellow dashed lines . Insets show a magnified view of apical 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

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 ( ) of spheroids or an in vivo WAE from a biopsy - injured mousecolon . of : ( H ) microvillar ( I ) from ( n = minimum of 5 per group ) . * p < 0 . 05 , * * p < 0 . 01 , * * * * p < 0 . 0001 by 1 - way ANOVA Tukey ' s post - .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 ( ) of spheroids or an in vivo WAE from a biopsy - injured mousecolon . ( J ) Higher power image of cytoplasm . Single mitochondria are outlined with a narrow blue dashed , 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

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 ) F - actin ( red ) . Nuclei are visualized with bisbenzimide ( blue ) . A similarly stained section of a small intestinal ulcer is shown for comparison . Arrowheads indicate apical . Scale bars , 50 µm .

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

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 ) F - actin ( red ) . Nuclei are visualized with bisbenzimide ( blue ) . A similarly stained section of a mouse villus is shown for comparison . Arrowheads indicate apical . Scale bars , 50 µm .

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

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 ) of average expression ± s . e . m . of Fabp1 , Ace2 , Maoa mRNAs ( n = 3 independent experiments ) . * p < 0 . 05 , * * p < 0 . 01 by one - way ANOVA Tukey ' s post - .

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 B-MEASURED_VAR 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

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 of spheroids stained for Ace2 ( green ) 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 B-MEASURED_VAR O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR 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 of EP4i - treated spheroids . ( F ) basal plasma membranes are outlined in orange solid lines , lateral plasma membranes are indicated with orange arrowheads , nuclei are outlined with wide yellow dashed lines . Insets show a magnified view of apical surface . ( G ) Higher power image of cytoplasm . Single mitochondria are outlined with a narrow blue dashed , 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

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 ) expressed as mean ± s . e . m . ( n = 3 independent experiments ) . * * p < 0 . 01by 1 - way ANOVA Tukey ' s post - .

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

O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Ptger4Δ / Δspheroids 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 Tukey ' s post - comparing genotypes within a treatment group . † † † p < 0 . 001 , † † † † p < 0 . 0001 by two - way ANOVA Sidak ' s post - comparing effects of treatments within a genotype .

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

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 . Scale bars , 200 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

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 ) of average expression ± s . e . m . of Cldn4 mRNA relative to DMSO treatment group ( n = 3 independent experiments ) . * p < 0 . 05 compared to DMSO group by unpaired t .

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

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 ( Nuc ) ( Cyt ) lysates . Lamin A / C Actin were used as loading controls for fractions , respectively ( n = 3 independent experiments ) . ( C ) Spheroids were cultured in , 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 B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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 indicated concentrations of CHIR 99021 . of average expression ± s . e . m . of Cldn4 ( E ) 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 Dunnett ' s post - .

O O O 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 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

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 of Ptger4Δ / Δspheroids cultured in differentiation medium with DMSO or 10 µM CHIR 99021 stained for β - catenin ( green ) 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 O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O

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 treated with conditioned mediums collected from L - WRN diluted as indicated . L - WRN CM had been produced in presence or absence of porcupine inhibitor C59 ( 10 - fold dilutions , 10 µM to 1 pM ) . * p < 0 . 05 , * * * p < 0 . 001 , * * * * p < 0 . 0001 by 1 - way ANOVA Dunnett ' s post - 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 . of average expression ± s . e . m . of Cldn4 ( I ) 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 Tukey ' s post - .

O O O O O O O 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 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

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

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

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 of wounds from Ptger4flox / flox ( Ptger4fl / fl ) Villin - Cre Ptger4flox / flox ( VilCrePtger4fl / fl ) mice four days post - biopsy injury . Scale bars , 750 µm . Wounds are outlined with a black dashed . Asterisk indicates fibrin clot . B , C of healing ( 1 - [ Day 4 wound / original wound ] x 100 ) ( B ) 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 - unpaired t - .

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

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

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

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

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 ) 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 29 KO neurons ) . Data information : graphs show mean ± , statistical was performed by unpaired two - Student ' s t ‐ n . s . - non - significant . * indicates p ≤ 0 . 05 , * * indicates p ≤ 0 . 01 , * * * indicates p ≤ 0 . 001 .

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 B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 in HA - BACE1 - eGFP - overexpressing WT KO neurons . Scale bar : 5µm .

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

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

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 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

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

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 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

(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 of BACE1 after 20 min of HA antibody in HA - BACE1 - mCherry - transfected WT 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 top panels , where analyzed BACE1 fraction is marked with red square .

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 B-CONTROLLED_VAR 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 B-MEASURED_VAR 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 of BACE1 after 20 min of HA antibody in HA - BACE1 - mCherry - transfected WT KO neurons , co - expressing either eGFP - RAB4 or eGFP - RAB4 - S22N , calculated as HA ( recycled ) / mCherry ( total ) signal ( 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 : graphs show mean ± , statistical 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 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 B-CONTROLLED_VAR 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 O O O O O O O O O O O O O O O O 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 of BACE1 after 20 min of HA antibody chase in HA - BACE1 - mCherry - transfected WT KO neurons , co - expressing either eGFP - RAB11 or eGFP - RAB11 - S22N , calculated as HA ( recycled ) / mCherry ( total ) signal ( 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 : graphs show mean ± , statistical was performed by two - way ANOVA n . s . - non - significant . * indicates p ≤ 0 . 05 , * * indicates p ≤ 0 . 01 , * * * indicates p ≤ 0 . 001 .

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 B-CONTROLLED_VAR 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 O O O O O O O O O O O O O O O O O O O O O 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 of BACE1 after 20 minutes of HA antibody uptake in HA - BACE1 - mCherry transfected WT 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 top panels where analyzed BACE1 fraction is marked with red square .

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 B-CONTROLLED_VAR 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 B-MEASURED_VAR 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 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 of BACE1 after 20 minutes of HA antibody uptake in HA - BACE1 - mCherry transfected WT KO neurons , co - expressing either eGFP - RAB4 or eGFP - RAB4 - S22N , calculated as HA ( internalized ) / mCherry ( total ) signal ( 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 : graphs show mean ± , statistical was performed by 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, 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 are increased in AP - 2µ KO neurons , when compared to WT set to 100 % ( KO : 129 . 65 ± 5 . 30 % , p = 0 . 005 , N = 4 biological replicates ) . ( N ) Surface versus total BACE1 is not altered in AP - 2µ KO neurons when normalized to WT set to 100 % ( KO : 83 . 02 ± 10 . 93 % , p = 0 . 109 , N = 4 biological replicates ) . Data information : graphs show mean ± , statistical was performed by one - sample Student ' s t ‐ in ( M , N ) . n . s . - non - significant . * indicates p ≤ 0 . 05 , * * indicates p ≤ 0 . 01 , * * * indicates p ≤ 0 . 001 .

O O O O O B-MEASURED_VAR O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 54 KO neurons , N = 4 biological replicates ) . Scale bar : 10 µm . Arrows indicate BACE1 accumulation within RAB5 - positive endosomes . Data information graphs show mean ± , statistical was performed by unpaired two - Student ' s t ‐ n . s . - non - significant . * indicates p ≤ 0 . 05 , * * indicates p ≤ 0 . 01 , * * * indicates p ≤ 0 . 001 .

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

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

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(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 AP - 2µ KO neurons . Scale bars : 20 µm , 5 µm ( inserts ) .

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


( F ) plot of BACE1 immunofluorescence along

(F) Line plot of BACE1 immunofluorescence along the neurites


of BACE1 colocalizing with 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 .

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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.


of BACE1 colocalizing with 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 : graphs show mean ± , statistical was performed by unpaired two - Student ' s t ‐ n . s . - non - significant . * indicates p ≤ 0 . 05 , * * indicates p ≤ 0 . 01 , * * * indicates p ≤ 0 . 001 .

O B-MEASURED_VAR 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 B-MEASURED_VAR 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

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

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(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 RAB7A in cortical sections of WT 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 : graphs show mean ± , statistical was performed by unpaired two - Student ' s t ‐ n . s . - non - significant . * indicates p ≤ 0 . 05 , * * indicates p ≤ 0 . 01 , * * * indicates p ≤ 0 . 001 .

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

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

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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 44 KO neurons , N = 4 biological replicates ) . Data information : graphs show mean ± , statistical was performed by unpaired two - Student ' s t ‐ n . s . - non - significant . * indicates p ≤ 0 . 05 , * * indicates p ≤ 0 . 01 , * * * indicates p ≤ 0 . 001 .

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(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 of WT AP - 2μ KO neurons transfected with BACE1 - mKeima - Red co - transfected with eBFP to reveal neuronal . Scale bars : 10µm , 2µm in zoomed .

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

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(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 ( A ) corresponding kymographs ( B ) from time - lapse videos of control neurons transfected with HA - BACE1 - eGFP 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 same arrowheads in ( B ) .

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

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(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 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 : graphs show mean ± , statistical was performed by unpaired two - Student ' s t ‐ n . s . - non - significant . * indicates p ≤ 0 . 05 , * * indicates p ≤ 0 . 01 , * * * indicates p ≤ 0 . 001 .

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(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 anterograde 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 KO = 32 neurons , N = 4 biological replicates ) . Data information : graphs show mean ± , statistical was performed by unpaired two - Student ' s t ‐ n . s . - non - significant . * indicates p ≤ 0 . 05 , * * indicates p ≤ 0 . 01 , * * * indicates p ≤ 0 . 001 .

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

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(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 AP - 2μ ΚΟ neurons , co - expressing 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 : graphs show mean ± , statistical 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 B-MEASURED_VAR O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR 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 B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 AP - 2μ KO neurons co - expressing HA - BACE1 - GFP tagRFP - LC3B . Scale bars : x = 2μm , y = 5s .

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(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 KO = 35 neurons , N = 4 biological replicates ) . Data information : graphs show mean ± , statistical was performed by unpaired two - Student ' s t ‐ 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 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.


( K ) Mobility of BACE1 carriers is significantly decreased in neurons overexpressing AP2 - αΑ Mut compared to neurons expressing AP2 - αΑ WT ( WT : 52 . 23 ± 4 . 16 % , Mut : 27 . 95 ± 3 . 31 , p = < 0 . 000 , WT = 26 Mut = 29 neurons , N = 4 biological replicates ) . Data information : graphs show mean ± , statistical was performed by unpaired two - Student ' s t ‐ n . s . - non - significant . * indicates p ≤ 0 . 05 , * * indicates p ≤ 0 . 01 , * * * indicates p ≤ 0 . 001 .

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(K) Mobility of BACE1 carriers is significantly decreased in neurons overexpressing the AP2-αΑ Mut compared to neurons expressing the AP2-αΑ WT (WT: 52.23±4.16%, Mut: 27.95±3.31, p=<0.000, WT=26 and Mut=29 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.


( L ) Representative corresponding kymographs from time - lapse videos of WT neurons transfected with either HA - BACE1 - GFP or HA - BACE1 - LL / AA - GFP . Scale bar : 5µm top panels , x = 2μm , y = 5s bottom panels .

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(L) Representative fluorescence images and corresponding kymographs from time-lapse videos of WT neurons transfected with either HA-BACE1-GFP or HA-BACE1-LL/AA-GFP. Scale bar: 5µm top panels, x=2μm, y=5s bottom panels.


( M ) Reduced mobility of BACE1 - LL / AA - positive vesicles , when compared to WT BACE1 ( WT : 38 . 97 ± 1 . 40 % , LL / AA : 19 . 89 ± 1 . 77 % , p < 0 . 000 , WT = 31 LL / AA = 33 neurons , N = 4 biological replicates ) . Data information : graphs show mean ± , statistical was performed by unpaired two - Student ' s t ‐ n . s . - non - significant . * indicates p ≤ 0 . 05 , * * indicates p ≤ 0 . 01 , * * * indicates p ≤ 0 . 001 .

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(M) Reduced mobility of BACE1-LL/AA-positive vesicles, when compared to WT BACE1 (WT: 38.97±1.40%, LL/AA: 19.89±1.77%, p<0.000, WT=31 and LL/AA=33 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.


( N ) Reduced velocity of BACE1 - LL / AA - positive vesicles , when compared to WT BACE1 ( WTAntero : 0 . 38 ± 0 . 03 μm / s , LL / AAAntero : 0 . 15 ± 0 . 03 μm / s , pWTAntero vs pLL / AAAntero < 0 . 000 ; WTRetro : 0 . 39 ± 0 . 03 μm / s , LL / AARetro : 0 . 25 ± . 03 μm / s , pWTRetro vs pLL / AARetro = 0 . 000 , WT = 31 LL / AA = 33 neurons , N = 4 biological replicates ) . Data information : graphs show mean ± , statistical was performed by unpaired two - Student ' s t ‐ n . s . - non - significant . * indicates p ≤ 0 . 05 , * * indicates p ≤ 0 . 01 , * * * indicates p ≤ 0 . 001 .

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(N) Reduced velocity of BACE1-LL/AA-positive vesicles, when compared to WT BACE1 (WTAntero: 0.38±0.03 μm/s, LL/AAAntero: 0.15±0.03 μm/s, pWTAntero vs pLL/AAAntero<0.000; WTRetro: 0.39±0.03 μm/s, LL/AARetro: 0.25±.03 μm/s, pWTRetro vs pLL/AARetro =0.000, WT=31 and LL/AA=33 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.


( A , B ) eGFP / mCherry signal is significantly increased in mCherry - APP - P1 - eGFP - expressing KO neurons comparing to WT ( WT : 0 . 94 ± 0 . 06 % , KO : 1 . 65 ± 0 . 18 , p = 0 . 000 , WT = 39 KO = 41 neurons , N = 5 biological replicates ) . Scale bars : 2μm . Data information : graphs show mean ± , statistical was performed by unpaired two - Student ' s t ‐ n . s . - non - significant . * indicates p ≤ 0 . 05 , * * indicates p ≤ 0 . 01 , * * * indicates p ≤ 0 . 001 .

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(A, B) The eGFP/mCherry signal intensity ratio is significantly increased in mCherry-APP-P1-eGFP-expressing KO neurons comparing to the WT (WT: 0.94±0.06%, KO: 1.65±0.18, p=0.000, WT=39 and KO=41 neurons, N=5 biological replicates). Scale bars: 2μm. 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.


Confocal of WT AP - 2μ ΚΟ entorhinal cortex immunostained with antibody recognizing total APP APP - CTFs . Scale bars : 100 µm ( C )

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Confocal images of WT and AP-2μ ΚΟ entorhinal cortex immunostained with antibody recognizing total APP and APP-CTFs. Scale bars: 100 µm (C)


Confocal of WT AP - 2μ ΚΟ entorhinal cortex immunostained with antibody recognizing total APP APP - CTFs . Scale bars : 20µm ( D ) .

Confocal images of WT and AP-2μ ΚΟ entorhinal cortex immunostained with antibody recognizing total APP and APP-CTFs. Scale bars: 20µm (D).


( E - G ) of APP - CTFs are significantly increased in AP - 2µ KO cortex compared to WT set to 100 % ( KOC99 : 156 . 65 ± 8 . 37 % , p = 0 . 004 , N = 4 biological replicates ; KOC83 : 154 . 79 ± 15 . 6 % , p = 0 . 012 ; N = 5 biological replicates ) . Data information : graphs show mean ± , statistical was performed by one - sample Student ' s t ‐ n . s . - non - significant . * indicates p ≤ 0 . 05 , * * indicates p ≤ 0 . 01 , * * * indicates p ≤ 0 . 001 .

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(E-G) Levels of APP-CTFs are significantly increased in AP-2µ KO cortex compared to the WT set to 100% (KOC99: 156.65±8.37%, p=0.004, N=4 biological replicates; KOC83: 154.79±15.6%, p=0.012; N=5 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.


( H ) of full - ( FL ) APP are unaltered in AP - 2µ KO cortex ( KOAPP FL : 106 . 64 ± 12 . 96 % , p = 0 . 315 , N = 6 biological replicates ) . Data information : graphs show mean ± , statistical was performed by one - sample Student ' s t ‐ n . s . - non - significant . * indicates p ≤ 0 . 05 , * * indicates p ≤ 0 . 01 , * * * indicates p ≤ 0 . 001 .

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(H) Levels of full-length (FL) APP are unaltered in AP-2µ KO cortex (KOAPP FL: 106.64±12.96%, p=0.315, N=6 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.


( I ) of Nicastrin ( NCT ) are unaltered in AP - 2µ KO

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(I) Levels of Nicastrin (NCT) are unaltered in the AP-2µ KO cortex


( J , K ) Intracellular of Aβ1 - 40 are significantly increased in AP - 2µ KO axons compared to WT ( WTAβ1 - 40 : 1 . 82 ± 0 . 12 , KOAβ1 - 40 : 2 . 45 ± 0 . 12 , p = 0 . 000 , WT = 24 KO = 24 neurons , N = 3 biological replicates ) . Scale bars , 5µm . Data information : graphs show mean ± , statistical was performed by unpaired two - Student ' s t ‐ n . s . - non - significant . * indicates p ≤ 0 . 05 , * * indicates p ≤ 0 . 01 , * * * indicates p ≤ 0 . 001 .

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(J, K) Intracellular levels of Aβ1-40 are significantly increased in AP-2µ KO axons compared to the WT (WTAβ1-40: 1.82±0.12, KOAβ1-40: 2.45±0.12, p=0.000, WT=24 and KO=24 neurons, N=3 biological replicates). Scale bars, 5µm. 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.