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 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 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 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

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

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 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

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 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

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 B-MEASURED_VAR 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

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 B-MEASURED_VAR 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

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

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 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

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 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 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 B-MEASURED_VAR 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

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 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

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 B-MEASURED_VAR 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

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 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

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

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 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 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

(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 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) 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 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 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

(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 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 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

(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 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 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 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 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 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 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 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 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 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 .

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 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 B-MEASURED_VAR 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 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 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 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 B-MEASURED_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 B-MEASURED_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 O 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 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 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 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 B-MEASURED_VAR 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 O O O O O O O O O O O O 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 .

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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 and 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 ) 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 B-MEASURED_VAR 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 B-MEASURED_VAR O O B-MEASURED_VAR 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 B-MEASURED_VAR 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 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 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 B-MEASURED_VAR 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 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 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 B-MEASURED_VAR O B-MEASURED_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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 .

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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 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 .

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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 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 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 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 .

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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 ) 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 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 O O O O O O O O O O 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 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 B-MEASURED_VAR 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 .

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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 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 .

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

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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 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 B-MEASURED_VAR O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR 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 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 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 .

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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 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.


( 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 .

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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 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 .

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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 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 .

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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 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 .

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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 ) 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 .

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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 images of WT and AP - 2μ ΚΟ entorhinal cortex immunostained with antibody recognizing total APP and 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 images of WT and AP - 2μ ΚΟ entorhinal cortex immunostained with antibody recognizing total APP and 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 ) 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 .

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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 ) 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 .

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

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


( 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 .

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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.


Intracellular levels of Aβ1 - 42 are significantly increased in AP - 2µ KO axons ( WTAβ1 - 42 : 0 . 65 ± 0 . 08 , KOAβ1 - 42 : 0 . 91 ± 0 . 10 , p = 0 . 046 , WT = 38 and KO = 38 neurons , N = 3 biological replicates ) . Scale bar : 5 µm .

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Intracellular levels of Aβ1-42 are significantly increased in AP-2µ KO axons (WTAβ1-42: 0.65±0.08, KOAβ1-42: 0.91±0.10, p=0.046, WT=38 and KO=38 neurons, N=3 biological replicates). Scale bar: 5 µm.


Intracellular levels of Aβ1 - 42 are significantly increased in AP - 2µ KO axons ( WTAβ1 - 42 : 0 . 65 ± 0 . 08 , KOAβ1 - 42 : 0 . 91 ± 0 . 10 , p = 0 . 046 , WT = 38 and KO = 38 neurons , N = 3 biological replicates ) . Scale bar : 5 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 .

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Intracellular levels of Aβ1-42 are significantly increased in AP-2µ KO axons (WTAβ1-42: 0.65±0.08, KOAβ1-42: 0.91±0.10, p=0.046, WT=38 and KO=38 neurons, N=3 biological replicates). Scale bar: 5 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.


( N ) Significantly increased ratio of Αβ1 - 42 / Αβ1 - 40 measured by ELISA in the media of cultured AP - 2μ KO neurons compared to the WT set to 100 % ( KO : 124 . 57 ± 10 . 97 % , p = 0 . 037 , N = 6 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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(N) Significantly increased ratio of Αβ1-42/ Αβ1-40 measured by ELISA in the media of cultured AP-2μ KO neurons compared to the WT set to 100% (KO:124.57±10.97%, p=0.037, N=6 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.


Accumulation of intracellular Aβ1 − 42 in neurons expressing the AP2 - α − WT ( WT : 1 . 14 ± 0 . 18 , WT = 28 and Mut = 33 neurons , N = 4 biological replicates ) . Scale bars : 20µm , 2µm ( inserts ) .

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Accumulation of intracellular Aβ1−42 in neurons expressing the AP2-α−WT (WT: 1.14±0.18 , WT=28 and Mut=33 neurons, N=4 biological replicates). Scale bars: 20µm, 2µm (inserts).


Accumulation of intracellular Aβ1 − 42 in neurons overexpressing the AP2 - α - Mut comparing to neurons expressing the AP2 - α − WT ( WT : 1 . 14 ± 0 . 18 , Mut : 2 . 06 ± 0 . 29 , p = 0 . 011 , WT = 28 and Mut = 33 neurons , N = 4 biological replicates ) . Scale bars : 20µm , 2µm ( inserts ) . 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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Accumulation of intracellular Aβ1−42 in neurons overexpressing the AP2-α-Mut comparing to neurons expressing the AP2-α−WT (WT: 1.14±0.18, Mut: 2.06±0.29, p=0.011, WT=28 and Mut=33 neurons, N=4 biological replicates). Scale bars: 20µm, 2µm (inserts). 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.


Accumulation of intracellular Aβ1 - 42 is rescued upon BACE1 knock - down ( WTScr : 2 . 76 ± 0 . 20 WTshBACE1 : 2 . 52 ± 0 . 12 27 to 39 neurons per condition , N = 3 biological replicates ) . Scale bar : 10µm .

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Accumulation of intracellular Aβ1-42 is rescued upon BACE1 knock-down (WTScr: 2.76±0.20 WTshBACE1: 2.52±0.12 27 to 39 neurons per condition, N=3 biological replicates). Scale bar: 10µm.


Accumulation of intracellular Aβ1 - 42 is rescued upon BACE1 knock - down in AP - 2µ KO neurons ( WTScr : 2 . 76 ± 0 . 20 , KOScr : 3 . 94 ± 0 . 21 ; WTshBACE1 : 2 . 52 ± 0 . 12 ; KOshBACE1 : 2 . 74 ± 0 . 21 ; pWTScr vs pKOScr = 0 . 000 , pKOscr vs pKOshBACE1 = 000 ; pWTscr vs pWTshBACE1 = 0 . 826 ; pWTshBACE1 vs pKOshBACE1 = 0 . 863 ; 27 to 39 neurons per condition , N = 3 biological replicates ) . Scale bar : 10µm . Data information : All graphs show mean ± SEM , statistical analysis was performed by two - way ANOVA in ( T ) n . s . - non - significant . * indicates p ≤ 0 . 05 , * * indicates p ≤ 0 . 01 , * * * indicates p ≤ 0 . 001 .

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Accumulation of intracellular Aβ1-42 is rescued upon BACE1 knock-down in AP-2µ KO neurons (WTScr: 2.76±0.20, KOScr: 3.94±0.21; WTshBACE1: 2.52±0.12; KOshBACE1: 2.74±0.21; pWTScr vs pKOScr=0.000, pKOscr vs pKOshBACE1=000; pWTscr vs pWTshBACE1=0.826; pWTshBACE1 vs pKOshBACE1=0.863; 27 to 39 neurons per condition, N=3 biological replicates). Scale bar: 10µm. Data information: All graphs show mean ± SEM, statistical analysis was performed by two-way ANOVA in (T) n.s.-non-significant. * indicates p≤0.05, ** indicates p≤0.01, *** indicates p≤0.001.


( U , V ) Levels of intracellular Aβ1 − 42 are significantly upregulated in neurons overexpressing the HA - BACE1 - LL / AA - GFP comparing to neurons expressing the HA - BACE1 - GFP ( BACE1 : 0 . 78 ± 0 . 90 , BACE1 - LL - AA : 1 . 99 ± 0 . 33 , p = 000 , 28 - 29 neurons per condition , N = 3 biological replicates ) . Scale bar , 50µm . 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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(U, V) Levels of intracellular Aβ1−42 are significantly upregulated in neurons overexpressing the HA-BACE1-LL/AA-GFP comparing to neurons expressing the HA-BACE1-GFP (BACE1: 0.78±0.90, BACE1-LL-AA: 1.99±0.33, p=000, 28-29 neurons per condition, N=3 biological replicates). Scale bar, 50µm. 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 ) AP - 2α levels normalized to GAPDH are significantly decreased in lysates of iPSC - derived neurons from late - onset AD patients carrying the TREM2 p . R47H ( AD - TREM2 - 2 and AD - TREM2 - 4 ) variant compared to healthy controls ( CON8 and CON9 ) set to 100 % ( ADAP - 2α : 52 . 42 ± 8 . 04 % , p = 0 . 020 , N = 3 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 .

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(A, B) AP-2α levels normalized to GAPDH are significantly decreased in lysates of iPSC-derived neurons from late-onset AD patients carrying the TREM2 p.R47H (AD-TREM2-2 and AD-TREM2-4) variant compared to healthy controls (CON8 and CON9) set to 100% (ADAP-2α: 52.42±8.04%, p=0.020, N=3 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 ) Confocal images of either AP - 2 WT ( AAV - GFPCamkIIα - labelled ) or AP - 2 KO ( AAV - Cre - GFPCamkIIα - labelled ) granule neurons in the dentate gyrus of 13 - week - old mice . Scale bars : 20µm , 2µm ( inserts ) .

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(C) Confocal images of either AP-2 WT (AAV-GFPCamkIIα-labelled) or AP-2 KO (AAV-Cre-GFPCamkIIα-labelled) granule neurons in the dentate gyrus of 13-week-old mice. Scale bars: 20µm, 2µm (inserts).


( D ) Loss of AP - 2µ significantly decreases the number of spines on granule neurons ( WT : 12 . 63 ± 0 . 88 , KO : 8 . 07 ± 0 . 45 , p = 0 . 004 , 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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(D) Loss of AP-2µ significantly decreases the number of spines on granule neurons (WT: 12.63±0.88, KO: 8.07±0.45, p=0.004, 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 , F ) Overlap coefficient between the presynaptic marker Bassoon and the postsynaptic marker PSD95 is significantly decreased in the entorhinal cortex of AP - 2µ KO mice compared to the WT ( WT : 0 . 45 ± 0 . 02 , KO : 0 . 33 ± 0 . 06 , p = 0 . 034 , N = 3 biological replicates ) . Scale bar , 750nm . 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 B-MEASURED_VAR O O O O B-MEASURED_VAR 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 O O O O O O O O O O O O O O O O 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) Overlap coefficient between the presynaptic marker Bassoon and the postsynaptic marker PSD95 is significantly decreased in the entorhinal cortex of AP-2µ KO mice compared to the WT (WT: 0.45±0.02, KO: 0.33±0.06, p=0.034, N=3 biological replicates). Scale bar, 750nm. 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.


Lost preference for the novel object ( nov ) comparing to the familiar object ( fam ) in AP - 2µ KO mice tested 24h after training in NOR Representative heatmaps are shown in ( G ) .

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

Lost preference for the novel object (nov) comparing to the familiar object (fam) in AP-2µ KO mice tested 24h after training in NOR Representative heatmaps are shown in (G).


Lost preference for the novel object ( nov ) comparing to the familiar object ( fam ) in AP - 2µ KO mice tested 24h after training in NOR ( WTfam : 27 . 51 ± 6 . 77 , WTnov : 72 . 49 ± 6 . 77 , pWTfam vs WTnov = 0 . 000 ; KOfam : 43 . 9 ± 13 . 12 , KOnov : 59 . 91 ± 13 . 12 , pKOfam vs KOnov = 0 . 469 , N = 7 WT and 8 KO mice ) . 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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Lost preference for the novel object (nov) comparing to the familiar object (fam) in AP-2µ KO mice tested 24h after training in NOR (WTfam: 27.51±6.77, WTnov: 72.49±6.77, pWTfam vs WTnov=0.000; KOfam: 43.9±13.12, KOnov: 59.91±13.12, pKOfam vs KOnov=0.469, N=7 WT and 8 KO mice). 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 ) AP - 2µ KO mice are significantly more active in the open field compared to the WT ( WT : 1663 ± 139 . 08 % , KO : 3976 ± 802 . 30 % , p = 0 . 020 , N = 7 WT and 8 KO mice ) . Representative heatmaps are shown in ( I ) . 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 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 O O O O O O O O O O O O O O O O O O O O O

(I, J) AP-2µ KO mice are significantly more active in the open field compared to the WT (WT: 1663±139.08%, KO: 3976±802.30%, p=0.020, N=7 WT and 8 KO mice). Representative heatmaps are shown in (I). 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 ) Overlap coefficient between Bassoon and PSD95 is rescued upon BACE1 KD in AP - 2µ KO neurons ( WTscr : 0 . 39 ± 0 . 02 ; KOscr : 0 . 24 ± 0 . 01 ; WTshBACE1 : 0 . 37 ± 0 . 02 ; KOshBACE1 : 0 . 33 ± 0 . 02 ; pWTscr vs pKOscr < 0 . 000 ; pKOscr vs pKOshBACE1 = 0 . 010 ; pWTscr vs pWTshBACE1 = 0 . 813 ; pWTshBACE1 vs pKOshBACE1 = 0 . 623 , 22 - 24 neurons for each condition , N = 3 ) . Scale bars : 10µm . 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 .

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(K, L) Overlap coefficient between Bassoon and PSD95 is rescued upon BACE1 KD in AP-2µ KO neurons (WTscr: 0.39±0.02; KOscr: 0.24±0.01; WTshBACE1: 0.37±0.02; KOshBACE1: 0.33±0.02; pWTscr vs pKOscr<0.000; pKOscr vs pKOshBACE1=0.010; pWTscr vs pWTshBACE1=0.813; pWTshBACE1 vs pKOshBACE1=0.623, 22-24 neurons for each condition, N=3). Scale bars: 10µm. 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.


Neuronal loss is rescued in the entorhinal cortex of AP - 2µ mice haploinsufficient for BACE1 ( AP - 2WT / BACEWT : 2 . 25 ± 0 . 08 N = 9 sections from N = 3 for each genotype ) . Scale bar : 100 μm .

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Neuronal loss is rescued in the entorhinal cortex of AP-2µ mice haploinsufficient for BACE1 (AP-2WT/BACEWT: 2.25±0.08 N=9 sections from N=3 for each genotype). Scale bar: 100 μm.