Composite profile of Rrm3 occupancy across the average ORF plotted as Rrm3 percentage of significant ChIP hits per segment . Each gene was divided into 10 equivalent segments from the start and end coordinates ( segments 2 - 10 ) . Two additional segments of the same size were considered upstream ( 5 ′ ) and downstream ( 3 ′ ) of each ORF ( segments 1 and 12 , respectively ) . The average signal log2 ratio for Rrm3 significant hits that map on each segment was plotted .

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

Composite profile of Rrm3 occupancy across the average ORF plotted as Rrm3 percentage of significant ChIP hits per segment. Each gene was divided into 10 equivalent segments from the start and end coordinates (segments 2-10). Two additional segments of the same size were considered upstream (5′) and downstream (3′) of each ORF (segments 1 and 12, respectively). The average signal log2 ratio for Rrm3 significant hits that map on each segment was plotted.


Composite profile of Rrm3 occupancy across the average ARS plotted as Rrm3 percentage of significant ChIP hits per segment . Each ARS was divided into 11 equivalent segments from the start and end coordinates . The average signal log2 ratio for Rrm3 significant hits that map on each segment was plotted .

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

Composite profile of Rrm3 occupancy across the average ARS plotted as Rrm3 percentage of significant ChIP hits per segment. Each ARS was divided into 11 equivalent segments from the start and end coordinates. The average signal log2 ratio for Rrm3 significant hits that map on each segment was plotted.


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

O O O O O O O O O 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 .

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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 .

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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 .

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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 .

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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 .

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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 .

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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 .

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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 .

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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 .

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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 .

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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 .

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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 .

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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 .

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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 .

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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 .

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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 .

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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 .

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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 .

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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 .

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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.


B Cryosections were subjected to immunofluorescence microscopy using appropriate excitation wavelengths and emission filters . White boxes indicate the magnified area in the lower panel . All images were taken as " multiple image alignments " . Scale bars represent 100 and 25 µm in the upper and lower panel , respectively .

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B Cryosections were subjected to immunofluorescence microscopy using appropriate excitation wavelengths and emission filters. White boxes indicate the magnified area in the lower panel. All images were taken as "multiple image alignments". Scale bars represent 100 and 25 µm in the upper and lower panel, respectively.


C Whole brain and midbrain lysates of adult mice were prepared as outlined ; . Total protein ( 20 µg ) was immunoblotted for MAD2 or α - Tubulin and compared to HEK - 293 cell lysates .

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C Whole brain and midbrain lysates of adult mice were prepared as outlined ;. Total protein (20 µg) was immunoblotted for MAD2 or α-Tubulin and compared to HEK-293 cell lysates.


D Z - stacks of individual TPH + / MAD2 + positive neurons were generated by confocal microscopy . Intensity profiles of an average projection were generated using ImageJ / Plot Profile software . Scale bar represents 20 µm .

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D Z-stacks of individual TPH+/MAD2+ positive neurons were generated by confocal microscopy. Intensity profiles of an average projection were generated using ImageJ/Plot Profile software. Scale bar represents 20 µm.


B - D GST pull - down experiments were conducted in duplicates using the indicated GST fusion proteins . Eluates ( 30 % ) were subjected to immunoblotting together with an aliquot ( 1 % ) of the input . Blots were stained with Ponceau S and MAD2 was detected by immunoblotting . Densitometric signals were quantified using ImageJ software . Results were compared using unpaired two - tailed t - tests ; * * * * p < 0 . 0001 , ns - not significant ; exact p - values are indicated ; error bars represent SD of 8 independent biological replicates ( n = 8 ) .

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B - D GST pull-down experiments were conducted in duplicates using the indicated GST fusion proteins. Eluates (30%) were subjected to immunoblotting together with an aliquot (1%) of the input. Blots were stained with Ponceau S and MAD2 was detected by immunoblotting. Densitometric signals were quantified using ImageJ software. Results were compared using unpaired two-tailed t-tests; ****p < 0.0001, ns - not significant; exact p-values are indicated; error bars represent SD of 8 independent biological replicates (n = 8).


E Total cell lysates ( 5 µg ) of indicated transfections were subjected to immunoblotting , using antibodies against YFP and MAD2 .

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E Total cell lysates (5 µg) of indicated transfections were subjected to immunoblotting, using antibodies against YFP and MAD2.


YFP - hMAD2 - constructs were generated Whole cell lysates of transfected HEK - 293 cells were used for GST pull - down experiments . One - way ANOVA with Dunnett ' s multiple posthoc comparison ; * * * p < 0 . 001 , ns - not significant ; error bars represent SD of 3 independent biological replicates ( n = 3 ) .

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YFP-hMAD2-constructs were generated Whole cell lysates of transfected HEK-293 cells were used for GST pull-down experiments. One-way ANOVA with Dunnett's multiple posthoc comparison; ***p < 0.001, ns - not significant; error bars represent SD of 3 independent biological replicates (n = 3).


A - B Immunofluorescence of p31comet and BubR1 in mouse dorsal raphe . White boxes indicate the magnified sections in the lower panels . Scale bars represent 100 and 25 µm in the upper and lower panels , respectively .

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A - B Immunofluorescence of p31comet and BubR1 in mouse dorsal raphe. White boxes indicate the magnified sections in the lower panels. Scale bars represent 100 and 25 µm in the upper and lower panels, respectively.


C Co - immunoprecipitation experiments using lysates from HEK - 293 cells stably expressing the indicated YFP - tagged transporters . Eluates ( 20 % ) as well as 0 . 1 % total cell lysate were analyzed by immunoblotting . The immunoblot for mouse anti - β2 - adaptin was subsequently used for incubation with rabbit anti - GFP primary and IRDye 800CW anti - rabbit secondary antibody . IRDye signals were detected using the LI - COR Odyssey CLx imaging system . Densitometric signals were quantified using ImageJ software .

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C Co-immunoprecipitation experiments using lysates from HEK-293 cells stably expressing the indicated YFP-tagged transporters. Eluates (20%) as well as 0.1% total cell lysate were analyzed by immunoblotting. The immunoblot for mouse anti-β2-adaptin was subsequently used for incubation with rabbit anti-GFP primary and IRDye 800CW anti-rabbit secondary antibody. IRDye signals were detected using the LI-COR Odyssey CLx imaging system. Densitometric signals were quantified using ImageJ software.


HEK - 293 cells stably expressing YFP - hSERT were transfected with indicated siRNAs ( " C " - ctrl siRNA ; " M " - MAD2 siRNA ) and subjected to co - immunoprecipitation . All signals were normalized to YFP - SERT . Results were compared using unpaired two - tailed t - tests ; * * * p < 0 . 001 , * * * * p < 0 . 0001 , ns - not significant ; calculated p - values are indicated ; error bars represent SD of 7 ( BubR1 ) , 8 ( β2 - adaptin ) , 5 ( p31comet ) and 8 ( HSP70 ) independent biological replicates .

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HEK-293 cells stably expressing YFP-hSERT were transfected with indicated siRNAs ("C" - ctrl siRNA; "M" - MAD2 siRNA) and subjected to co-immunoprecipitation. All signals were normalized to YFP-SERT. Results were compared using unpaired two-tailed t-tests; ***p < 0.001, ****p < 0.0001, ns - not significant; calculated p-values are indicated; error bars represent SD of 7 (BubR1), 8 (β2-adaptin), 5 (p31comet) and 8 (HSP70) independent biological replicates.


A Radioactive substrate uptake was conducted KM and Vmax were determined by fitting the data to the equation of a rectangular hyperbola . Representative curves are shown . Error bars represent SEM . For comparison of Vmax paired two - tailed t - test was conducted ; * * p < 0 . 01 , ns - not significant ; exact p - value is indicated . Data are derived from 6 independent biological replicates . Lysates were prepared from an aliquot of the cells , which were used for the uptake assay , and subjected to immunoblotting to verify MAD2 knockdown . Immunodetection of α - tubulin was used as a loading control .

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A Radioactive substrate uptake was conducted KM and Vmax were determined by fitting the data to the equation of a rectangular hyperbola. Representative curves are shown. Error bars represent SEM. For comparison of Vmax paired two-tailed t-test was conducted; **p < 0.01, ns - not significant; exact p-value is indicated. Data are derived from 6 independent biological replicates. Lysates were prepared from an aliquot of the cells, which were used for the uptake assay, and subjected to immunoblotting to verify MAD2 knockdown. Immunodetection of α-tubulin was used as a loading control.


B Cell surface biotinylation using HEK - 293 cells stably expressing YFP - hSERT was performed Whole cell lysates and streptavidin - conjugated fractions ( i . e . , surface protein ) were subjected to immunoblotting .

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B Cell surface biotinylation using HEK-293 cells stably expressing YFP-hSERT was performed Whole cell lysates and streptavidin-conjugated fractions (i.e., surface protein) were subjected to immunoblotting.


C - D HEK - 293 cells stably expressing YFP - hSERT were transfected with the indicated siRNAs , imaged and analyzed Representative maximum intensity projections of Z - stacks are shown . Scale bars represent 50 and 20 µm in full size images and zoomed areas , respectively . Intracellular SERT - signal was measured using ImageJ software . The dot plot in panel D shows values ( presented as Log10 to account for the large variation in the signal ) from individual cells ( Ctrl siRNA n = 112 cells ; MAD2 siRNA n = 116 cells ) deriving from 3 independent biological replicates , the median and the interquartile range . The statistically significant difference was assessed by an unpaired two - tailed t - test , the p - value is indicated .

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C - D HEK-293 cells stably expressing YFP-hSERT were transfected with the indicated siRNAs, imaged and analyzed Representative maximum intensity projections of Z-stacks are shown. Scale bars represent 50 and 20 µm in full size images and zoomed areas, respectively. Intracellular SERT-signal was measured using ImageJ software. The dot plot in panel D shows values (presented as Log10 to account for the large variation in the signal) from individual cells (Ctrl siRNA n = 112 cells; MAD2 siRNA n = 116 cells) deriving from 3 independent biological replicates, the median and the interquartile range. The statistically significant difference was assessed by an unpaired two-tailed t-test, the p-value is indicated.


E - H HEK - 293 cells stably expressing YFP - hSERT were transfected with the indicated siRNA , subsequently co - transfected with plasmids encoding mCherry - Rab5 ( panel E ) or mCherry - Rab7A ( panel G ) Manders ' overlap coefficients for the fraction of mCherry overlapping YFP were calculated for multiple individual images using ImageJ / JACoP . Number of analyzed images : mCh - Rab5 / ctrl - siRNA = 38 ; mCh - Rab5 / MAD2 - siRNA = 33 ( Panel F ) ; mCh - Rab7A / ctrl - siRNA = 13 ; mCh - Rab7A / Mad2 - siRNA = 14 ( panel H ) . Statistically significant differences were assessed by unpaired two - tailed t - test , the p - values were below 0 . 0001 . Error bars represent SD of 3 independent biological replicates ( n = 3 ) . Scale bars represent 50 and 20 µm in full size images and zoomed areas , respectively .

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E - H HEK-293 cells stably expressing YFP-hSERT were transfected with the indicated siRNA, subsequently co-transfected with plasmids encoding mCherry-Rab5 (panel E) or mCherry-Rab7A (panel G) Manders' overlap coefficients for the fraction of mCherry overlapping YFP were calculated for multiple individual images using ImageJ/JACoP. Number of analyzed images: mCh-Rab5/ctrl-siRNA = 38; mCh-Rab5/MAD2-siRNA = 33 (Panel F); mCh-Rab7A/ctrl-siRNA = 13; mCh-Rab7A/Mad2-siRNA = 14 (panel H). Statistically significant differences were assessed by unpaired two-tailed t-test, the p-values were below 0.0001. Error bars represent SD of 3 independent biological replicates (n = 3). Scale bars represent 50 and 20 µm in full size images and zoomed areas, respectively.


B Immunofluorescence images of rat primary dorsal raphe neurons were analyzed using ImageJ / JACoP software . Bars indicate Manders ' overlap coefficients for the fraction of SERT overlapping with the denoted marker protein . One - way ANOVA and Tukey ' s multiple comparisons test ; * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 . Number of analyzed cells : Rab11A n = 20 ; Rab7A n = 11 ; Rab4 n = 8 ; LAMP1 n = 12 ; Rab5 n = 14 ; beta - COP n = 7 ; TGN38 n = 5 ; GM130 n = 11 ; SEC24C n = 11 . Error bars represent SEM .

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B Immunofluorescence images of rat primary dorsal raphe neurons were analyzed using ImageJ/JACoP software. Bars indicate Manders' overlap coefficients for the fraction of SERT overlapping with the denoted marker protein. One-way ANOVA and Tukey's multiple comparisons test; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Number of analyzed cells: Rab11A n = 20; Rab7A n = 11; Rab4 n = 8; LAMP1 n = 12; Rab5 n = 14; beta-COP n = 7; TGN38 n = 5; GM130 n = 11; SEC24C n= 11. Error bars represent SEM.


E Primary rat dorsal raphe neurons were cultured After 14 days , cells were infected with lentiviral particles encoding either scramble - shRNA or shRNA directed against MAD2 . Five days after infection , cells were fixed in acetone / methanol ( 1 : 1 ) and subjected to immunofluorescence with the indicated primary and secondary antibodies . Confocal images were captured on a Nikon A1 laser scanning confocal microscope at either 20 × ( Fig . 5 E , left panel ) or 60 × ( Fig . 5 E , right panel ) magnification , representative images are shown . Due to imaging conditions the optical section thickness is increased at 20 × compared to 60 × magnification . Calibration bars in images showing rMAD2 represent arbitrary fluorescence units . Scale bars represent 50 and 10 µm for 20 × and 60 × magnification , respectively .

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E Primary rat dorsal raphe neurons were cultured After 14 days, cells were infected with lentiviral particles encoding either scramble-shRNA or shRNA directed against MAD2. Five days after infection, cells were fixed in acetone/methanol (1:1) and subjected to immunofluorescence with the indicated primary and secondary antibodies. Confocal images were captured on a Nikon A1 laser scanning confocal microscope at either 20× (Fig. 5 E, left panel) or 60× (Fig. 5 E, right panel) magnification, representative images are shown. Due to imaging conditions the optical section thickness is increased at 20× compared to 60× magnification. Calibration bars in images showing rMAD2 represent arbitrary fluorescence units. Scale bars represent 50 and 10 µm for 20× and 60× magnification, respectively.


F Surface and intracellular SERT - signal from scramble - and MAD2 - shRNA infected neurons ( n = 40 and n = 30 , respectively ) was quantified within regions of interest ( ROIs ) using ImageJ software and the resulting ratios compared using an unpaired two - tailed t - test , the p - value was below 0 . 0001 ( n = 4 biological replicates ) . Error bars represent SD . The right part provides 4 additional representative images of both : scramble - shRNA and MAD2 - shRNA expressing serotonergic neurons ( SERT : orange , nuclei : blue ) . Scale bars represent 30 µm .

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F Surface and intracellular SERT-signal from scramble- and MAD2-shRNA infected neurons (n = 40 and n = 30, respectively) was quantified within regions of interest (ROIs) using ImageJ software and the resulting ratios compared using an unpaired two-tailed t-test, the p-value was below 0.0001 (n = 4 biological replicates). Error bars represent SD. The right part provides 4 additional representative images of both: scramble-shRNA and MAD2-shRNA expressing serotonergic neurons (SERT: orange, nuclei: blue). Scale bars represent 30 µm.


( A ) Immunofluorescence analysis of HeLa cells transfected with either a non - target dsRNA ( NT ) or one of the three siRNAs targeting LETM1 ( siR1 , siR2 or siR3 ) and labeled with anti - TOM20 antibody . In siR1 treated cells , the mitochondria formed a ' honeycomb ' of swollen distinct organelles ; siR3 resulted in giant organelles with a central region distinguished by reduced TOM20 ; siR2 , produced relatively little swelling and the mitochondrial network was generally well preserved . The pronounced swelling induced by siR1 significantly increased circularity ( p = 2 . 32E - 58 ) ( ImageJ analysis ) , siR1 circularity = 0 . 683 ± 0 . 012 compared with 0 . 414 ± 0 . 008 for the NT ; where 1 = a perfect circle . Over 150 mitochondria were quantified in each cell type ; data are means ± SEM . Scale bar represents 12 µm in the main images and 4 µm in offset magnification

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(A) Immunofluorescence analysis of HeLa cells transfected with either a non-target dsRNA (NT) or one of the three siRNAs targeting LETM1 (siR1, siR2 or siR3) and labeled with anti-TOM20 antibody. In siR1 treated cells, the mitochondria formed a 'honeycomb' of swollen distinct organelles; siR3 resulted in giant organelles with a central region distinguished by reduced TOM20; siR2, produced relatively little swelling and the mitochondrial network was generally well preserved. The pronounced swelling induced by siR1 significantly increased circularity (p = 2.32E-58) (ImageJ analysis), siR1 circularity = 0.683 ±0.012 compared with 0.414 ±0.008 for the NT; where 1 = a perfect circle. Over 150 mitochondria were quantified in each cell type; data are means ± SEM. Scale bar represents 12 µm in the main images and 4 µm in offset magnification


( B ) 35S labeling of de novo mitochondrial protein synthesis of HeLa cells transfected as in ( A ) . Polypeptide assignments flank the gel images . Coomassie stained gels are used as loading controls and immunoblots indicate the efficiency of LETM1 knockdown

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(B) 35S labeling of de novo mitochondrial protein synthesis of HeLa cells transfected as in (A). Polypeptide assignments flank the gel images. Coomassie stained gels are used as loading controls and immunoblots indicate the efficiency of LETM1 knockdown


( D ) Representative immunoblots for the OXPHOS proteins NDUFB8 and COII of HeLa cells transfected with NT or siLETM1 ( siR1 , siR2 or siR3 ) . Vinculin and GAPDH are shown as loading controls . The mean relative abundances for respiratory subunits COII and NDUFB8 are shown beneath the blots . Data are expressed as mean ± SEM of n = 8 independent experiments

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(D) Representative immunoblots for the OXPHOS proteins NDUFB8 and COII of HeLa cells transfected with NT or siLETM1 (siR1, siR2 or siR3). Vinculin and GAPDH are shown as loading controls. The mean relative abundances for respiratory subunits COII and NDUFB8 are shown beneath the blots. Data are expressed as mean ± SEM of n=8 independent experiments


( E ) Mitochondrial oxygen consumption rate ( OCR ) measured using a Seahorse flux analyzer before ( basal ) and after the addition ( maximal ) of the uncoupler FCCP , in HeLa cells treated with NT or siLETM1 ( siR1 or siR2 ) . Data are means ± SEM of n = 5 experiments

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(E) Mitochondrial oxygen consumption rate (OCR) measured using a Seahorse flux analyzer before (basal) and after the addition (maximal) of the uncoupler FCCP, in HeLa cells treated with NT or siLETM1 (siR1 or siR2). Data are means ± SEM of n=5 experiments


( A ) LETM1 expression was suppressed in HeLa cells by transfection with targeted siRNAs ( siR1 , siR2 or siR3 ) . A non - target dsRNA ( NT ) served as control . Cells were fixed and immunolabeled with anti - DNA antibody ( green ) . A higher magnification of selected mtDNA foci is shown beside each picture . ( B ) Quantification of cells in ( A ) displaying mtDNA abnormalities . At least 50 cells per siRNA were counted from 4 ( siR2 ) and 5 ( siR1 or siR3 ) independent experiments . Data are expressed as means ± SEM . * * * P < 0 . 001 ( One - way ANOVA )

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(A) LETM1 expression was suppressed in HeLa cells by transfection with targeted siRNAs (siR1, siR2 or siR3). A non-target dsRNA (NT) served as control. Cells were fixed and immunolabeled with anti-DNA antibody (green). A higher magnification of selected mtDNA foci is shown beside each picture. (B) Quantification of cells in (A) displaying mtDNA abnormalities. At least 50 cells per siRNA were counted from 4 (siR2) and 5 (siR1 or siR3) independent experiments. Data are expressed as means ± SEM. ***P<0.001 (One-way ANOVA)


( C ) HeLa cells were labeled with bromouridine ( BrU ) for 60 mins , 144 h after transfection with NT or siR2 and stained with anti - BrdU ( green ) and anti - GRSF1 ( red ) antibodies . Cell nuclei were stained with DAPI ( blue )

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(C) HeLa cells were labeled with bromouridine (BrU) for 60 mins, 144 h after transfection with NT or siR2 and stained with anti-BrdU (green) and anti-GRSF1 (red) antibodies. Cell nuclei were stained with DAPI (blue)


( D ) HeLa cells were labeled with anti - DNA ( green ) and anti - GRSF1 ( red ) antibodies 144 h after transfection with NT or siR3 . Cell nuclei were stained blue with DAPI

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(D) HeLa cells were labeled with anti-DNA (green) and anti-GRSF1 (red) antibodies 144 h after transfection with NT or siR3. Cell nuclei were stained blue with DAPI


( A ) Steady state levels of LETM1 protein in fibroblasts from controls ( C1 - C3 ) , WHS LETM1 + / - ( S1 , S3 , S4 ) and S5 the sole case of WHS in this study whose deletion does not encompass LETM1 ( boxed in blue ) . GAPDH is shown as a loading control

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(A) Steady state levels of LETM1 protein in fibroblasts from controls (C1-C3), WHS LETM1+/- (S1, S3, S4) and S5 the sole case of WHS in this study whose deletion does not encompass LETM1 (boxed in blue). GAPDH is shown as a loading control


( B ) Immunofluoresence analysis of control ( C1 ) , and WHS LETM1 + / - S1 - S4 , fibroblasts with anti - TOM20 antibody . Scale bars are 8 µm in the main images and 3 µm in offset merged magnifications

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(B) Immunofluoresence analysis of control (C1), and WHS LETM1+/- S1-S4, fibroblasts with anti-TOM20 antibody. Scale bars are 8 µm in the main images and 3 µm in offset merged magnifications


( C ) Electron micrographs of controls ( C1 and C2 ) and WHS LETM1 + / + ( S5 ) cells ; side panels show zooms . Scale bar : 0 . 2 µm . Black arrows indicate the areas shown offset at higher magnification ( D ) Electron micrographs of WHS LETM1 + / - S1 , S3 and S4 cells . Black arrows indicate elongated and branched mitochondria ; side panels show magnifications . Scale bar : 0 . 2 µm

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(C) Electron micrographs of controls (C1 and C2) and WHS LETM1+/+ (S5) cells; side panels show zooms. Scale bar: 0.2 µm. Black arrows indicate the areas shown offset at higher magnification (D) Electron micrographs of WHS LETM1+/- S1, S3 and S4 cells. Black arrows indicate elongated and branched mitochondria; side panels show magnifications. Scale bar: 0.2 µm


( A ) Representative immunoblots of mitochondrial lysates of HEK cells separated on a 20 - 42 . 5 % iodixanol gradients probed for LETM1 , PDH and TFAM

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(A) Representative immunoblots of mitochondrial lysates of HEK cells separated on a 20-42.5% iodixanol gradients probed for LETM1, PDH and TFAM


( B ) Left panel : immunoblot analysis of total PDH protein levels and the inactive phosphorylated ( repressed form ) , PDHS293 , in control ( C1 , C3 ) , WHS LETM1 + / - ( S1 , S3 , S4 ) and WHS LETM1 + / + ( S5 ) fibroblasts . Center and Right panels : Immunoblot analysis of PDK3 in control ( C1 - C3 ) , WHS LETM1 + / - ( S1 , S3 and S4 ) and WHS LETM1 + / + ( S5 ) fibroblasts

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(B) Left panel: immunoblot analysis of total PDH protein levels and the inactive phosphorylated (repressed form), PDHS293, in control (C1, C3), WHS LETM1+/- (S1, S3, S4) and WHS LETM1+/+ (S5) fibroblasts. Center and Right panels: Immunoblot analysis of PDK3 in control (C1-C3), WHS LETM1+/- (S1, S3 and S4) and WHS LETM1+/+ (S5) fibroblasts


( C ) Left panel : change in cell confluence of control ( C1 - C3 ) , WHS LETM1 + / - ( S1 - S4 ) and WHS LETM1 + / + ( S5 ) fibroblasts grown in BHB over the course of 188 h . Data are expressed as means ± SEM of n = 3 independent experiments . Right panel : images of control and WHS LETM1 + / - S4 cells growing either in the presence of 25 mM glucose ( HG ) or BHB ( KB ) for 6 days . Scale bar 300 µm

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(C) Left panel: change in cell confluence of control (C1-C3), WHS LETM1+/- (S1-S4) and WHS LETM1+/+ (S5) fibroblasts grown in BHB over the course of 188 h. Data are expressed as means ± SEM of n=3 independent experiments. Right panel: images of control and WHS LETM1+/- S4 cells growing either in the presence of 25 mM glucose (HG) or BHB (KB) for 6 days. Scale bar 300 µm


( D ) Confocal images of control cells ( C1 ) grown for 24 h in the presence of BHB and immunolabeled with anti - TOM20 ( red ) and anti - DNA ( green ) , and DAPI stained ( blue )

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(D) Confocal images of control cells (C1) grown for 24 h in the presence of BHB and immunolabeled with anti-TOM20 (red) and anti-DNA (green), and DAPI stained (blue)


( E ) Proportion of cells that displayed mtDNA aggregation during the KB treatment ( panel i - control cells ( C1 , C2 ) , and WHS LETM1 + / + ( S5 ) fibroblasts ; panel ii - LETM1 + / - ( S1 , S2 , S4 ) fibroblasts ) . At least 50 cells per cell line were counted at the indicated time points . Data are expressed as mean ± SEM of n = 3 independent experiments

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(E) Proportion of cells that displayed mtDNA aggregation during the KB treatment (panel i - control cells (C1, C2), and WHS LETM1+/+ (S5) fibroblasts; panel ii - LETM1+/- (S1, S2, S4) fibroblasts). At least 50 cells per cell line were counted at the indicated time points. Data are expressed as mean ± SEM of n=3 independent experiments


( a ) Expression of H2B - mCherry under control of the CLV3 promoter in 14 - d - old seedlings . Whole - mount immunostaining using α - mCherry antibodies and laser scanning microscopy ( scale bar 10 μm ) .

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(a) Expression of H2B-mCherry under control of the CLV3 promoter in 14-d-old seedlings. Whole-mount immunostaining using α-mCherry antibodies and laser scanning microscopy (scale bar 10 μm).


( c ) A representative example for enrichment of CLV3 transcript in mCherry - positive nuclei determined by qRT - PCR and normalized to wt ( N = 1 ) .

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(c) A representative example for enrichment of CLV3 transcript in mCherry-positive nuclei determined by qRT-PCR and normalized to wt (N = 1).


( a ) Expression of CLV3 , mCherry , TEL2 , PAN , and the meristem marker genes STM and KNAT1 . Asterisks indicate timepoints of significantly different expression ( Wald test , Benjamini & Hochberg corrected , q < 0 . 05 ) between stem and non - stem cells for each time point ( N = 2 ) . + = stem cell nuclei ; - = non - stem cell nuclei , E = nuclei from embryos , D7 / 14 / 35 = nuclei from 7 / 14 / 35 day - old plants , S14 = nuclei from 14 d - old above - ground seedlings .

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(a) Expression of CLV3, mCherry, TEL2, PAN, and the meristem marker genes STM and KNAT1. Asterisks indicate timepoints of significantly different expression (Wald test, Benjamini & Hochberg corrected, q<0.05) between stem and non-stem cells for each time point (N = 2). + = stem cell nuclei; - = non-stem cell nuclei, E = nuclei from embryos, D7/14/35 = nuclei from 7/14/35 day-old plants, S14 = nuclei from 14 d-old above-ground seedlings.


( c ) Number of DEGs between stem and non - stem cells at each timepoint . The banded portion of the bars indicates the number of transcription factor genes ( also in parenthesis ) .

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(c) Number of DEGs between stem and non-stem cells at each timepoint. The banded portion of the bars indicates the number of transcription factor genes (also in parenthesis).


( b ) Expression of individual significantly up - regulated genes marked with # in ( a ) . Asterisks indicate timepoints of significantly different expression ( Wald test , Benjamini & Hochberg corrected , q < 0 . 05 ) between stem and non - stem cells for each time point ( N = 2 ) . + = stem cells ; - = non - stem cells , E = nuclei from embryos , D7 / 14 / 35 = nuclei from 7 / 14 / 35 day - old plants , S14 = nuclei from 14 d - old above - ground seedlings .

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(b) Expression of individual significantly up-regulated genes marked with # in (a). Asterisks indicate timepoints of significantly different expression (Wald test, Benjamini & Hochberg corrected, q<0.05) between stem and non-stem cells for each time point (N = 2).+ = stem cells; - = non-stem cells, E = nuclei from embryos, D7/14/35 = nuclei from 7/14/35 day-old plants, S14 = nuclei from 14 d-old above-ground seedlings.


( a ) Box plot summarizing differential expression of unambiguously mapped individual TEs , compared with all protein - coding genes , or housekeeping genes . Box plots outline the interquartile range ( IQR ) with the median and whiskers + / - 1 . 5 IQR . For all p - values , see also Appendix Table EV6 . * * indicate a p - value smaller than 1 . 01e - 11 ( two sided non paired Wilcoxon rank sum test ) .

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(a) Box plot summarizing differential expression of unambiguously mapped individual TEs, compared with all protein-coding genes, or housekeeping genes. Box plots outline the interquartile range (IQR) with the median and whiskers +/- 1.5 IQR . For all p-values, see also Appendix Table EV6. ** indicate a p-value smaller than 1.01e-11 (two sided non paired Wilcoxon rank sum test).


( a ) Increased expression of TEs in stem cells of D7 seedlings according to qRT - PCR . Box plots outline the interquartile range ( IQR ) with the median and whiskers + / - 1 . 5 IQR . N = 2 ( ATLANTYS3 , ATCOPIA39 , ATCOPIA49 , ATCOPIA29 , ATGP1 - 1 ) , N = 3 ( VANDAL6 , ATGP2 , ATGP1 - 2 , ATLINE1 , ATGP1 - 3 ) , N = 4 ( ATHILA6A , ATCOPIA83 ) , N = 5 ( VANDAL12 , ATHILA3 , ATCOPIA22 ) , and N = 6 for CLAVATA . * p < 0 . 01 , * * p < 0 . 001 ( t - test ) .

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(a) Increased expression of TEs in stem cells of D7 seedlings according to qRT-PCR. Box plots outline the interquartile range (IQR) with the median and whiskers +/- 1.5 IQR. N = 2 (ATLANTYS3, ATCOPIA39, ATCOPIA49, ATCOPIA29, ATGP1-1), N = 3 (VANDAL6, ATGP2, ATGP1-2, ATLINE1, ATGP1-3), N = 4 (ATHILA6A, ATCOPIA83), N = 5 (VANDAL12, ATHILA3, ATCOPIA22), and N = 6 for CLAVATA. *p<0.01, **p<0.001 (t-test).


( b ) Heatmap of TE expression in several silencing mutants determined by qRT - PCR relative to a housekeeping gene . To be able to display data in log - scale also for samples with zero expression , a value of 1 . 0e - 6 ( factor 10 smaller than the smallest value ) was added to all values . Data for nuclei and CLV3 : : H2BmCherry are the same as in ( a ) . For seedling samples , N = 2 ( ATCOPIA49 , ATHILA3 ) , N = 3 ( ATCOPIA39 , VANDAL6 , ATGP2 , ATLINE1 , ATCOPIA29 , ATGP1 - 1 ) , and N = 4 ( ATLANTYS3 , VANDAL12 , ATGP1 - 2 , ATHILA6A ) .

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

(b) Heatmap of TE expression in several silencing mutants determined by qRT-PCR relative to a housekeeping gene. To be able to display data in log-scale also for samples with zero expression, a value of 1.0e-6 (factor 10 smaller than the smallest value) was added to all values. Data for nuclei and CLV3::H2BmCherry are the same as in (a). For seedling samples, N = 2 (ATCOPIA49, ATHILA3), N = 3 (ATCOPIA39, VANDAL6, ATGP2, ATLINE1, ATCOPIA29, ATGP1-1), and N = 4 (ATLANTYS3, VANDAL12, ATGP1-2, ATHILA6A).


( a ) CG , CHG , and CHH methylation at chromosome 3 in stem and non - stem cells . The inset for CHG methylation shows a magnification of the pericentromeric region .

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(a) CG, CHG, and CHH methylation at chromosome 3 in stem and non-stem cells. The inset for CHG methylation shows a magnification of the pericentromeric region.


( a ) DNA methylation in % at CHG ( left ) and CHH ( right ) sites in TE with increased expression at D7 , compared between developmental stages ( D7 / 14 / 35 = 7 / 14 / 35 day - old plants ) and nuclei of stem ( + ) and non - stem ( - ) cells .

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(a) DNA methylation in % at CHG (left) and CHH (right) sites in TE with increased expression at D7, compared between developmental stages (D7/14/35 = 7/14/35 day-old plants) and nuclei of stem (+) and non-stem (-) cells.


( b ) Genomic location of the TEs ( blue ) from ( a ) on the five chromosomes . Dark grey bars indicate the location of centromeres and blue lines indicate global TE density .

(b) Genomic location of the TEs (blue) from (a) on the five chromosomes. Dark grey bars indicate the location of centromeres and blue lines indicate global TE density.


( a ) DNA methylation differences between stem and non - stem cells and their enrichment within DMRs of different epigenetic mutants using permutation tests ( data from Stroud , Greenberg , Feng , Bernatavichute and Jacobsen 2013 ) . Mutants are in alphabetical order according to the gene acronym . Red indicates an enrichment of methylated DNA in the respective context and blue a depletion .

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(a) DNA methylation differences between stem and non-stem cells and their enrichment within DMRs of different epigenetic mutants using permutation tests (data from Stroud, Greenberg, Feng, Bernatavichute and Jacobsen 2013). Mutants are in alphabetical order according to the gene acronym. Red indicates an enrichment of methylated DNA in the respective context and blue a depletion.


( B ) Microarray heat map showing the upregulation in expression of the 11 candidate genes in β cell - ablated versus control islets . Igfbp1a and b were the genes whose expression increased the most after β - cell ablation .

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(B) Microarray heat map showing the upregulation in expression of the 11 candidate genes in β cell-ablated versus control islets. Igfbp1a and b were the genes whose expression increased the most after β-cell ablation.


( D - E ) Representative images at 6 dpf of Tg ( ins : kaede ) ; Tg ( ins : CFP - NTR ) transgenic larvae that had been injected at the 1 - 2 - cell stage with transposase mRNA ( control ) or transposase mRNA + bactin : igfbp1a ( bactin : igfbp1a ) , subjected to β - cell ablation by metronidazole ( MTZ ) during 3 - 4 dpf , and subsequently allowed to regenerate for 2 days . The GFP + heart ( arrowhead ) visualizes successful integration of the construct . Islets are indicated by white arrows . Scale bars : 100 μm . ( F ) Quantification of β - cell regeneration at 6 dpf in control ( n = 23 ) , bactin : igfbp1a - overexpressing ( n = 13 ) , and bactin : igfbp1b - overexpressing ( n = 8 ) Tg ( ins : kaede ) ; Tg ( ins : CFP - NTR ) larvae ; * * * P = 0 . 0002 , ns = nonsignificant ( P = 0 . 3106 ) .

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

(D-E) Representative images at 6 dpf of Tg(ins:kaede);Tg(ins:CFP-NTR) transgenic larvae that had been injected at the 1-2-cell stage with transposase mRNA (control) or transposase mRNA + bactin:igfbp1a (bactin:igfbp1a), subjected to β-cell ablation by metronidazole (MTZ) during 3-4 dpf, and subsequently allowed to regenerate for 2 days. The GFP+ heart (arrowhead) visualizes successful integration of the construct. Islets are indicated by white arrows. Scale bars: 100 μm. (F) Quantification of β-cell regeneration at 6 dpf in control (n=23), bactin:igfbp1a-overexpressing (n=13), and bactin:igfbp1b-overexpressing (n=8) Tg(ins:kaede);Tg(ins:CFP-NTR) larvae; ***P=0.0002, ns= nonsignificant (P=0.3106).


( G ) Immunohistochemistry showing Igfbp1 protein expression in 6 dpf Tg ( ins : GFP ) following β - cell ablation between 3 - 4 dpf . Scale bar : 50 μm .

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(G) Immunohistochemistry showing Igfbp1 protein expression in 6 dpf Tg(ins:GFP) following β-cell ablation between 3-4 dpf. Scale bar: 50 μm.


( H ) Both liver - specific ( lfabp promoter ; n = 46 ) and widespread ( bactin promoter ; n = 18 ) overexpression of igfbp1a increase β - cell regeneration when compared to control ( n = 77 ) .

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(H) Both liver-specific (lfabp promoter; n=46) and widespread (bactin promoter; n=18) overexpression of igfbp1a increase β-cell regeneration when compared to control (n=77).


( I - M ) Quantification of β cells with or without β - cell ablation and igfbp1a overexpression by confocal microscopy , which detects even weakly insulin - expressing β cells . Scale bars : 15 μm . See also Figure EV1 .

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(I-M) Quantification of β cells with or without β-cell ablation and igfbp1a overexpression by confocal microscopy, which detects even weakly insulin-expressing β cells. Scale bars: 15 μm. See also Figure EV1.


( A - C ) Igfbp1a promotes β cell regeneration , rather than β cell survival . To trace β cells , we exposed Tg ( ins : kaede ) larvae to UV light ( which causes the existing Kaede protein to switch from emitting green fluorescence to emitting red fluorescence ) just before ablating the β cells by MTZ treatment from 3 - 4 dpf . After regeneration , the newly formed β cells are green , whereas the β cells that survived ablation are yellow ( overlap of green and red ) . ( A & amp ; amp ; B ) Representative confocal images at 6 dpf of control and bactin : igfbp1a - overexpressing Tg ( ins : kaede ) ; Tg ( ins : CFP - NTR ) transgenic larvae ; arrows indicate surviving ( yellow ) β cells . Scale bars : 10 μm . ( C ) Quantification of β cell regeneration ( green bars ) and β cell survival ( yellow bars ) per larva at 6 dpf . * * * P < 0 . 001 : n = 20 larvae in the control group ; n = 27 larvae in the bactin : igfbp1a - overexpressing group .

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(A-C) Igfbp1a promotes β cell regeneration, rather than β cell survival. To trace β cells, we exposed Tg(ins:kaede) larvae to UV light (which causes the existing Kaede protein to switch from emitting green fluorescence to emitting red fluorescence) just before ablating the β cells by MTZ treatment from 3-4 dpf. After regeneration, the newly formed β cells are green, whereas the β cells that survived ablation are yellow (overlap of green and red). (A&amp;B) Representative confocal images at 6 dpf of control and bactin:igfbp1a-overexpressing Tg(ins:kaede);Tg(ins:CFP-NTR) transgenic larvae; arrows indicate surviving (yellow) β cells. Scale bars: 10 μm. (C) Quantification of β cell regeneration (green bars) and β cell survival (yellow bars) per larva at 6 dpf. ***P<0.001: n=20 larvae in the control group; n=27 larvae in the bactin:igfbp1a-overexpressing group.


( D - F ) Igfbp1a does not promote δ - cell regeneration . We treated control and bactin : igfbp1a - overexpressing Tg ( sst : flag - NTR ) ; Tg ( sst : dsRed ) larvae with MTZ from 3 - 4 dpf to ablate δ cells , and then allowed them to regenerate until 6 dpf . ( D & amp ; amp ; E ) Representative confocal images at 6 dpf of control and bactin : igfbp1a - overexpressing larvae showing comparable number of δ cells after 2 days of regeneration . Scale bars : 15 μm . ( F ) Quantification of the total number of δ cells per δ - cell ablated larva at 6 dpf compared to the baseline number of δ cells in non - ablated control larvae . P = 0 . 2325 . n = 13 in the control group , n = 7 in the bactin : igfbp1a group .

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(D-F) Igfbp1a does not promote δ-cell regeneration. We treated control and bactin:igfbp1a-overexpressing Tg(sst:flag-NTR);Tg(sst:dsRed) larvae with MTZ from 3-4 dpf to ablate δ cells, and then allowed them to regenerate until 6 dpf. (D&amp;E) Representative confocal images at 6 dpf of control and bactin:igfbp1a-overexpressing larvae showing comparable number of δ cells after 2 days of regeneration. Scale bars: 15 μm. (F) Quantification of the total number of δ cells per δ-cell ablated larva at 6 dpf compared to the baseline number of δ cells in non-ablated control larvae. P=0.2325. n=13 in the control group, n=7 in the bactin:igfbp1a group.


( G ) Other Igfbps do not promote β - cell regeneration . We injected Tg ( ins : kaede ) ; Tg ( ins : CFP - NTR ) transgenics with either transposase mRNA ( control ; n = 31 ) or transposase mRNA + one of six different igfbps , igfbp2a ( n = 25 ) , igfbp2b ( n = 25 ) , igfbp3 ( n = 26 ) , igfbp5a ( n = 17 ) , igfbp6b ( n = 34 ) , and igfbp7 ( n = 17 ) , treated them with MTZ from 3 - 4 dpf to ablate their β cells , and then quantified their β cells after 2 days of regeneration , at 6 dpf ; ns = nonsignificant ; ( igfbp2aP = 0 . 986 , igfbp2bP = 0 . 037 , igfbp3P = 0 . 979 , igfbp5aP = 0 . 999 , igfbp6bP = 0 . 997 , and igfbp7P = 0 . 999 ) .

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

(G) Other Igfbps do not promote β-cell regeneration. We injected Tg(ins:kaede);Tg(ins:CFP-NTR) transgenics with either transposase mRNA (control; n=31) or transposase mRNA + one of six different igfbps, igfbp2a (n=25), igfbp2b (n=25), igfbp3 (n=26), igfbp5a (n=17), igfbp6b (n=34), and igfbp7 (n=17), treated them with MTZ from 3-4 dpf to ablate their β cells, and then quantified their β cells after 2 days of regeneration, at 6 dpf; ns=nonsignificant; (igfbp2aP=0.986, igfbp2bP=0.037, igfbp3P=0.979, igfbp5aP=0.999, igfbp6bP=0.997, and igfbp7P=0.999).


( H ) Tg ( pcsk1 : GFP ) is expressed in regenerating β cells within the islet ( arrowheads ) , but not outside the islet ( arrow ) , at 6 dpf in larvae overexpressing igfbp1a . Scale bar : 15 μm .

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(H) Tg(pcsk1:GFP) is expressed in regenerating β cells within the islet (arrowheads), but not outside the islet (arrow), at 6 dpf in larvae overexpressing igfbp1a. Scale bar: 15 μm.


( I & amp ; amp ; J ) Free - glucose levels during β - cell regeneration in control ( bactin : mCherry ) and bactin : igfbp1a - overexpressing Tg ( ins : kaede ) ; Tg ( ins : CFP - NTR ) larvae ( I ) , as well as in Tg ( ins : kaede ) ; Tg ( ins : CFP - NTR ) larvae injected in the pericardial sac at 4 dpf with 1 ng of recombinant mouse Igfbp1 ( J ) . We treated larvae with MTZ from 3 - 4 dpf to ablate their β cells , and monitored their free - glucose levels at 3 - 7 dpf . Free - glucose levels were significantly lower after genetic igfbp1a overexpression or Igfbp1 - protein injection ( red lines ) than in controls ( black lines ) at 7 and 6 dpf , respectively . Baseline reference levels of free glucose throughout development are shown for a different set of larvae without β - cell ablation . n = 24 larvae ( four pools of six larvae ) per data point .

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

(I&amp;J) Free-glucose levels during β-cell regeneration in control (bactin:mCherry) and bactin:igfbp1a-overexpressing Tg(ins:kaede);Tg(ins:CFP-NTR) larvae (I), as well as in Tg(ins:kaede);Tg(ins:CFP-NTR) larvae injected in the pericardial sac at 4 dpf with 1 ng of recombinant mouse Igfbp1 (J). We treated larvae with MTZ from 3-4 dpf to ablate their β cells, and monitored their free-glucose levels at 3-7 dpf. Free-glucose levels were significantly lower after genetic igfbp1a overexpression or Igfbp1-protein injection (red lines) than in controls (black lines) at 7 and 6 dpf, respectively. Baseline reference levels of free glucose throughout development are shown for a different set of larvae without β-cell ablation. n=24 larvae (four pools of six larvae) per data point.


( A - C ) Tg ( ins : GFP ) ; Tg ( tp1 : H2B - mCherry ) ; Tg ( ins : Flag - NTR ) transgenics , with or without Tg ( bactin : igfbp1a ) , were treated with MTZ from 3 - 4 dpf to ablate the β cells , and were then allowed to regenerate from 4 - 6 dpf . Representative confocal images at 6 dpf of control ( A ) and Tg ( bactin : igfbp1a ) ( B ) larvae showing a modest number of ins + tp1 + co - expressing cells , indicated by arrows , after 2 days of regeneration . Scale bars : 15 μm . ( C ) Quantification of the total number of β cells ( green bars ) at 6 dpf , and of β cells expressing Tg ( tp1 : H2B - mCherry ) , i . e . , of ductal origin ( yellow bars ) . * * * * P < 0 . 0001 , ns = nonsignificant . n = 23 larvae in the control group , n = 17 larvae in the Tg ( bactin : igfbp1a ) group .

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(A-C) Tg(ins:GFP);Tg(tp1:H2B-mCherry);Tg(ins:Flag-NTR) transgenics, with or without Tg(bactin:igfbp1a), were treated with MTZ from 3-4 dpf to ablate the β cells, and were then allowed to regenerate from 4-6 dpf. Representative confocal images at 6 dpf of control (A) and Tg(bactin:igfbp1a) (B) larvae showing a modest number of ins+ tp1+ co-expressing cells, indicated by arrows, after 2 days of regeneration. Scale bars: 15 μm. (C) Quantification of the total number of β cells (green bars) at 6 dpf, and of β cells expressing Tg(tp1:H2B-mCherry), i.e., of ductal origin (yellow bars). ****P<0.0001, ns= nonsignificant. n=23 larvae in the control group, n=17 larvae in the Tg(bactin:igfbp1a) group.