EMBO/soda-nel huggingface dataset:
Prostaglandin E2 promotes intestinal repair through an adaptive cellular response of the epithelium (2016)
Figure 3

Figure 3. Spheroids differentiated with dmPGE2 or EP4i are composed of post-mitotic intestinal epithelial cells.

A Schematic of method used to generate stem cell, enterocyte and WAE cell groups.

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.

C, D Graphs showing the top five most significant pathways (C) and gene ontology cellular component terms (D) associated with Clusters 1 and 4.

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.

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.
Prostaglandin E2 promotes intestinal repair through an adaptive cellular response of the epithelium (2016)
Figure 4

Figure 4. In vitro generated wound-associated epithelial cells resemble their in vivo counterparts.

A, B Graphs showing the top five most significant pathways (A) and gene ontology cellular component terms (B) associated with Clusters 5 and 6.

C Graph of the top twelve significantly enriched pathways in in vivo colonic WAE cells.

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.

G-J Representative transmission electron microscopy (TEM) images of spheroids or an in vivo WAE cell from a biopsy-injured mouse colon. (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. 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. (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.
Prostaglandin E2 promotes intestinal repair through an adaptive cellular response of the epithelium (2016)
Figure 5

Figure 5. Inhibition of the prostaglandin receptor EP4 produces enterocytes in vitro.

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

D, E Graphs showing the top five most significant pathways (D) and gene ontology cellular component terms (E) associated with Cluster 3.

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.
Prostaglandin E2 promotes intestinal repair through an adaptive cellular response of the epithelium (2016)
Figure 6

Figure 6. PGE2-EP4 signaling controls a differentiation switch between wound-associated epithelial cells and enterocytes.

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/Δ spheroids 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.
Prostaglandin E2 promotes intestinal repair through an adaptive cellular response of the epithelium (2016)
Figure 7

Figure 7. Nuclear β-catenin signaling without canonical Wnt signaling suppresses enterocyte and promotes wound-associated epithelial cell differentiation.

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. (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.

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/Δ spheroids 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.

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.
Prostaglandin E2 promotes intestinal repair through an adaptive cellular response of the epithelium (2016)
Figure 8

Figure 8. Intestinal epithelium deficient for Ptger4 lacks wound-associated epithelial cells and has delayed wound repair following biopsy injury.

A Representative whole-mount images of wounds from Ptger4flox/flox (Ptger4fl/fl) and Villin-Cre Ptger4flox/flox (VilCre Ptger4fl/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. (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.
USP1 deubiquitinates Akt to inhibit PI3K-Akt-FoxO signalling in the muscle during prolonged starvation (2020)
Figure 1

Figure 1. USP1 is a deubiquitinating enzyme for Akt.

(A) Akt is deubiquitinated during fasting. Left: Soluble fractions of TA muscles from fed and fasted mice were analyzed by SDS-PAGE and immunoblot using Akt antibody. Right: densitometric measurement of presented blots (n=3). * p<0.05 vs. fed by one-tailed t-test. Data are represented as mean ±SEM.

(B) Akt was immunoprecipitated from the soluble fraction of muscles from fed or fasted (2d) mice. Percipitated were analyzed by immunoblotting using an anti-ubiquitin conjugates. Mouse IgG was used as a control for non-specific binding.

(C) Scheme for DUBs screening experiment to identify the enzyme that directly deubiquitinates Akt in vitro.

)D( Ubiquitinated Akt is a substrate for USP1 in vitro. Top: Akt was immunoprecipitated from the soluble fraction of TA muscle from fed mice, and was subjected to an in vitro deubiquitination by a panel of purified DUBs arrayed in a multi-well plate. The dividing line indicates the removal of an intervening lane for presentation purposes. Bottom: Densitometric measurements of presented blots. Data is presented as the ratio between ubiquitinated Akt to total Akt in each well.

(E) shRNA-mediated knockdown of USP1 in Hela cells. Soluble extracts were analyzed by immunoblotting.

(F) USP1 deubiquitinates Akt during fasting in vivo. Left: Soluble fractions of TA muscles transfected with shLacz control or shUSP1 from fed and fasted mice were analyzed by SDS-PAGE and immunoblotting using anti-Akt. The actin blot serves as a loading control. Right: densitometric measurements of presented blots (n=3). Data is presented as ratio of ubiquitinated Akt to total Akt normalized to actin. * p<0.05 vs. fed by one-tailed t-test. Data are represented as mean ±SEM.

(G) USP1 removes K63-linked polyubiquitin chains on Akt in vivo. Akt was immunoprecipitated from the soluble fraction of muscles expressing USP1(C90S) or control plasmids. Mouse IgG was used as a control for non-specific binding. Protein precipitates were subjected to immunoblotting with an antibody against K63-linked polyubiquitin conjugates.

(H) USP1 removes polyubiquitin chains linked to K8 on Akt. Akt was immunoprecipitated from the soluble fraction of TA muscles transfected with USP1(C90S) , HA-Akt(K8R) or control from fasted mice, and protein precipitates were analyzed by immunoblotting with anti-Akt antibody. Mouse IgG was used as a control for non-specific binding. Right: Densitometric measurements of presented blots. Data is presented as the ratio between ubiquitinated Akt to total Akt in each lane (n=2).
USP1 deubiquitinates Akt to inhibit PI3K-Akt-FoxO signalling in the muscle during prolonged starvation (2020)
Figure 2

Figure 2. Deubiquitination by USP1 reduces Akt phosphorylation at T308 and PI3K-Akt-FoxO signaling.

(A) Inhibition of USP1 increases PI3K-Akt-FoxO signaling during fasting. Soluble fractions of normal and atrophying muscles expressing shLacz or USP1(C90S) were analyzed by SDS-PAGE and immunoblot. The black line indicate the removal of intervening lanes for presentation purposes. Right: densitometric measurements of presented pAkt (T308), pAkt (S473), and Akt blots. Data is presented as ratio of phosphorylated Akt to total Akt (n=3). * p<0.05 vs. fed by one-tailed t-test. Data are represented as mean ±SEM.

(B) Downregulation of USP1 reduces MuRF1 and Atrogin1 expression during fasting. Quantitative RT-PCR of mRNA preparations from atrophying and control muscles expressing shLacz or shUSP1 (80% transfection efficiency) using primers for MuRF1 and Atrogin1. Data are plotted as the mean fold change relative to fed control. n = 4. *, P < 0.05 vs. shLacz in fed. #, P < 0.05 vs. shLacz in fasting by one-tailed t-test. Data are represented as mean ±SEM.

(C( Downregulation of USP1 markedly reduces muscle fiber atrophy. Cross-sectional areas of 500 fibers transfected with shUSP1 (that express GFP, green bars) vs. 500 nontransfected fibers (black bars) in the same muscle. n = 5 mice. A representative image is shown; Laminin staining is in red; Bar, 50μm.

(D) Downregulation of USP1attenuates the loss of muscle mass during fasting. shUSP1 was delivered to more than 60% of muscle fibers. Mean weights of electroporated muscles are plotted as the percent weight loss. n = 6. #, P < 0.05 vs. shLacz in fed; *, P < 0.05 vs. shLacz in fasting by one-tailed t-test. Data are represented as mean ±SEM.

(E) USP1 inhibition significantly increases glucose tolerance in mice during fasting. Left: mice injected i.p. with specific USP1 Inhibitor (12ug/gr body weight) or saline during fasting (2d) were subjected to glucose tolerance test. Blood glucose levels were measured at the indicated time points following glucose injection (1 mg/gr body weight). Data is depicted in a graph as mg/dL glucose (n=3). * p<0.05, ** p<0.005, *** p<0.0005 vs. mice injected with saline by one-tailed t-test. Data are represented as mean ±SEM. Right: soluble fractions of muscles from injected mice were analyzed by SDS-PAGE and immunoblot.
USP1 deubiquitinates Akt to inhibit PI3K-Akt-FoxO signalling in the muscle during prolonged starvation (2020)
Figure 3

Figure 3. The phosphatase PHLPP1 reduces Akt phosphorylation at S473 in fasting.

(A) During fasting, downregulation of PHLPP1 enhances Akt phosphorylation at S473. Soluble fractions of muscles transfected with shLacz or shPHLPP1 from fasted mice (2d) were analyzed by immunoblotting. Right: densitometric measurements of presented blots. Data is presented as ratio of shPHLPP1 to shLacz (n=3 for shLacz and n=4 for shPHLPP1). * p<0.05 vs. shLacz in fasting by one-tailed t-test. Data are represented as mean ±SEM.

(B) PHLPP1 downregulation increases rates of protein synthesis during fasting. Mice were injected with puromycin, and soluble fractions of electroporated muscles were analyzed by immunoblotting using puromycin antibody. Analysis of right (R) and left (L) limbs for each mouse is shown.

(C) Downregulation of PHLPP1 markedly reduces muscle fiber atrophy. Cross-sectional areas of 500 fibers transfected with shPHLPP1 (that express GFP, green bars) vs. 500 nontransfected fibers (black bars) in the same muscle. n = 4 mice. A representative image is shown; Laminin staining is in red; Bar, 50μm.

(D) USP1/UAF1 interact with PHLPP1 in normal muscle. USP1 was immunoprecipitated from the soluble fraction of normal muscles. Mouse IgG was used as a control for non-specific binding. Precipitates were analyzed by immunoblotting.
USP1 deubiquitinates Akt to inhibit PI3K-Akt-FoxO signalling in the muscle during prolonged starvation (2020)
Figure 4

Figure 4. Akt is recruited to USP1-PHLPP1-TSC1 complex in fasting.

(A) Scheme of affinity purification technique to isolate components that bind Akt. Four immunoprecipitation experiments of USP1, PHLPP1, UAF1 and FoxO from muscle homogenates during fasting were performed in parallel to non-specific mouse IgG control, and protein precipitates were analyzed by mass spectrometry.

(B) Mass spectrometry data obtained in (A) is presented using Venny diagram tool. Only proteins that were at least threefold more abundant in the immunoprecipitation samples than in IgG control are listed.

(C( In fasting, Akt sediments to glycerol gradient fractions containing USP1/UAF1, PHLPP1, and TSC1/TSC2. Muscle homogenates from fed or fasted mice were separated on 10%-40% glycerol gradient, and alternate fractions were analyzed by SDS-PAGE and immunoblot. Red rectangle marks fractions containing USP1/UAF1, PHLPP1, TSC1 and TSC2 in fed conditions.

(D) During fasting, Akt is recruited to USP1-PHLPP1-TSC1 complex. TSC1 was immunoprecipitated from the soluble fraction of muscles expressing shLacz or USP1(C90S) from fed or fasted mice. Mouse IgG was used as a control for non-specific binding. Precipitates were analyzed by immunoblotting. Black lines indicate the removal of intervening lanes for presentation purposes.

(E) During fasting, inhibition of USP1 reduces rates of protein synthesis. Mice were injected with puromycin, and soluble fractions of electroporated muscles were analyzed by immunoblotting using puromycin antibody. Right: Densitometric measurement of the presented blot. n = 3. *, P < 0.05 vs. shLacz by one-tailed t-test. Data are represented as mean ±SEM.

(F) Inhibition of USP1 results in TSC1 accumulation. Soluble fraction of muscles expressing shLacz or USP1(C90S) from fasted mice were analyzed by SDS-PAGE and immunoblotting. Right: Densitometric measurement of the presented blot. n = 3. *, P < 0.05 vs. shLacz. Data is presented as protein content (AU) in USP1(C90S) vs. shLacz by one-tailed t-test. Data are represented as mean ±SEM.

(G) mTOR inhibition with rapamycin results in reduced USP1 protein content and enhanced Akt phosphorylation at T308. However, a simultaneous downregulation of USP1 is required to promote TSC1 accumulation. Soluble fraction of muscles expressing shLacz or gUSP1 (in vivo CRISPR) from fed and fasted mice injected i.p. with rapamycin (6mg/kg body weight) or saline were analyzed by SDS-PAGE and immunoblot.
USP1 deubiquitinates Akt to inhibit PI3K-Akt-FoxO signalling in the muscle during prolonged starvation (2020)
Figure 5

Figure 5. Dab2 facilitates Akt recruitment to USP1-PHLPP1-TSC1 complex.

(A) During fasting, inhibition of Dab2-Akt association reduces the interaction of Akt with USP1-PHLPP1-TSC1 complex. Left: TSC1 was immunoprecipitated from the soluble fraction of muscles expressing shLacz or Dab2-DN from fed or fasted mice. Mouse IgG was used as a control for non-specific binding. Precipitates were analyzed by immunoblotting. Right: densitometric measurement of PHLPP1 and TSC1 blots, and data is presented as PHLPP1/TSC1 ratio.

(B) Dab2 promotes Akt deubiquitination in vivo. Left: Akt was immunoprecipitated from the soluble fraction of muscles expressing shLacz or Dab2-DN from fed and fasted mice using a specific antibody. Mouse IgG was used as a control for non-specific binding. Protein precipitates were analyzed by Western blot analysis using Akt antibody. Right: Densitometric measurements of presented blots. Data is presented as the ratio between ubiquitinated Akt to total Akt in each lane (n=2).

(C( Dab2 promotes cleavage of K63-linked polyubiquitin chains on Akt in vivo. Akt was immunoprecipitated from the soluble fraction of muscles expressing shLacz or Dab2-DN from fed and fasted mice using a specific antibody. Mouse IgG was used as a control for non-specific binding. Protein precipitates were subjected to immunoblotting with an antibody against K63-linked polyubiquitin conjugates.

(D) Inhibition of Dab2-Akt association enhances Akt phosphorylation in fasting. Soluble fraction of TA muscles transfected with shLacz or Dab2-DN from fed and fasted mice were analyzed by SDS-PAGE and immunoblotting using the indicated antibodies. Right: densitometric measurement of presented blots. Data are mean ±SEM, n=3. * p<0.05. vs. shLacz in fed conditions, by one-tailed t-test.

SUPPLEMENTAL INFORMATION TITLES AND LEGENDS