EMBO/soda-nel huggingface dataset:
Loss of TMEM106B and PGRN leads to severe lysosomal abnormalities and neurodegeneration in mice (2020)
Figure 12

Figure 12. FTLD related pathological changes in Tmem106b-/-Grn-/- mice.

(A, B) Western blot analysis of p62, ubiquitin (Ub), TDP-43 and p-TDP-43 in RIPA- and urea-soluble fractions from spinal cord (C5-C8) and brain of 5-month-old WT, Tmem106b-/-, Grn-/-, and Tmem106b-/-Grn-/- mice. Spinal cord: n=3; brain: n=4. Data presented as mean ± SEM. One-way ANOVA tests with Bonferroni's multiple comparisons: *, p<0.05; **, p<0.01***, p<0.001, ****, p<0.0001.

(C, D) Immunostaining of p62 and ubiquitin in the spinal cord sections from 5-month-old WT, Tmem106b-/-, Grn-/-, and Tmem106b-/-Grn-/- mice. The number of p62 or Ub positive puncta was quantified. n=3. Data presented as mean ± SEM. One-way ANOVA tests with Bonferroni's multiple comparisons: ***, p<0.001, ****, p<0.0001. Scale bar = 10 µm.

Expanded View Figure Legends
miR-181a/b downregulation:a mutation-independent therapeutic approach for Inherited Retinal Diseases (2022)
Figure 1

Figure 1: miR-181a/b-1 downregulation slows down retinal degeneration in P347S mice at p30.

A) qRT-PCR analysis reveals increased levels of most of the miR-181a/b targets in the eyes of P347S/miR-181a/b+/- vs P347S animals. N=4 animals/genotype.

B-C') Immunofluorescence analysis of active-caspase 3 in the ONL of P347S and P347S/miR-181a/b+/-.

D, E) TUNEL analysis in the ONL of P347S and P347S/miR-181a/b+/-.

F) Quantification of immunofluorescence analysis in (B-C') (N=4 eyes P347S and N=6 eyes P347S/ miR-181a/b+/).

G) Quantification of TUNEL analysis in (D, E) (N=3 eyes/genotype).

Data information: Scale bars are 50 μm. Data are presented as mean ± SEM. Student's t-test unpaired.
miR-181a/b downregulation:a mutation-independent therapeutic approach for Inherited Retinal Diseases (2022)
Figure 2

Figure 2: miR-181a/b-1 downregulation ameliorates the retinal phenotype in P347S mice at p30.

A-C) ERG response (a-wave in A and b-wave in B), recorded in scotopic conditions, of WT, P347S and P347S/miR-181a/b+/- animals at p30; N≥18 eyes. Data are presented as mean ± SD. Two-way ANOVA test. Representative curves are reported in (C).

D-P) Immunofluorescence analysis showed amelioration of Rhodopsin localization (D-F'; white arrowheads); C-arrestin expression (G-I') and PR outer segment (OS) and inner segment (IS) structures, as determined, respectively, by PNA (J-L'); and Phalloidin staining (M-O'') in P347S/miR-181a/b+/- vs P347S eyes at p30. M''-O'' show higher magnification of M-O. Scale bars 25 μm in D-O'; 5μm in M''-O''. Fluorescence densitometry quantification of each staining is reported in (P), N=3 eye/genotype for each staining. WT vs P347S p values are reported in red, P347S vs P347S/miR-181a/b+/- p values are reported in black. Data are presented as mean ± SEM. Student's t-test, unpaired.
miR-181a/b downregulation:a mutation-independent therapeutic approach for Inherited Retinal Diseases (2022)
Figure 3

Figure 3: The effect of miR-181a/b-1 downregulation in P347S mice is still detectable at p90.

A-C) ERG response (a-wave in A and b-wave in B), recorded in scotopic conditions, of P347S and P347S/miR-181a/b+/- animals at p90; N≥20 eyes/genotype. Data are presented as mean ± SD. Two-way ANOVA test. Representative curves are reported in (C).

D-L) Immunofluorescence analysis of Rhodopsin (D-E'), C-arrestin (F-G'), PNA (H-I'); and Phalloidin staining (J-K'') in P347S and P347S/miR-181a/b+/- retinas at p90. J''-K'' show higher magnification of J-K. Scale bars 25 μm in D-K'; 10μm in J''-K''. Fluorescence densitometry quantification of each staining is reported in (L), N=3 eye/genotype/staining. Data are presented as mean ± SEM. Student's t-test, unpaired.

M) Measurement of P347S and P347S/miR-181a/b+/- ONL thickness (N≥3 eyes/genotype; Data are presented as mean ± SEM. Student's t-test, unpaired).

N) Graphical representation of the results of OKR analysis by the optokinetic tracking assays reported as cycles/degree. Visual acuity is preserved in P347S/miR-181a/b+/- animals with respect to P347S (N=4 animals/genotype). Data are presented as mean ± SEM. Student's t-test, unpaired.
miR-181a/b downregulation:a mutation-independent therapeutic approach for Inherited Retinal Diseases (2022)
Figure 4

Figure 4: miR-181a/b-1 downregulation improves the mitochondrial phenotype in the retina of P347S mice.

A) Gene Ontology analysis [Cellular component (CC) terms] shows a significant downregulation of mitochondria-related gene pathways in P347S vs WT retinas at p12, as assessed by transcriptome analysis.

B-F) Electron microscopy analysis shows an increase of mitochondrial fragmentation in P347S PRs with respect to WT and amelioration of the mitochondrial phenotype in P347S/miR-181a/b+/- vs P347S PRs at p12 and p30 (N=2 animals/genotype). M, mitochondria.

G-I) The quantitative analysis of mitochondrial phenotype is expressed as measurement of the mitochondria perimeter (G), mitochondria number (H) and as the ratio of mitochondria area per the analyzed field area (I). N=2 animals/genotype.

Data information: Scale bars are 1μm. Data are presented as mean ± SD. One-way ANOVA test.
miR-181a/b downregulation:a mutation-independent therapeutic approach for Inherited Retinal Diseases (2022)
Figure 5

Figure 5: miR-181a/b-1 downregulation decreases Drp1 levels via regulation of the Jak2/Stat3 pathway. A) WB analysis of Drp1, one of the key proteins involved in the mitochondrial fission pathway, in the optic cup of WT, P347S DMSO-treated, P347S/miR-181a/b+/- DMSO-treated and of P347S/miR-181a/b+/- FEDRATINIB-treated (an inhibitor of the JAK2/STAT3 pathway) mice at p30.

B) Quantification revealed that Drp1 protein levels are increased in P347S with respect to WT, and partially rescued in P347S/miR-181a/b+/-. Data are normalized to Gapdh. N ≥ 4 eye/genotype. Please note that all compared bands from WT, P347S, P347S/miR-181a/b+/- and P347S/miR-181a/b+/- with FEDRATINIB samples are from the same blots, which were cropped and shown organized in the panel for the sake of data presentation clarity (see source data).

C-D) qRT-PCR analysis reveals decreased levels of the Drp1 transcript and increased levels of Stat3 and Jak2 transcripts in the eyes of P347S/miR-181a/b+/- vs those of P347S animals. N≥4 eyes/genotype.

E) qRT-PCR analysis on ex vivo retinas reveals that the treatment with FEDRATINIB counter-rescued the upregulation of Irf1 and the downregulation of Drp1 transcript levels in P347S/miR-181a/b+/- animals. N≥4 eyes/genotype

F) Quantification of WB in A reveals decreased levels of Drp1 in P347S/miR-181a/b+/- DMSO-treated vs P347S and increased levels in P347S/miR-181a/b+/- FEDRATINIB-treated ex-vivo retinas. N≥4 eyes/genotype.

Data information: Data are presented as mean of Fold Change ± SEM. Student's t-test, unpaired.
miR-181a/b downregulation:a mutation-independent therapeutic approach for Inherited Retinal Diseases (2022)
Figure 6

Figure 6. AAV2/8-Sponge-miR-181a/b delivery ameliorates the retinal phenotype of P347S mice.

A-C) ERG response (b-wave) reported as Delta (∆) amplitude in P347S animals injected at p4 with AAV2/8.CMV.GFP-Sponge-miR-181a/b, AAV2/8.CMV.RHO-Sponge-miR-181a/b, AAV2/8.VMD2.GFP-Sponge-miR-181a/b vectors with respect to the corresponding controls (AAV2/8.CMV.GFP, AAV2/8.RHO.GFP and AAV2/8.VMD2.GFP; red dotted line) at p30 (A; N≥18 eyes/treatment, for each promoter vector) and p70 (B; N≥15 eyes/treatment, for each promoter vector). P values of comparison between AAV2/8.CMV.RHO-Sponge-miR-181a/b-injected eyes and corresponding control (AAV2/8.RHO.GFP) are shown in red, whereas p values of comparison between AAV2/8.CMV.GFP-Sponge-miR-181a/b-injected eyes and corresponding controls (AAV2/8.CMV.GFP) are shown in black. Data are presented as mean of Delta (∆) amplitude ± SD. Two-way ANOVA test. P30 representative curves at 20 candles are reported in (C).

(D-U) Immunofluorescence analysis showed amelioration of Rhodopsin localization (p30 D-G', p70 L-O'; white arrowheads) and C-arrestin (p30 H-K', p70 P-S'; white arrowheads) expression in Sponge-miR-181a/b-injected eyes vs the corresponding controls also highlighting improvement of the OS structure. At both time points the effect of amelioration was more significant and evident when the AAV2/8.CMV.GFP-Sponge-miR-181a/b was used. Scale bars are 25 μm. Fluorescence densitometry quantification of each staining is reported in T for p30 and in U for p70, N=3 eye/treatment/staining. Data are presented as mean of Fold Change ± SEM. Student's t-test, paired.
miR-181a/b downregulation:a mutation-independent therapeutic approach for Inherited Retinal Diseases (2022)
Figure 7

Figure 7. AAV2/8-Sponge-miR-181a/b delivery ameliorates the retinal phenotype of rd10 mice.

A-I') Immunofluorescence analysis on central retinal sections at p30 showed ONL thickness increase, amelioration of Rhodopsin localization (A-D') and C-arrestin expression (E-H') in AAV2/8.CMV.GFP-Sponge-miR-181a/b-injected vs AAV2/8.CMV.GFP-injected rd10 eyes. Scale bars are 25 μm. Fluorescence densitometry quantification of each staining is reported in I, N=3 eye/treatment/staining. Data are presented as mean of Fold Change ± SEM. Student's t-test, paired.

(J-K) Analysis of retinal function based on pupillary light responses (PLR) Water Maze tests. (J) PLR showed a significantly higher pupil constriction in rd10 mice injected with AAV2/8.CMV.GFP-Sponge-miR-181a/b than in AAV2/8.CMV.GFP-injected rd10 eyes, at both 10 lux and 100 lux intensity stimuli (N≥6 eyes/treatment and N=9 eyes/treatment, respectively). Data are presented as mean ± SEM. Student's t-test, unpaired. (K) Water Maze test highlighted a shorter latency in reaching the platform in AAV2/8.CMV.GFP-Sponge-miR-181a/b-injected animals respect to control-injected ones (N≥6 eyes/treatment). Data are presented as mean ± SD. ANOVA test. S1 is for Session1 and S2 is for Session2.
Modeling population size independent tissue epigenomes by ChIL-seq with single thin sections (2021)
Figure 1

Figure 1: Epigenomic profiling using a single tissue section.

(A) Schematic diagram of the ChIL protocol for tissue.

(B-D) Immunofluorescent images of mouse liver (B), testis (C) and heart (D). H3K27ac, PolII-S5P, and laminin were stained with specific primary antibodies and visualized using fluorescent labeled anti-mouse ChIL-probe (red: H3K27ac and PolIIS5P) and anti-rabbit IgG (green: laminin). DNA was counterstained with Hoechst 33342. Scale Bar: 20 µm (left images), 10 µm (right images).

(E) Genome browser images of ChIL-seq for H3K27ac and PolII-S5P and bulk tissue RNA-seq data at the Alb locus in liver tissues.

(F) Library complexity of ChIL data. Poisson represents an ideal case of the uniform probability of obtaining reads from the mouse genome, whereas preseq refers to the future/past predictions of a species discovery curve of sequenced reads using Preseq(Daley & Smith, 2013a). Black circle indicates the read number we sequenced for this prediction.

(G) Breakdown of mapped reads at the annotated genomic regions. Gene body: 3'-UTR, exon, intron, 5'-UTR; Others: ncRNA, miRNA, snoRNA, and pseudogenes. The proportions of the annotated region on the mouse genome are shown as "Genome" at the bottom lane.

(H) Genome-wide correlation at 10 kbp bins. Hierarchical clustering of Pearson's correlation coefficient of log-transformed ChIL-seq counts is shown.
Modeling population size independent tissue epigenomes by ChIL-seq with single thin sections (2021)
Figure 2

Figure 2: Upstream factor identification through enhancer analysis using ChIL-seq.

(A) Tissue specificity of identified enhancers by H3K27ac ChIL-seq. The odds ratios of hits in the reference tissue-specific enhancer list identified by bulk-tissue ChIP-seq data(Shen et al, 2012) are shown. Odds is defined in the Materials and Methods. The cells enclosed by black squares indicate the maximal odds ratios (i.e., maximal specificity) for each row.

(B) The IGV tracks of H3K27ac ChIL-seq at identified tissue-specific enhancers of Rxra, Gnat3, Eps8, and a house-keeping gene of Actb loci are shown with the replicates.

(C) Specific motif enrichment analysis was conducted using chromVAR(Schep et al, 2017). Hierarchical clustering of deviation-Z scores of three replicates of each tissue is shown.

(D) Super-enhancer identification. Tissue-specific enhancers are identified, so that they are listed more than twice (twice: blue, all: red) in the top 5% in all enhancer candidates and are not in the SEs of other tissues. Grey shades indicate the top 5% of tag count in enhancer candidates.

(E) Immunofluorescent images of mouse liver sections. Tissues were stained with anti-Hnf4ɑ antibody and visualized by a fluorescent-labeled anti-mouse ChIL probe. DNA was counterstained with Hoechst 33342. Scale bar: 200 µm (top), 10 µm (bottom).

(F) Hnf4ɑ binds to the SE at Alb gene loci.

(G) Gene set enrichment analysis of Hnf4ɑ-bound genes (top), and their rate of Hnf4ɑ-bound genes in sliding windows of 100 genes (bottom).
Modeling population size independent tissue epigenomes by ChIL-seq with single thin sections (2021)
Figure 3

Figure 3: PolII ChIL-seq detect active genes in tissue.

(A) Dynamic ranges of bulk-tissue RNA-seq and PolII S5P ChIL-seq. The cumulative proportion in total mapped reads at genes (red: ChIL-seq, blue: RNA-seq) were compared. Genes are ordered by the read counts on the exons for RNA-seq and on +/-750 bp from TSS for ChIL-seq, respectively.

(B) Signal intensities of ChIL-seq correlated with the expression levels of genes. The lines indicate the average CPM of each expression group at TSS. The expression groups were assigned with respect to the expression levels (TPM) of genes.

(C) Coverage of expressed genes by PolII S5P ChIL-seq peaks. The stacked bar chart shows the proportions of detected genes in the RNA-seq only (RNA-seq: blue), PolII S5P ChIL-seq only (ChIL: red) and both (Common: green).

(D) Higher expression levels at PolII S5P ChIL-seq peaks. The expression levels of all expressed genes (TPM > 0) are shown.

(E) The tissue-specific genes identified by PolII S5P ChIL-seq. The IGV tracks of all replicates of PolII S5P ChIL-seq are shown at each specific gene (Trf, Myh6, and Meig for the liver, heart, and testis, respectively). Actb is also shown as the ubiquitously expressed gene in the three tissues.
Modeling population size independent tissue epigenomes by ChIL-seq with single thin sections (2021)
Figure 4

Figure 4: Statistical modeling of the traveling ratio reveals the independent dynamics between population and transcriptional regulation in regenerating skeletal muscle tissues.

(A) Immunofluorescent images of the mouse tibialis anterior muscle on the indicated days after CTX treatment. The images of anti-mouse ChIL probe for PolII-S5P (red) and anti-rabbit IgG for laminin (green) are shown. Scale bar: 20 µm. Refer to Figure S6A for more frequent time points.

(B) PolII S5P ChIL-seq signal of the marker genes of mature skeletal muscle (Acta1) and macrophages (Cd68).

(C) Proportion of sequenced reads (%UMI) occupied by the representative cell types in muscle regeneration. The single cell data (GSE143437) by De Micheli et al.(De Micheli et al) was re-analyzed. See Figure S6B for the detailed cell-type annotations.

(D) Extraction of independent dynamics of the population and transcriptional regulation. Change in PolII distribution at the gene loci: a gene (blue) was transcriptionally activated (red nuclei) following the stimuli, while population size was unchanged. Change in the height of PolII distribution: a type of cells (yellow) was grown after the stimuli, while the transcriptional activity was maintained.

(E) Estimated mean (95% confidence interval) of TR and the CPM of PolII S5P ChIL-seq at TSS. Representative genes of mature skeletal muscle cells and immune cells are shown.

(F) Bulk-tissue expression levels (TPM) of the representative genes.

(G) Different activities of two major cell-types in muscle regeneration. Scatter plots of log2FC of day-3 vs. day-0 of TR (x-axis) and the TSS-level (y-axis) are shown: immune cell marker genes (left); myogenic genes: right. Colors indicate significance in TR and TSS-level based on |log2FC| > 1 (two-fold) and FDR < 0.1.

(H) Activities of major cell types in muscle regeneration. The colors of the heatmap show the log2FC to day 0 (uninjured) of TR and TSS levels. Representative genes with significant changes in TR are shown.

(I) The dynamics of the biological process in muscle regeneration and the participating cell types. Genes were assigned to five groups (C1-5) based on highest time point of TR. OR indicates the specificity of participation to the biological processes.
Proteasome mapping reveals sexual dimorphism in tissue-specific sensitivity to protein aggregations (2020)
Figure 2

Figure 2: Spinal cord and intestine show sexual dimorphism in Ub-K48 inked proteins and proteasome assembly.

A-D Western blot analysis for K48Ub-linked proteins in spinal cord (A) and intestine (C) lysates from male and female mice. (B,D) Quantification of panels A and C, respectively. Values were normalized to actin as a loading control. Each central line and error bar indicate mean +/- SEM of a biological replicates.

E) Curve of correlation between chymotrypsin-like activity and Ub-K48 linked proteins in the intestine, where variability between mice is observed.

F) Native gel followed by western of β5 subunit to distinguish unassembled catalytic core (CP) or assembled with regulatory core (RP) in male (n=5) and females (n=5) mice in intestine.

G) Native gel followed by western blot of β5 subunit to distinguish unassembled catalytic core (CP) or assembled with regulatory core (RP) in male (n=5) and female (n=5) spinal cord.

H) Quantification of the ratio of assembled (RP-CP) versus unassembled (CP) proteasome in the intestine of males and females. Each central line and error bar indicate mean +/- SEM of a biological replicates (n=5 per group).

I) Quantification of the ratio of RP-CP versus CP proteasome in the spinal cord of males and females. Each central line and error bar indicate mean +/- SEM of a biological replicates (n=5 per group).

J) Correlation curve between chymotrypsin-like activity and RP-CP/CP ratio in individual female (purple) and male (blue) intestine.

K) Correlation curve between chymotrypsin-like activity and RP-CP/CP ratio in individual female (purple) and male (blue) spinal cord.

Data information: * indicates p<0.05 by unpaired two-tailed students t-test comparing male and female samples.
Prostaglandin E2 promotes intestinal repair through an adaptive cellular response of the epithelium (2016)
Figure 1

Figure 1. Prostaglandin E2 induces differentiation of intestinal epithelial stem cells to wound-associated epithelial cells.

A Schematic of spheroid differentiation method. L-WRN CM, conditioned medium from an L cell line engineered to secrete Wnt3a, R spondin 3 and Noggin. Y-27632, ROCK inhibitor. EGF, epidermal growth factor.

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. (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). (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. (E) Quantification of the average expression ± s.e.m. of Cldn4 mRNA relative to the 0 nM treatment group (n = 3 independent experiments). (F) Quantification of average spheroid area ± s.e.m. relative to spheroids treated with 0 nM Iloprost (n = 4 images from two independent experiments). ***p<0.001, ****p<0.0001 by one-way ANOVA and Dunnett's post-test.
Prostaglandin E2 promotes intestinal repair through an adaptive cellular response of the epithelium (2016)
Figure 2

Figure 2. PGE2 signals through the prostaglandin receptor EP4 to induce differentiation of wound-associated epithelial cells.

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. (C) Quantification of average spheroid area ± s.e.m. relative to spheroids treated with DMSO alone (n = 4 images from two independent experiments). (D) Quantification of the average expression ± s.e.m. of Cldn4 mRNA relative to stem cell spheroids (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.

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) and (I) average expression ± s.e.m. of Cldn4 mRNA (n = 3 independent experiments) relative to EP4i-treated Ptger4flox/flox spheroids.

J-L Human ileal 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) and (L) the average expression ± s.e.m. of CLDN4 mRNA (n = 3 independent donor lines) relative to stem spheroids.