EMBO/soda-nel huggingface dataset:
An RNA-binding atypical tropomyosin recruits kinesin-1 dynamically to oskar mRNPs (2016)
Figure 6

Figure 6: Accumulation of oskar mRNPs around the NE

(A-C) Localization of EmGFP-Tm1-I (A) oskar mRNA (B) and Khc (C) around the nurse cell nuclear envelope (magenta, WGA staining).

(D-F) Mean distribution profile of Khc (D) mKate2-Tm1-I, EmGFP-Tm1-I (E) and oskar mRNA (F) around the NE of nurse cells (marked with a dotted vertical line). Genotypes are indicated as follows: wild-type control with solid black line (D-F), oskar RNA null with solid grey line (D, E), Tm1eg9/Tm1eg9 with dotted black line (D, F) Lines indicate mean and 95% confidence intervals. We analysed the accumulation of mKate2-Tm1-I instead of EmGFP-Tm1-I due to unexpected GFP expression from the oskar0 allele.

(G) Khc accumulation around the NE. To calculate accumulation the signal intensity measured at the position of the peak observed in the wild-type control (D, arrowhead, 356±17.6 nm away from NE) was divided by the signal intensity 2 s.d. away (d, arrow, at 356+2*410 nm). P values of pairwise Mann-Whitney U tests against wild-type control or Tm1gs rescued with EmGFP-Tm1-I are indicated above the boxplots. Numbers indicate the number of nuclei and the number of egg-chambers (in brackets) analysed.

(H-K) Khc accumulation around the NE of nurse cells over-expressing EmGFP-Tm1-I within Tm1eg9/Tm1eg9 (H), and oskar RNA null egg-chambers (I) expressing either the oskar 3’ UTR (J) or oskar Δi(1,2,3) (K).

Scale bars are 10 µm (A-C), 5 µm (H-K).
An RNA-binding atypical tropomyosin recruits kinesin-1 dynamically to oskar mRNPs (2016)
Figure 7

Figure 7: Effects of EmGFP-Tm1-I overexpression on oskar mRNA localization

(A-E) Mean oskar mRNA distribution (green, detected by conventional FISH) within oocytes in which oskar mRNA is substituted by oskar Δi(1,2,3) mRNA (A, B), oskar 3’UTR mRNA (D) and that (in addition) over-express EmGFP-Tm1-I (B, D and E). Wild-type control (C) and oocytes expressing Khc RNAi and EmGFP-Tm1-I (E). Magenta indicates standard deviation of the distribution of oskar mRNA. Scale bar is 10% of total oocyte length (C).

(F) Position of the oskar mRNA center of mass relative to the geometric center of the oocyte (dotted horizontal line) along the anteroposterior (AP) axis. Posterior pole is the top of the chart. P values of pairwise Mann-Whitney U tests are indicated. Numbers indicate number of oocytes analysed.
A subpopulation of itch sensing neurons marked by Ret and Somatostatin expression (2016)
Figure 1

Figure 1. Ret-eGFP is Expressed in Multiple Sensory Neuron Subsets

(A−L) Ret-positive neurons largely overlap with markers for myelinated neurons (NF200), non-peptidergic nociceptors (IB4) and C-fiber low-threshold mechanoreceptors (TH). However some Ret positive neurons are negative for these markers (indicated by the arrows in D,H and L) suggesting the existence of a further subset of Ret+ neurons. (M) Quantification of the proportion of Ret-eGFP positive neurons in DRG . (N) Quantification of Ret-eGFP co-expression with other markers. (n=2278 cells from 3 mice) Scale bars, 50 μm.
A subpopulation of itch sensing neurons marked by Ret and Somatostatin expression (2016)
Figure 2

Figure 2. Multiple Ret-positive Subpopulations as Determined by Flow Cytometric Analysis of Dissociated Sensory Neurons from Avil-Cre::Ret+/eGFP Mice

(A) Flow cytometric analysis of dissociated sensory neurons plotted according to their level of endogenous Ret-eGFP expression and IB4 binding (also indicated in Figure EV1). There are 5 well-defined subsets, 2 of which do not bind to IB4 but display a differential level of eGFP intensity (termed Ret-eGFPLo:IB4Neg, A, and Ret-eGFPHi:IB4Neg ,B, respectively). The other 3 subsets bind IB4 and display a range of eGFP intensities, termed Ret-eGFPLo:IB4Lo, C, Ret-eGFPHi:IB4Lo, D, and Ret-eGFPHi:IB4Hi, E. Percent of total events are indicated in each box. (B) No correlation between the median cell size of different Ret+ subsets and eGFP intensity or IB4 binding. The graph shows the forward scatter values plotted against the normalized number of cells, displayed as the percent of Max. (C−D) Variations in endogenous eGFP intensity are clearly visible with fluorescent microscopy. (C) Cultured neurons and (D) DRG section from Avil-Cre::Ret+/eGFP mice displaying native eGFP fluorescence and stained with IB4. Different levels of eGFP intensity (high -indicated by arrows and low -indicated by arrowheads) are detected across IB4+ and IB4- cells. Scale bar, 50 μm.
A subpopulation of itch sensing neurons marked by Ret and Somatostatin expression (2016)
Figure 3

Figure 3. Transcriptional profiling of Ret-eGFPHi:IB4Neg and Ret-eGFPLo:IB4Neg Sensory Neurons.

(A) Ret-eGFPHi:IB4Neg and Ret-eGFPLo:IB4Neg neurons display distinct expression profiles. A volcano plot of fold change expression in Ret-eGFPLo:IB4Neg versus Ret-eGFPHi:IB4Neg against probability. Ret has higher expression in the Ret-eGFPHi:IB4Neg subset, which also shows an up regulation of the Ret co-receptor Gfra2 and Fam38b, encoding for the mechanosensitive ion channel Piezo2. The Ret-eGFPLo:IB4Neg subset, displays an array of molecules previously associated with itch (marked in red). (B) Itch-associated transcripts are enriched in the Ret-eGFPLo:IB4Neg population compared to all DRG neurons. Volcano plot of fold change expression in Ret-eGFPLo:IB4Neg neurons versus sorted neurons from Avil-Cre::R26tdRFP mice against probability. Molecules linked to itch perception are significantly upregulated in the Ret-eGFPLo:IB4Neg subset. (C) Gene expression within the Ret-eGFPLo:IB4Neg population is confirmed by triple comparison between Ret-eGFPLo:IB4Neg, Ret-eGFPHi:IB4Neg and Avil-Cre::R26tdRFPdatasets. (D) Validation of differential microarray screening by quantitative RT-PCR. Transcripts encoding ion channels, receptors, neuropeptides, signaling molecules and transcription factors were selected for quantitative RT-PCR. Differential expression between Ret-eGFPLo:IB4Neg and Ret-eGFPHi:IB4Neg populations correlates with microarray analysis (n=3).
A subpopulation of itch sensing neurons marked by Ret and Somatostatin expression (2016)
Figure 4

Figure 4. SstCre Driven Expression in DRG and Skin.

(A−H) SstCre mediated recombination of the Ret locus drives eGFP expression in sensory neurons that do not bind to IB4 or co-express NF200 or TH. Triple immunostaining of DRG from Sst-Cre::ReteGFP/+ mice with RetGFP (A), IB4 (B) and NF200 (C), and RetGFP (E), IB4 (F) and TH (G). (I) Quantification of SstCre::Ret-eGFP expression in DRG. (J) Quantification of co-expression of SstCre::Ret-eGFP with neuronal markers (n=8827 cells from 3 mice). (K−M) Peripheral projections of sensory neurons from Sst-Cre::Rosa26SnapCaaX mice terminate in the hairy skin as free nerve endings. Non-innervated hair follicles are indicated by arrows. Double labelling using TMRStar to label SNAP-tag (K) and DAPI (L). Scale bars, 50 μm.
A subpopulation of itch sensing neurons marked by Ret and Somatostatin expression (2016)
Figure 5

Figure 5. Flow cytometric and transcriptome analysis of Sst-Cre::ReteGFP versus Ret-eGFPHi:IB4Neg sensory neurons.

(A-B) Contour diagram of dissociated sensory neurons from Sst-Cre::ReteGFP/+ (A) and Avil-Cre::ReteGFP/+neurons (B), plotted according to their native eGFP expression and IB4 binding. Only one subset of Ret-eGFP cells can be defined within the Sst-Cre::ReteGFP/+ dissociated DRG neurons. This displays the same range of eGFP fluorescence as the Ret-eGFPLo:IB4Neg population and doesn’t bind to IB4. (C) Quantitative RT-PCR analysis of selected transcripts in the Sst-Cre::ReteGFP (darker colors) and Ret-eGFPHi:IB4Neg (lighter colors) subsets displayed as differential expression compared to the Ret-eGFPHi:IB4Neg subset. Almost all the genes that are up-regulated in the Ret-eGFPLo:IB4Neg compared to the Ret-eGFPHi:IB4Neg subset are also enriched in the Sst-Cre::ReteGFP population (n=3)
A subpopulation of itch sensing neurons marked by Ret and Somatostatin expression (2016)
Figure 6

Figure 6. Diphtheria toxin mediated ablation of SstCre positive DRG neurons.

(A-G) Ablation of Sst-Cre::AviliDTR positive sensory neurons by injection of diphtheria toxin (DTX). Immunostaining of DRG from Sst-Cre::AviliDTR mice with an antibody against the diphtheria toxin receptor and IB4 in the absence of diphtheria toxin (A-C) and after systemic injection of diphtheria toxin (D-F). (G) Quantification of diphtheria toxin receptor positive neurons before and after injection of diphtheria toxin. (H-K) Examples of calcium flux in dissociated DRG upon application of histamine (H), chloroquine (I), IL-31 (J) and 5HT1F agonist Ly344864 (K). (L) Quantification of percent responding neurons to the indicated pruritogens before or after systemic injection of diphtheria toxin in Sst-Cre::AviliDTR mice. Scale bars, 50 μm, asterisk denotes p<0.05, n=4-6 mice, Student’s t-test, error bars indicate SEM.
A subpopulation of itch sensing neurons marked by Ret and Somatostatin expression (2016)
Figure 7

Figure 7. Reduced scratching behavior in Sst-Cre::AviliDTR mice after diphtheria toxin mediated ablation.

(A-B) Nociceptive withdrawal thresholds in control (AviliDTR without the Cre) and Sst-Cre::AviliDTR mice after systemic diphtheria toxin injection. No significant differences in thermal (A) or mechanical (B) withdrawal reflexes after ablation of SstCre positive neurons. (C-F) Scratching behavior after a single intradermal injection of the indicated pruritogen observed for 1 hour. All compounds evoked a significant increase in the number of scratching bouts compared to vehicle alone in control (AviliDTR) or Sst-Cre::AviliDTR mice. Diphtheria toxin injection in control mice (AviliDTR) had no effect on scratching behavior evoked by histamine (C), chloroquine (D), IL-31 (E) or 5HT1F agonist Ly344864 (F) but significantly reduced responses to IL31, or Ly344864 in Sst-Cre::AviliDTR mice. n-numbers indicated in brackets, asterisk denotes p<0.05, two way RM ANOVA, Holm-Sidak multiple comparison. Error bars indicate SEM.
Gut microbiota-CRAMP axis shapes intestinal barrier function and immune responses in dietary gluten-induced enteropathy (2021)
Figure 1

Figure 1. Epithelial CRAMP production is defective in dietary gluten-induced enteropathy (GIE).

(A) Animal protocol.

(B) Serum CRAMP determination by ELISA (n = 8).

(C, D) Western blot of pro-form CRAMP and mature peptide in duodenum and ex vivo duodenal epithelial cells.

(E) Localization and expression of CRAMP (red), E-cadherin (green), F4/80 (green) and Ly6G (green) in duodenum by immunofluorescent staining. Representative photomicrographs of individual and merged staining were shown. Nuclei were stained with DAPI (blue). Scale bar: 50 μm.

Data information: Data in B were representative and were the mean ± SD from three independent experiments. Data (C-E) were representative from three independent experiments. P values were calculated by unpaired two-tailed t-test for comparison of two groups (B).
Gut microbiota-CRAMP axis shapes intestinal barrier function and immune responses in dietary gluten-induced enteropathy (2021)
Figure 2

Figure 2. CRAMP deficiency potentiates GIE.

(A) Animal protocol.

(B) Changes in body weight relative to starting weight during 6 weeks (n = 6).

(C) Left, representative images of duodenal damage by H&E staining. Scale bar: 200 μm. Right, graph depicted the ratio of the morphometric assessment of villus height to crypt depth (n = 6).

(D) Western blot and densitometry analyses of duodenal tight junction proteins (claudin-1, occludin, ZO-1 and ZO-2; n = 6).

(E) Western blot and densitometry analysis of duodenal zonulin (n = 6).

(F) Intestinal permeability was assessed by measuring FITC-dextran (n = 6).

Data information: Data (B-F) were representative and were the mean ± SD from three independent experiments. P values were calculated by one-way ANOVA followed by Tukey's post hoc test for multiple comparisons..
Gut microbiota-CRAMP axis shapes intestinal barrier function and immune responses in dietary gluten-induced enteropathy (2021)
Figure 3

Figure 3. Replenishing duodenal CRAMP is protective against GIE.

(A) Animal protocol.

(B) Duodenal CRAMP determination by ELISA (n = 6).

(C) Changes in body weight relative to starting weight during 6 weeks (n = 6).

(D) Left, representative images of duodenal damage by H&E staining. Scale bar: 200 μm. Right, graph depicted the ratio of the morphometric assessment of villus height to crypt depth (n = 6).

(E) Western blot and densitometry analyses of duodenal tight junction proteins (claudin-1, occludin, ZO-1 and ZO-2; n = 6).

(F) Western blot and densitometry analysis of duodenal zonulin (n = 6).

(G) Intestinal permeability was assessed by measuring FITC-dextran (n = 6).

Data information: Data (B-G) were representative and were the mean ± SD from three independent experiments. P values were calculated by one-way ANOVA followed by Tukey's post hoc test for multiple comparisons.
Gut microbiota-CRAMP axis shapes intestinal barrier function and immune responses in dietary gluten-induced enteropathy (2021)
Figure 4

Figure 4. CRAMP counteracts gliadin-triggered inflammatory signaling to protect epithelial function.

(A) Western blot and densitometry analyses of gliadin-triggered inflammatory signaling [p-EGFR (Tyr1068), p-Akt, Akt, MyD88, TRAF6, p-p65 and p65] in epithelial cells ex vivo (n = 6).

(B) Western blot and densitometry analyses of gliadin-triggered inflammatory signaling [p-EGFR (Tyr1068), p-Akt, Akt, MyD88, TRAF6, p-p65, p65 and zonulin] in vitro (n = 6).

Data information: Data (A and B) were representative and were the mean ± SD from three independent experiments. P values were calculated by one-way ANOVA followed by Tukey's post hoc test for multiple comparisons.
Gut microbiota-CRAMP axis shapes intestinal barrier function and immune responses in dietary gluten-induced enteropathy (2021)
Figure 5

Figure 5. CRAMP modulates intestinal immune disorder in GIE.

(A) Il15 expression in duodenal epithelial cells ex vivo (n = 8).

(B) The frequency of NKG2D+ IELs among CD3+CD4+ population (n = 8).

(C) The frequency of CX3CR1+ macrophages in CD45+CD11b+ population and ALDH activity in CD45+CD11b+CX3CR1+ macrophages in lamina propria (n = 8).

(D) The percentages of three subsets of CD45+CD11c+ DCs and ALDH activity in CD45+CD11c+ DCs in lamina propria (n = 8).

(E-G) The percentages of (E) Foxp3+ Treg, (F) T-bet+ Th1 and (G) RORγt+ Th17 among CD3+CD4+ population in lamina propria were shown by flow cytometry (n = 8).

Data information: Data in A were representative and were the mean ± SD from two independent experiments. Data (B-G) were representative and were the median ± interquartile range from three independent experiments. P values were calculated by one-way ANOVA followed by Tukey's post hoc test for multiple comparisons.
Gut microbiota-CRAMP axis shapes intestinal barrier function and immune responses in dietary gluten-induced enteropathy (2021)
Figure 6

Figure 6. Gut dysbiosis contributes to CRAMP degradation.

(A) The unweighted (left) and weighted (right) UniFrac distances based on OTU abundance (NC: gluten-free, M: gluten; n = 6).

(B) The taxonomic composition distribution at phylum level (n = 6).

(C) The taxonomic composition distribution at genus level (n = 6).

(D) Duodenal colonizing P. aeruginosa and A. muciniphila as determined by RT-qPCR using bacterial-specific species gene primers (n = 6).

(E) The production of LasB (a protease of P. aeruginosa) was examined in duodenum (n = 6).

(F) Dot plot of Pearson correlation coefficients among CRAMP, P. aeruginosa and A. muciniphila at species level. The size of each point represented the correlation coefficient and the color represented positive (red) or negative (purple) relationship (n = 6).

(G) Animal protocol.

(H) Duodenal colonizing PA14 and PAO1 as determined by RT-qPCR using P. aeruginosa-specific species gene primer (n = 9).

(I) Western blot and densitometry analyses of pro-form CRAMP and mature peptide in duodenum (n = 8).

Data information: Data (D, E, H, I) were representative and were the mean ± SD from three independent experiments. P values were calculated by unpaired two-tailed t-test for comparison of two groups (B-E) or one-way ANOVA followed by Tukey's post hoc test for multiple comparisons (H, I).
Gut microbiota-CRAMP axis shapes intestinal barrier function and immune responses in dietary gluten-induced enteropathy (2021)
Figure 7

Figure 7. Pseudomonas aeruginosa depletion restores CRAMP production and dampens GIE.

(A) Animal protocol.

(B) The levels of CRAMP in serum (n = 8).

(C) The levels of CRAMP in duodenum (n = 6).

(D) Representative images of intestinal damage by H&E staining. Scale bar: 200 μm. The graph depicted the ratio of the morphometric assessment of villus height to crypt depth (n = 6).

(E) Western blot and densitometry analyses of tight junction proteins (claudin-1 and occludin) and zonulin in duodenum (n = 6).

(F) Intestinal permeability was assessed by measuring FITC-dextran (n = 6).

(G) The frequency of NKG2D+ IELs among CD3+CD4+ population (n = 8).

(H) The frequency of CX3CR1+ macrophages among CD45+CD11b+ population (n = 8).

(I) The percentages of three subsets of CD45+CD11c+ DCs (n = 8).

(J-L) The percentages of (J) Foxp3+ Treg, (K) T-bet+ Th1 and (L) RORγt+ Th17 among CD3+CD4+ population in lamina propria were shown by flow cytometry (n = 8).

Data information: Data (B-F) were representative and were the mean ± SD from three independent experiments. Data (G-L) were representative and were the median ± interquartile range from three independent experiments. P values were calculated by one-way ANOVA followed by Tukey's post hoc test for multiple comparisons.

Expanded view figure legends
Histological Hallmarks and Role of Slug/PIP Axis in Pulmonary Hypertension Secondary to Pulmonary Fibrosis (2020)
Figure 1

Figure 1: Pulmonary vascular remodeling pattern in PF-PH patients is distinct from patients with PF alone. (A) Masson's trichrome representative images of pulmonary fibrosis and quantification. (B) Relative transcript expression of Fibronectin, (C) Collagen I and (D) Collagen III to GAPDH. (E) Masson's trichrome representative images of vascular wall thickening and quantification. (F) Masson's trichrome representative images and correlation between lung fibrosis and vascular wall thickness in PF and PF-PH patients. Out of the 14 PF and PF-PH patients, 3 of them had no non-fibrotic area and therefore were excluded for comparison between non-fibrotic and fibrotic areas (n=11/group; Ashcroft score non-fibrosis: 0-2; mild-fibrosis: 3-5; severe-fibrosis >5) (* compared to non-fibrotic areas of the same group of patients). (G) Masson's trichrome representative images of vascular wall thickening in non-fibrotic area (Ashcroft score 0-2) and fibrotic area (Ashcroft score >2) and quantification in PF and PF-PH. The number of samples per group for each experiment are written within each bar graph. Statistical test: panels A, E, F, G: ANOVA; panels B, C, D: t-test (* vs. Ctrl, † vs. PF, ! vs. PAH, $ vs. PF-PH; **** p<0.0001, †††† p<0.0001, !!!! P<0.0001, $p<0.05, $$$$p<0.0001).
Histological Hallmarks and Role of Slug/PIP Axis in Pulmonary Hypertension Secondary to Pulmonary Fibrosis (2020)
Figure 2

Figure 2: PF-PH patients exhibit increased proliferation of vascular wall cells and increased Slug expression. (A) Representative images of Ki67-positive pulmonary vascular endothelial cells (EC, vWF in green) and smooth muscle cells (SMC, aSMA in white) and quantification (* vs PF, *p<0.05, ***p<0.001). Relative expression of Slug mRNA (B) and protein (C) normalized to GAPDH in PF and PF-PH patients. (D) Representative images of Slug (red) expression in bronchial epithelial cells (EpC), fibroblasts (Fib) (vimentin in green) macrophages (Mф, CD68 in green) pulmonary vascular endothelial cells (EC, von Willebrand Factor in green) and pulmonary vascular smooth muscle cells (SMC, aSMA in white); and quantification. (E) Relative CD68 mRNA expression normalized to GAPDH (F) Representative images of Slug (red) expression in macrophages (Mф , CD68 in green) in non-fibrotic and fibrotic areas of the lung in PF and PF-PH patients and quantification. The number of samples per group for each experiment are included within each bar graph. Statistical test: panels A to D: t-test; panel E ANOVA (* vs. PF; * p<0.05)
Histological Hallmarks and Role of Slug/PIP Axis in Pulmonary Hypertension Secondary to Pulmonary Fibrosis (2020)
Figure 3

Figure 3: Online lung microarray analysis revealed PIP as one of the Slug transcriptional targets mediating vascular cell proliferation in PF-PH. (A) Heatmap of the 20 genes up-regulated in GSE24988 microarray and known to be Slug transcriptionnal target. (B) Venn Diagram showing the overlap between Slug targets implicated in cell proliferation and known to be extracellular. (C) Heatmap of the seven transcriptional targets of Slug known to promote proliferation and to be extracellular. Relative expression of Prolactin-induced protein (PIP) mRNA (D) and protein (E) expression normalized to GAPDH in PF and PF-PH patients. (F) Representative images of PIP immunohistochemistry showing its extracellular localization and quantification. Proliferation assay on healthy fibroblast (G) Pulmonary arterial EC (H) and on healthy pulmonary arterial SMC (I and J) in the presence or absence of PIP protein. The number of samples per group for each experiment are included within each bar graph. Statistical test: panel D to F: t-test; panel G to J: ANOVA (* p<0.05, **p<0.01 ***p<0.001).
Histological Hallmarks and Role of Slug/PIP Axis in Pulmonary Hypertension Secondary to Pulmonary Fibrosis (2020)
Figure 4

Figure 4: An animal model recapitulating most of the histological hallmarks of PH secondary to PF. (A) Masson's trichrome representative images of lung fibrosis and quantification two weeks after PBS or bleomycin instillation. Quantification of (B) fibronectin, (C) Collagen I and (D) Collagen III mRNA. (E) Schematic of experimental protocol. Animals were divided into 4 groups: control (Ctrl), pulmonary fibrosis (PF), pulmonary hypertension (PH) and PF secondary to PH (PF-PH). At day 0, rats in PF and PF-PH groups received an intra-tracheal instillation of Bleomycin (2.5mg/Kg), whereas Ctrl and PH groups received a PBS instillation. At day 14, PH and PF-PH groups received a subcutaneous injection of monocrotaline (MCT, 60mg/kg), whereas Ctrl and PF groups received a subcutaneous injection of PBS. (F) Right ventricular systolic pressure (RVSP) and (G) Right ventricular hypertrophy index following the experimental protocol. (H) Masson's trichrome representative images and quantification of Ashcroft score in the experimental model. Relative expression of fibronectin (I), collagen I (J) and collagen III (K) mRNA normalized to GAPDH. (L) Masson's trichrome representative images and quantification of vascular remodeling. (M) Masson's trichrome representative images and quantification of the vascular remodeling between fibrotic and non-fibrotic areas in the experimental model. The number of samples per group for each experiment are included within each bar graph. Statistical test : panel A to D : t-test; panel F to M : ANOVA (* vs. Ctrl, † vs. PF, ! vs. PH; * p<0.05, **p<0.01, *** p<0.001, ****p<0.0001, † p<0.05, †††p<0.001. †††† p<0.0001, ! P<0.05, !!!! p<0.0001).