EMBO/soda-nel huggingface dataset:
TMEM16A expression in cholinergic neurons of medial habenula mediates anxiety-related behaviors (2019)
Figure 3

Fig 3. TMEM16A deficiency reduced the frequency of spontaneous firing of the mHb neurons. A. Immunohistochemical staining of 40-µm coronal brain sections with an anti-TMEM16A antibody. The TMEM16A signals in the mHb of TMEM16A cKO mice were compared to the ones of littermate controls. TMEM16A colocalized with the cholinergic marker ChAT in the mHb. The specific deletion of TMEM16A in the mHb is evident. B. An illustration of a brain slice (sagittal view) showing the site of electrophysiological recordings. C. Representative traces demonstrating the spontaneous activity of the mHb neurons in a cell-attached mode from TMEM16A cKO mice compared to the one from littermate controls. D. A summary bar graph showing the frequencies of spontaneous firing of the mHb neurons in TMEM16A cKO (4 mice) and littermate controls (The numbers inside each bar indicate the numbers of recorded cells from 4 mice each, paired Student t-test, data are presented as mean values ± s.e.m.). E. Representative averaged traces of sAPs of the mHb neurons with the pipette solution (40 mM Cl- and 0.5 mM EGTA) from TMEM16A cKO mice and littermate controls. F. Summary bar graphs showing the frequency, half-width, and AHP slope of the sAPs of mHb neurons from TMEM16A cKO mice and littermate controls (The numbers inside each bar indicate the numbers of recorded cells, unpaired Student t-test, data are presented as mean values ± s.e.m.).
TMEM16A expression in cholinergic neurons of medial habenula mediates anxiety-related behaviors (2019)
Figure 4

Fig 4. TMEM16A deficiency reduced the frequency of spontaneous firing of the IPN neurons. A. Immunohistochemical images of the IPN region from control and TMEM16A cKO mice. The c-Fos signal was greatly reduced in TMEM16A cKO mice. Scale bar = 200 μm. B. A summary bar graph showing reduced c-Fos expression in the IPN in TMEM16A cKO mice. (NCTL=7 (n=42), NcKO=6 (n=36), unpaired Student t-test, data are presented as mean values ± s.e.m.). C. An illustration of a brain slice (sagittal view) to show the site of electrophysiological recordings. D. Representative traces of sEPSCs of neurons in the medial part of the IPN showing a reduced frequency in TMEM16A cKO mice compared to that of littermate controls. E & F. Summary bar graphs of the frequency and amplitude of the sEPSCs of IPN neurons in TMEM16A cKO (cKO) mice and littermate controls (CTL). Data are presented as mean values ± s.e.m. (paired Student t-test). The numbers inside each bar indicate the numbers of recorded cells.
Hepatic PPARα function and lipid metabolic pathways are dysregulated in polymicrobial sepsis (2019)
Figure 1

Fig 1. Hepatic PPARα signaling is disturbed at a genome-wide level during sepsis. (A-D) RNA-seq of liver 10h post Sham or CLP. Mice (n = 3/group) underwent a sham or CLP operation and were injected with GW7647 (10 µg/g) 6h post-surgery, after 4h (total of 10h), livers were isolated and RNA was prepared. (A) Scatter plot showing log fold change (LFC) of all GW7647-upregulated genes (LFC > 0.8 and P < 0.05) in sham versus their LFC 10h after CLP. The red line represents the diagonal, the black line represents the real slope (0.3115) of the data. (B) Venn diagram depicting the amounts of genes upregulated (up) or downregulated (dn) by GW7647 in sham and CLP mice (LFC > 0.8 or < -0.8 and P < 0.05). (C) Top enriched gene ontology (GO) terms for genes that are downregulated in CLP mice without stimulation compared to unstimulated sham controls (LFC < -0.8 and P < 0.05). Composite of 3 datasets; CLP1 (6h after CLP), CLP2 (8h after CLP) and CLP3 (10h after CLP). Analysis was performed with the Enrichr tool. (D) Heatmap of differentially expressed genes in sham mice after GW7647 treatment, involved in β-oxidation of fatty acids (unit scale bar = log2 of the normalized counts). (E-F) Confirmation of RNA-seq data via qPCR on pure hepatocytes isolated via flow cytometry-based sorting (n = 3/group). (E) Ppara and (F) Hmgcs2 mRNA expression is shown as relative expression, normalized to housekeeping genes Hprt and Rpl. P-values were calculated using 2-way ANOVA analysis.
Hepatic PPARα function and lipid metabolic pathways are dysregulated in polymicrobial sepsis (2019)
Figure 2

Fig 2. PPARα levels are decreased in the liver during sepsis and correlate with disease severity. (A) Mice (n = 4/group, data are representative of 2 experiments) underwent a sham (with or without starvation) or CLP operation and liver was isolated on several timepoints post-surgery for RNA preparation and qPCR. Ppara mRNA expression is shown as relative expression, normalized to housekeeping genes Hprt and Rpl. P-values were calculated using 1-way ANOVA analysis. (B) PPARA protein levels were analyzed in livers 24h after sham or CLP by Western Blot using actin as a loading control. (C) Quantification of PPARA western blot. P-value was calculated via 2-way Student's t-test (n = 4/group). (D) Pearson correlation between log fold change (LFC) Ppara expression levels and body temperature 24h post-sepsis (n= 38, r = 0.6875, combined data of 4 independent experiments). (E) Liver Hmgcs2 mRNA expression at different timepoints post-sepsis, expression is shown as relative expression, normalized to housekeeping genes Hprt and Rpl. P-values were calculated using 1-way ANOVA analysis (n = 4/group, data are representative of 2 experiments). (F) Oxygen consumption rates (OCR) of liver tissue explants 24h post sham or CLP. Liver tissue was isolated 24h post-surgery and OCR was measured via Seahorse with BSA or palmitic acid (PA) as a substrate for 42 min. n = 4/group. (G) Visualization of T42(min) OCR, p-values were calculated using 2-way ANOVA analysis.
Hepatic PPARα function and lipid metabolic pathways are dysregulated in polymicrobial sepsis (2019)
Figure 4

Fig 4. Lipids accumulate in liver and kidney during sepsis and cause lipotoxicity. (A-B) Immunofluorescent images of liver and kidney 24h after sham (with or without starvation) or CLP (n= 6-7/group, combined data of 2 independent experiments). White scale bar = 20µm. (A) Cryosections were stained with Acti-stain (green), Hoechst (blue), and LipidTOX (red). Z-stacks were generated in 5-10 areas scattered across the entire tissue section. (B) The amount of lipid droplets (LDs)/cell and average size of LDs (represented by voxel counts) were calculated for each Z-stack. Averages of the amount and size of LDs were converged for each mouse and biological replicates are depicted in the table as mean ± SEM. P-values were calculated using 1-way ANOVA tests and can be found in Table EV1. (C-D) Quantification of lipid peroxidation by determination of (C) MDA and (D) 4-HNE concentrations in liver homogenates 24h post-surgery in sham and CLP mice as described in methods (n = 6-7/group, combined data of 2 independent experiments). P-values were calculated with 1-way ANOVA tests. (E) Apoptosis in liver paraffin-fixated sections 24h after sepsis, measured by TUNEL staining, and presented as % PI positive cells/µm² tissue area. (n= 6/7 mice/group, combined data of 2 independent experiments). P-values were calculated with 1-way ANOVA tests.
Hepatic PPARα function and lipid metabolic pathways are dysregulated in polymicrobial sepsis (2019)
Figure 5

Fig 5: The PPARα agonist Pemafibrate reduces mortality of sepsis by stimulating PPARα signaling and improving metabolic parameters. Mice were pre-treated with Pemafibrate (1mg/kg) or vehicle (0,9% NaCl) for 1 week before being subjected to CLP. (A) Survival was monitored during 9 days, after which no further deaths occurred. Survival curve was analyzed via a Log rank test. Combined data of 3 experiments, n = 20/group. (B) Liver samples were isolated 24h after CLP (n= 5-7/group, combined data of 2 independent experiments), mRNA was prepared and gene expression levels of Ppara was analyzed via qPCR. Gene expression values are shown as relative expression, normalized to housekeeping genes Hprt and Rpl. P-values were calculated via 2-way ANOVA test. (C-D) Plasma was isolated 24h after sepsis and (C) FFA concentration and (D) glycerol concentration were determined as described in the method section. P-values were calculated with 2-way ANOVA tests. n = 5-7/group, combined data of 2 independent experiments. Arrows represent the % of decrease caused by pemafibrate treatment during sepsis. (E) Immunofluorescent images of cryosections of liver 24h post-surgery that were stained with Acti-stain (green), hoechst (blue) and LipidTOX (red). Z-stacks were generated in 5-10 areas scattered across the entire tissue section. White scale bar = 20µm. (F) The amount of lipid droplets (LDs)/cell and average size of LDs (represented by voxel counts) were calculated for each Z-stack. Averages of the amount and size of LDs were converged for each mouse and biological replicates are depicted in the table as mean ± SEM. P-values were calculated using two-way Student's t-tests. n = 6/group, combined data of 2 independent experiments. (G-H) Quantification of lipid peroxidation by determination of MDA concentration in (G) liver and (H) kidney homogenates 24h post-surgery in sham and CLP mice (n= 6-7/group, combined data of 2 independent experiments ), as described in methods. P-values were calculated with 2-way ANOVA tests. Arrows represent the % of decrease caused by pemafibrate treatment during sepsis. (I) Oxygen consumption rates (OCR) of liver tissue explants 24h post sham or CLP (vehicle or pemafibrate treated) after supplementation of palmitic acid (PA), measured via Seahorse. Visualization of T42(min) OCR. P-values were calculates using 2-way ANOVA analysis. One experiment, n = 3/group.
Hepatic PPARα function and lipid metabolic pathways are dysregulated in polymicrobial sepsis (2019)
Figure 6

Fig 6: Pemafibrate reduces tissue damage during sepsis. (A-F) Mice were pre-treated with pemafibrate (1mg/kg) or vehicle (0,9% NaCl) for 1 week before being subjected to sham or CLP. (A-C) Plasma was collected 24h post-surgery and (A) Alanine-amino transferase (ALT), (B) creatinine concentrations, and (C) IL-6 levels were measured as described in methods. P-values were calculated with 2-way ANOVA tests. n = 6-7/group, combined data of 2 independent experiments. (D) Systemic bacterial load (CFU/ml blood) 24h post-sepsis in vehicle or pemafibrate treated mice. P-values were calculated with 2-way Student's t-test. (E-F) Apoptosis in (E) liver and (F) kidney paraffin-fixated sections 24h after sepsis, measured with TUNEL staining and depicted as % of PI positive cells/µm² tissue area. P-values were calculated with 2-way ANOVA tests. n = 6-7/group, combined data of 2 independent experiments. (G) Pemafibrate (1 mg/kg) or vehicle (0,9% NaCl) was administered at different timepoints before and after the induction of sepsis and survival was monitored during 9 days, after which no further deaths occurred. Survival curve was analyzed via Log rank tests, p-values can be found in Table EV1. Combined data of 3 independent experiments, n = 15/group. (H) Mice were injected with the PPARα antagonist GW6471 (10µg/g) or vehicle (DMSO) 3h pre- and 3h post-CLP. Survival was monitored during 9 days, after which no further deaths occurred. Survival curve was analyzed via a Log rank test. Combined data of 2 independent experiments, n = 16/group.
DNA damage response at telomeres boosts the transcription of SARS-CoV-2 receptor ACE2 during aging (2021)
Figure 1

Figure 1 - ACE2 expression increases during aging in mouse and human lungs.

A RT-qPCR detection of Ace2 mRNA expression levels in lungs from young (2-3 months) and old (22-24 months) mice (n = 7 mice per group).

B Representative microphotographs and quantitative analyses of ACE2 immunohistochemical staining in lungs from young (2 months) and old (22 months) mice (n = 3 mice per group). Scale bar 200 µm.

C Representative microphotographs and quantitative analyses of ACE2 immunohistochemical staining in lung parenchyma of young (20-35 years old) and old (60-80 years old) humans (n = 4-7 individuals per group). Scale bar, 200 µm.

D Double-marker immunofluorescence and quantitative analyses of ACE2 intensity level in type II pneumocytic pro-SP-C-positive and CD31-positive endothelia in lungs from young and old mice (n = 3-6 mice per group). Scale bar, 100 µm. a.u. = arbitrary units.

E Double-marker immunofluorescence and quantitative analyses of ACE2 intensity level in type II pneumocytic TTF1-positive and CD31-positive endothelia in lungs from young and old humans (n = 4-7 individuals per group). Scale bar, 100 µm. a.u. = arbitrary units.

F Single-cell transcriptomic data from aging tissues in the mouse lung from Tabula Muris Senis. Upper left panel: uniform manifold approximation and projection (UMAP) of cell identity with ATII cells represented as red dots. Upper right panel: dot plot of Ace2 expression according to age (young = 3 months; old = 30 months). Dot size represents fraction of cells expressing a given gene. Lower panel: UMAP of Ace2 expressing cells depicted as blue dots.

Data information: In (A-E), data are presented as mean ± s.e.m. *P<0.05, ﻿***P<0.001 Two-sided unpaired Student's t-test.
DNA damage response at telomeres boosts the transcription of SARS-CoV-2 receptor ACE2 during aging (2021)
Figure 2

Figure 2 - ACE2 levels increase upon telomere shortening in human cells and in lungs of G3 Terc-/- mice.

A RT-qPCR detection of ACE2 mRNA expression levels in early passages (PD 34-37) and late passages (PD 61-64) human normal fibroblasts (BJ) (n = 3-4 replicates per group).

B ddPCR detection of ACE2 mRNA expression levels in early passages (PD 22.37) and late passages (PD 140) human bronchial epithelial cells (HBEC) (n = 6 replicates per group).

C Representative images and quantitative analyses of ACE2 immunohistochemical staining in lungs of age-matched wild type and G3 Terc-/- mice (n = 3-4 mice per group). Scale bar, 200 µm.

D Double-marker immunofluorescence and quantitative analyses of ACE2 intensity level in type II pneumocytic pro-SP-C-positive and CD31-positive endothelia in lungs of age-matched wild type and G3 Terc-/- mice (n = 3-4 mice per group). Scale bar, 100 µm. a.u. = arbitrary units.

Data information: In (A-D), data are presented as mean ± s.e.m. *P<0.05, ﻿***P<0.001, ﻿****P<0.0001. Two-sided unpaired Student's t-test.
DNA damage response at telomeres boosts the transcription of SARS-CoV-2 receptor ACE2 during aging (2021)
Figure 3

Figure 3 - DDR activation induces ACE2 expression in cultured cells and in vivo.

A Immunofluorescence showing gammaH2AX foci in MEFs Trf2F/F at the indicated time points following 4OHT treatment and consequent TRF2 knockout. Scale bar, 25 µm.

B RT-qPCR detection of Ace2 mRNA expression levels in MEFs Trf2F/F treated as in A (n = 3 independent experiments).

C Immunofluorescence showing gammaH2AX foci in HeLa shTRF2 cells at the indicated time points following doxycycline treatment and consequent TRF2 knockdown. Scale bar, 25 µm.

D RT-qPCR detection of ACE2 mRNA expression levels in HeLa shTRF2 cells treated as in C (n = 6 independent experiments).

E Representative immunofluorescence images of 53BP1 staining in liver from Trf2F/F mice treated with tamoxifen (to induce TRF2 loss and telomere uncapping) or vehicle. The animals as been injected also with PBS. Scale bar, 10 µm.

F RT-qPCR detection of Ace2 mRNA expression levels in livers of mice treated as in E (n = 5-7 mice per group).

Data information: In (B, D, F), data are presented as mean ± s.e.m. *P<0.05, ﻿**P<0.01. Two-sided unpaired Student's t-test (F) or two-way paired ANOVA (B,D).
DNA damage response at telomeres boosts the transcription of SARS-CoV-2 receptor ACE2 during aging (2021)
Figure 4

Figure 4 - DDR activation mediates transcriptional upregulation of ACE2 gene.

A Gene set enrichment analysis showing enriched pathways from the MSigDB_Hallmark gene-set library. Bars are colored according to p value, with significantly enriched pathways (P < 0.05) in blue. Combined score: combined value of both the p-value and z-score.

B Relative luciferase activity in HeLa shTRF2 following ionizing radiation (5 Gy) or TRF2 knockdown upon doxycycline-induced shTRF2 expression (n=3 independent experiments). Error bars represent the s.e.m. ﻿*P<0.05. Two-way paired ANOVA.
DNA damage response at telomeres boosts the transcription of SARS-CoV-2 receptor ACE2 during aging (2021)
Figure 5

Figure 5 - Selective inhibition of telomeric DDR decreases ACE2 expression in cultured cells and in vivo.

A Immunofluorescence showing gammaH2AX foci in Trf2F/F MEFs at the indicated time points following 4OHT treatment and consequent TRF2 knockout and treated with DMSO or ATMi. Scale bar, 25 µm.

B RT-qPCR detection of Ace2 mRNA expression levels in MEFs Trf2F/F treated as in A (n = 3 independent experiments).

C Representative immunofluorescence images of 53BP1 staining in liver from Trf2F/F mice treated with tamoxifen (to induce telomere uncapping) or vehicle and injected with the indicated ASOs or PBS as control. Scale bar, 10 µm.

D RT-qPCR detection of Ace2 mRNA levels in livers of mice treated as in C (n = 5-8 mice per group).

E Representative microphotographs and quantitative analyses of ACE2 immunohistochemical staining in lungs of age-matched wild type and G3 Terc-/- mice, treated with the indicated ASOs (n = 4-9 mice per group). Scale bar, 200 µm.

F Double-marker immunofluorescence and quantitative analyses of ACE2 intensity level in type II pneumocytic pro-SP-C-positive cells in lungs of age-matched wild type and G3 Terc-/- mice, treated with the indicated ASOs (n = 4-9 mice per group). Scale bar, 100 µm. a.u. = arbitrary units.

Data information: In (B, D-F), data are presented as mean ± s.e.m. *P<0.05, **P<0.01, ***P<0.001, ***P<0.0001. Two-way paired (B) or unpaired (D-F) ANOVA.
Regulation of cGAS activity by RNA-modulated phase separation (2022)
Figure 2

Figure 2. hcGAS interacts with RNA in cells.

A. Hoechst 33342 and pyronin Y staining of HEK293T cells showing colocalization of the hcGAS-GFP granules and RNAs (n=3, biological replicates, data from one representative independent biological replicate are shown). Expression of the hcGAS-GFP was induced by doxycycline at a concentration of 0.1 µg/mL. HEK293T cells that express YFP were used as the negative controls. The scale bars represent 10 µm.

B and C. Fluorescence recovery after photo-bleaching (FRAP) assay of hcGAS-GFP granules in HEK293T cells (n=4, biological replicates, data from one representative independent biological replicate are shown). B) Images (left 1-6) of a 293T cell showing the hcGAS-GFP signal in the cell. The selected areas are indicated with circles and arrows and are numbered in the left-most image. Areas 1, 2, and 3 were photo-bleached at the time indicated blow images. Areas 4 and 5 were not photo-bleached and were used as the controls. Signal in area 6 was taken as background. The yellow fluorescence image on the right showing the pyronin Y-stained RNA signal, which colocalizes with the hcGAS-GFP signal in the cytoplasm. The scale bars represent 5 µm. C) Quantification of the fluorescence intensity changes in the 6 selected areas of the FRAP assay indicated in panel "B".

D. A schematic diagram showing the HeLa cell cytoplasm extraction and fractionation procedure. To release the cytoplasm without disrupting the nuclear membrane, the cell membranes were disrupted by suspension in a hypotonic buffer and passing the cells through a 29G needle three times. The hypotonic buffer contains 10 mM HEPES at pH 7.5, 5 mM KCl and 3 mM MgCl2.

E. OptiPrep density gradient analyses of the HeLa cell cytoplasm extracts and purified hcGAS with and without tRNA (n=3, biological replicates, data from one representative independent biological replicate are shown).

F. Western blot analysis of the OptiPrep gradient fractions with or without DNase and RNase treatments.

G. The three images on the left showing fluorescence signals of endogenous cGAS and RNA in HeLa cells (n=13, biological replicates, data from one representative independent biological replicate are shown). The scale bar represents 10 µm. The scatterplot on the right plots the signal intensities of cGAS versus RNA at each pixel in the indicated cytoplasmic area (yellow circle).

H. The three images on the left showing the fluorescence signals of mCherry and RNA in HEK293T cells (n=9, biological replicates, data from one representative independent biological replicate are shown). The scale bar represents 10 µm. The scatterplot on the right plots signal intensities of mCherry versus RNA at each pixel in the indicated cytoplasmic area (yellow circle).

I. Pearson's correlation coefficient (CC) analysis of the fluorescence signals of cGAS vs. RNA (n=13, biological replicates), and mCherry vs. RNA (n=9, biological replicates). The median values were calculated and used for evaluation of significance. ***P < 0.001 (two-tailed Mann-Whitney test).
A placental mammal-specific microRNA cluster acts as a natural brake for sociability in mice (2018)
Figure 1

Fig. 1: Increased sociability in miR379-410 ko mice.

a, Ultrasonic vocalizations (USV) in isolated miR379-410 ko mouse pups emitted on postnatal day P3, P6, P9 and P12. Left panel: total number of ultrasonic vocalizations (USV) in isolated miR379-410 wt and ko mouse pups emitted on average (P 3, 6, 9 and 12) when isolated from the mother, wt n=13 (male n=9, female n=4), ko n=20 (male n=11, female n=9), t31=2.274, *p=0.0301, unpaired Student's t-test. Right panel: developmental course for total number of USV, wt n=13 (male n=9, female n=4), ko n=20 (male n=11, female n=9), (development: F3,87=25.463, p<0.001; genotype: F1,29=4.979, p=0.03; development x genotype: F3,87=1.403, p= 0.22, rm-ANOVA); P 9: t31=2.096, *p=0.044; unpaired Student`s t-test.

b, Reciprocal social interaction in juvenile miR379-410 wt- and ko mice pairs (P23). Aberrant time spending in social interaction activity in miR379-410 ko-pairs compared to their control wt littermate pairs. Left panel: wt-pairs male n=13, ko-pairs male n=10, t21=2.433, *p=0.0240, unpaired Student's t-test. Middle panel: wt-pairs female n=15, ko-pairs female n=13, t26=1.873, ns p=0.0723, unpaired Student's t-test. Right panel: pooled data of wt-pairs n=28 (male n=13, female n=15), ko-pairs n=23 (male n=10, female n=13), t49=2.969, **p=0.005; unpaired Student`s t-test.

c, Emitted USVs during social interaction activity in genotype-matched pairs in juvenile mice (P23). Left panel: wt-pairs male n=13, ko-pairs male n=15, t21=2.704, *p=0.0133, unpaired Student's t-test. Middle panel: wt-pairs female n=15, ko-pairs female n=13, t26=2.869, **p=0.0081, unpaired Student's t-test. Right panel: pooled data of wt-pairs n=28 (male n=13, female n=15), ko-pairs n=23 (male n=10, female n=13), t49=3.992, ***p<0.001; unpaired Student`s t-test.

d, Social preference index in adolescent mice - defined as the ratio of time sniffing a stranger mouse vs. an object - is shown. Left panel: wt male n=26, ko male n=26, t50=1.075, ns p=0.2876, unpaired Student's t-test. Middle panel: wt female n=27, ko female n=30, t55=2.017, *p=0.0486, unpaired Student's t-test. Right panel: pooled data of wt n=53 (male n=26, female n=27), ko n=56 (male n=26, female n=30), t107=2.283, *p=0.0244; unpaired Student`s t-test.

Data information: Data are presented as mean ± s.e.m.