EMBO/soda-nel huggingface dataset:
Epithelial response to IFN-γ promotes SARS-CoV-2 infection (2021)
Figure 3

Fig. 3 SARS-CoV2 induces IFN-γ gene expression signature and promotes differentiation

A) Gene set enrichment analysis of the transcriptome data from uninfected vs. 48 h SARS-CoV-2 infected organoids was performed and the GO terms with the most significant positive correlation are displayed.

B) Gene set enrichment analysis of the transcriptome data comparing uninfected vs. 48 h SARS-CoV-2 infected organoids and the "Hallmark interferon gamma response" gene set. p-values were calculated using the fgsea package from https://www.bioconductor.org with 5000 permutations.

C) Analysis of discordance and concordance of transcriptomic responses (DISCO) between untreated vs. SARS-CoV-2 infected and untreated vs IFN-γ treated organoids. The 50 most regulated genes are highlighted; grey: discordant and black: concordant.

D) Relative expression of selected enterocyte-specific genes upon SARS-CoV-2 infection, normalized to GAPDH. Differences in gene expression were assessed using the linear model 'lmFit' and 'makeContrasts' in limma.

E) Immunofluorescence for KRT20 (green) in human colon organoids cultured in basic medium and either non-infected (left) or 48 h after SARS-CoV-2 infection (right), indicating a strong increase in KRT20 expression after viral infection. Scale bar: 50 µm. Quantification of fluorescence intensity below (n=8). Data are presented as mean +/- SD ,****: p≤0.0001, as determined by Student's t-test (more details see Appendix Table S2).

F) qPCR data displaying the increase of INF-γ target genes (CXCL11, IFIT3) (upper) and enterocyte marker genes (KRT20, ACE2) (lower) after SARS-CoV-2 infection, validating the results from the microarray analysis (n=3). Data are presented as mean +/- SD ,****: p≤0.0001, as determined by Student's t-test (more details see Appendix Table S2).
SFRP1 modulates astrocyte to microglia cross-talk in acute and chronic neuroinflammation (2021)
Figure 1

Figure 1. Astrocytes upregulate SFRP1 expression upon LPS stimulation.

A-D) Confocal image analysis of cryostat sections from adult CX3CR1+/GFP;Sfrp1+/βgal (A,B); Sfrp1+/βgal (C) and CX3CR1+/GFP;Sfrp1-/- (D) and mouse brains three days after intra-cortical infusion of saline or LPS. Sections were immunostained for βgal (magenta, green in C) and Iba1 (green in A; red in C) or Sfrp1 (magenta) and GFP (green, B and D), and GFAP (cyan in A;B, D, red in C). Arrowheads indicate βgal/GFAP (A) and Sfrp1/GFP co-localization (B). No Sfrp1 protein is detected in the null mice independently of the treatment (two bottom lines). Scale bar 25μm.

E) ELISA determination of Sfrp1 levels in brain extracts from 3 months-old wt and Sfrp1-/- mice. WT mice were injected either with saline or LPS. Three days after injection the region around the injected side (10 mm3 cortical cube) was isolated and SFRP1 content compared with that present in similar region of non-injected or Sfrp1-/- mice used as negative control (n=5 mice for each group). Error bars represent Standard Error. Statistical significance: ns P>0.5 ****P<0.0001; One-way ANOVA followed by Bonferroni multiple comparisons test.
SFRP1 modulates astrocyte to microglia cross-talk in acute and chronic neuroinflammation (2021)
Figure 2

Figure 2. SFRP1 is sufficient and required to enhance glial cell activation upon LPS treatment.

A) Coronal cryostat sections of LV-IRES-Gfp or LV-Sfrp1-IRES-Gfp infected brains 1-month post-infusion, immunostained for SFRP1 (magenta) Iba1 (green) GFAP (cyan), or TauP (red). Lv, lateral ventricle. Scale bar 100μm.

B) The graph shows the normalised level of GFAP, Iba1 and CD45 immunoreactivity (IR) and the area occupied by TauP signal (n=24 acquisitions, white dots; N=3 mice, black dots, for each group), normalized to LV-IRES-Gfp infected brains. Error bars represent Standard Error. Statistical significance: *P<0.05; **P<0.01; ****P<0.0001 by two-sided Student's t-test.

C, D) Coronal sections from wt and Sfrp1-/- mouse brains three days after infusion of saline or LPS, immunostained for GFAP (cyan, C) or CD45 (magenta, D). The images are high power views of the somatosensory cortex (lower power view in Fig EV1D). Scale bar 60μm.

E, F) The graphs show the normalised levels of immunoreactivity (IR) for GFAP (E) and CD45 (F, P=0,006) present in cortical sections (n=24 acquisitions white dots; from N=3 animals, black dots, per group) from wt and Sfrp1-/- animals infused with saline or LPS. Bars represent Standard Error. Statistical significance calculated per biological replicas is indicated in green and that based on number of acquisitions in black. ** or ## P<0.01; *** or ### P<0.001; **** or #### P<0.0001 by two-way ANOVA followed by Bonferroni's multiple comparisons test. * and # indicate significance between genotypes and treatments, respectively.
SFRP1 modulates astrocyte to microglia cross-talk in acute and chronic neuroinflammation (2021)
Figure 3

Figure 3. Sfrp1-/- mice develop a milder form of EAE.

A, B) Time course analysis and severity of the symptoms in wt and Sfrp1-/- mice after EAE induction. In A means are depicted with black (wt, n=19) and cyan (Sfrp1-/-, n=19) lines. In B data are expressed as % of the total number of analysed animals (n=19 per genotype).In A, Error bars represent Standard Error.

C) Wt and Sfrp1-/- mice immunized with MOG and sacrificed 16 days post immunization. Cryostat sections of the thoracic spinal cords were stained with antibodies against CD4, Iba1, GFAP and MBP. Images show the region dorsal fasciculus. White arrowheads point to the disruption of the pia surface in wt. Scale bar 100μm.

D) Quantification of CD4+ infiltrated lymphocytes, Iba1 normalised immunoreactivity, MBP normalised immunoreactivity and MBP immune-reactive area in the dorsal fasciculus of spinal cord sections from wt and Sfrp1-/- mice 16 days after immunization (n=16 acquisitions, white dots; from N=4 animals, black dots, per genotype). Error bars represent Standard Error. Statistic refers to number of acquisitions.

Data information: *P<0.05, **P<0.01, ***P<0.001 by Kolmogorov-Smirnov test followed by Mann-Whitney U nonparametric test comparing mice of same day after immunization (A) or two-sided Student's t-test (D).
SFRP1 modulates astrocyte to microglia cross-talk in acute and chronic neuroinflammation (2021)
Figure 4

Figure 4. SFRP1 amplifies LPS-induced microglial activation and modifies their cytokine secretion.

A) Representative cytometry plots of CD11b+ populations present in the cortex of CX3CR1GFP/+ or CX3CR1GFP/+;Sfrp1-/- mice 3 days after saline or LPS intraventricular infusion. Gating was set for isolating the following myeloid cell populations: CD11b+;CD45lo;GFP+ surveying microglia, CD11b+;CD45+;GFP+ activated microglia and CD11b+;CD45+;GFP- infiltrated monocytes.

B) Quantification of the percentage of activated microglia and infiltrated monocyte present in Sfrp1-/- and controls brains 3 days after infusion of saline or LPS (n=5 animals per genotype and condition). Error bars represent Standard Error. Statistical significance: **P<0.01 by two-way ANOVA followed by Bonferroni's multiple comparisons test.

C) Quantification of the total fluorescence from phagocytized pHrodo-labelled E. coli bioparticles and percentage of phagocyting cells in the CD11b+;GFP+ microglial population (n=3-5 animals per genotype and condition). Error bars represent Standard Error. Statistical significance: **P<0.01, ***P<0.001 by two-way ANOVA followed by Bonferroni' s multiple comparisons test.

D) ELISA determination of SFRP1 levels present in the media of microglia (n=4 cultures per genotype), astrocytes (n=4 cultures per genotype) or mixed astrocytes and microglial (2:1, n=7 cultures per genotype) cultures derived from wt or Sfrp1-/- pups exposed for 24h to saline or LPS (1μg/ml). Error bars represent Standard Error. Statistical significance: *P<0.05; ***P<0.001; ****P<0.0001; One-way ANOVA followed by Bonferroni Test.

E, F) Secretory profile of cytokines present in the medium of microglia, astrocytes or mixed astrocytes and microglial (2:1) culture as above (n=5-6 cultures type -microglia, astrocytes or mixed cultures- and genotype). The content of IFNγ, TNFα, IL1β, IL4, IL6 and IL10 was determined by Multiplex ELISA. Values are represented in absolute concentration. Error bars represent Standard Error. Statistical significance: ns P>0.05; *P<0.05; ***P<0.001; ****P<0.0001 by two-way ANOVA followed by Bonferroni's multiple comparisons test.
SFRP1 modulates astrocyte to microglia cross-talk in acute and chronic neuroinflammation (2021)
Figure 5

Figure 5. SFRP1 enhances microglial cells' transcriptional response to LPS treatment.

A) Principal component analysis with the 1000 most variable genes from microglial cells isolated from CX3CR1GFP/+ and CX3CR1GFP/+;Sfrp1-/- mouse brains three days after saline or LPS intra-cerebro-ventricular infusion. The analysis depicts sample clusterization by genotype and treatment.

B, C) Volcano plots of differential gene expression from CX3CR1GFP/+ (B) or CX3CR1GFP/+;Sfrp1-/- (C) microglial cell in response to LPS. Data are represented as Log2 Fold Change vs -Log10 adjusted p-value by the Wald test corrected for multiple comparisons by the Benjamini and Hochberg method. Blue vertical lines represent an increase of 75 and 400% in the expression levels respectively. Green horizontal lines represent a 0.05 or 0.01 adjusted p-value of statistical significance respectively. Genes with an expression change higher than 75% and an adjusted p-value lower than 0.05 are highlighted in dark red (+FC) and dark blue (-FC). Genes with an expression change higher than 400% and an adjusted p-value lower than 0.01 are coloured in light red (+FC) and light blue (-FC).

D) Scatterplot of differential expressed genes in CX3CR1GFP/+ and CX3CR1GFP/+;Sfrp1-/- microglia; the red line represents linear regression (slope 0.704014 p-value: < 2.2e-16 by F-test followed by Bonferroni's correction) of the differential expression, indicating response attenuation. Coloured dots represent response which are exclusive for each genotype as represented in E.

E) Venn diagram showing the extent of differential gene expression in response to LPS in CX3CR1GFP/+ or CX3CR1GFP/+;Sfrp1-/- microglial cells. Fold change (75%) and adjusted p-value < 0.05 cut-off by the Wald test corrected for multiple comparisons by the Benjamini and Hochberg method.

F) Heatmap showing fold changes of regularized log transformed gene-level RNA-seq counts with hierarchical clustering of samples and differentially expressed genes in response to LPS in CX3CR1GFP/+ or CX3CR1GFP/+;Sfrp1-/- microglial cells.
SFRP1 modulates astrocyte to microglia cross-talk in acute and chronic neuroinflammation (2021)
Figure 6

Figure 6. SFRP1 induces HIF target genes' up-regulation during neuroinflammation

A) Ingenuity Pathway Analysis network representation of NF-κB and HIF downstream targets for CX3CR1GFP/+ and CX3CR1GFP/+;Sfrp1-/- microglial cells. Coloured by Z-score alteration of their expression after LPS stimulus.

B) Integrative Genomics Viewer transcription profile of represented genes in microglial cells isolated for saline and LPS treated CX3CR1GFP/+ and CX3CR1GFP/+;Sfrp1-/- mouse brains. Scale bar: 2 Kbp.

Legends to Expanded View Figures
Neonatal neuronal WWOX gene therapy rescues Wwox null phenotypes (2021)
Figure 1

Figure 1. Restoration of mWwox in neurons improves growth of Wwox null mice and extends their life span.

Physical appearance of wild type (WT), Wwox null injected either with AAV9-hSynI-GFP (the control virus) or AAV9-hSynI-mWwox-IRES-GFP virus at P17.

Graph showing the body weight of WT, KO and KO+ AAV-mWwox at different days after birth (n= 4 per group).

Kaplan-Meier survival graph indicates prolonged life span of Wwox knockout mice injected with AAV9-hSynI-mWwox [total n=18, spontaneously dead n=6, mice taken out for electrophysiology/electron microscopy/analysis, are shown in yellow, n=12] compared to mice injected with AAV9-hSynI-GFP (n=6) or the non-injected (n=8 (P <0.0001, Log-rank Mantel-Cox test).

Immunofluorescence images showing the expression of WWOX (red) in brain tissue (cortex) at P17 from WT and Wwox null mice that were injected with AAV9-hSynI-mWwox (2x1010). Neurons are labelled with anti-NeuN antibody (green). The square white box shows the magnified area. White arrows show the transgene expression in NeuN positive cells.

Graph showing the percentage of NeuN and WWOX double positive cells in different parts of the brain (Cortex, Hippocampus and Cerebellum) from KO mice injected with AAV-mWwox at P17. NeuN and WWOX double positive cells were calculated from 3 identical sagittal sections of the AAV-mWwox injected mice (n=3) brains.

Expression of EGFP (green) is shown in brain tissue (cortex) of Wwox null injected with AAV9-hSynI-GFP (2x1010).

Data information: Error bars represent ±SD. Scale bars D) 20 µm, F) 100 µm
Neonatal neuronal WWOX gene therapy rescues Wwox null phenotypes (2021)
Figure 2

Figure 2. Restoration of hWWOX in neurons reverse the phenotypes of Wwox null mice and extends their life span.

Physical appearance of wild type (WT), Wwox null (KO) or KO injected with AAV-hWWOX (2x1010) at P19.

Graph showing body weight of WT, KO and KO+AAV-hWWOX at different days after birth. n=3 mice per genotype.

Kaplan-Meier survival graph indicates prolonged life span of Wwox knockout mice injected with AAV9-hWWOX [total n=16, alive n=6, spontaneously dead n=6, 4 mice (shown in yellow) were taken out for analysis) compared to the non-injected (n=8)] (P <0.0001, Log-rank Mantel-Cox test).

D-F. Immunofluorescence images showing the expression of WWOX (magenta) in cortex (D), Hippocampus (E) and Cerebellum (F) at P19 from WT and Wwox null mice that were injected with AAV-hWWOX (2x1010) or Wwox null alone. Neurons are labelled with anti-NeuN antibody (green). The square white box shows the magnified area.

Graph represents the percentage of neurons showing the transgene (NeuN and WWOX positive) expression at different parts (cortex, hippocampus and cerebellum) of the KO brain at P19 after AAV injection. NeuN and WWOX double positive cells were calculated from 3 identical sagittal sections of the AAV-hWWOX injected mice (n=3) brains.

Data information: Error bars represent ±SD. Scale bars D), E) 100 µm, F) 50 µm
Neonatal neuronal WWOX gene therapy rescues Wwox null phenotypes (2021)
Figure 3

Figure 3. Neuronal restoration of WWOX reduces epileptic activity and astrogliosis in KO.

Representative traces of cell-attached recordings performed in WT (blue), KO (red) and KO injected with either with AAV9-hSynI-mWwox (KO+A-mW shown in purple) or AAV9-hSynI-hWWOX (KO+A-hW shown in green) pups at P18-21 days. Traces represent spontaneous neocortical activity (action potentials shown). The panels represent 12 s recording with an inset presenting a zoom-in of an 0.5 s interval.

The graph shows the recordings (P18-21) averages over 30 neurons from WT (n=3), 30 neurons from KO+A-mWwox (n=3), 30 neurons from KO+A-hWWOX (n=3), and 45 neurons from KO (n=3).

Representative traces of cell-attached recordings performed in a WT (blue) and a KO+A-mWwox (purple) adult mice (6 months old).

The graph shows the averages over 60 neurons from WT adult mice (n=3) and 60 neurons from KO+A-mWwox adult mice (n=3). There was no significant change observed between the average firing rate of the WT and the KO+A-mWwox adult mice.

Immunofluorescence staining with anti-GFAP (shown in green) and anti-Iba1 (shown in magenta) antibodies showing the expression of astrocytes and microglia at P19 from WT, KO and KO+A-hWWOX. The square white box shows the magnified area from CA3 regions of hippocampus from all the genotypes.

F, G. Graphs represents the quantification of GFAP (F) and Iba1 (G) positive cells from the images shown in E. The number of GFAP or Iba1 cells were counted from 3 identical sagittal sections from CA3 region per group (n=3 per each genotype).

H. Immunocytochemical images showing astrocytes (anti-GFAP) and microglia (anti-Iba1) in CA3 region of hippocampus from WT (n=3) and rescued (KO+AAV-hWWOX) (n=3) mice at 9 months. Magnified area is shown with square box.

I, J. Graphs represents the quantification of GFAP (I) and Iba1 (J) positive cells from the images shown in H. The number of GFAP or Iba1 cells were counted from 3 identical sagittal sections (paraffin embedded) from CA3 region per group (n=3 per each genotype).

Data information: Error bars represent ±SD. Scale bars E) and H) 50 µm. n.s non-significant (**P <0.01, ***P <0.001, ****P <0.0001, Student's t-test).
Neonatal neuronal WWOX gene therapy rescues Wwox null phenotypes (2021)
Figure 4

Figure 4. WWOX restoration in neurons improves myelination by promoting OPCs differentiation in Wwox null.

Images of whole brain sagittal sections that were immunolabelled with anti-MBP at P17 from indicated mice (n=3 for each group). MBP staining (shown in gray) in cortex (green dotted line represents the border of the cortex), hippocampus and cerebellum is presented (magnified from the top panel).

Sagittal section of the brain (P17) tissues immunostained for CC1 (green) and PDGFRα (magenta). Images showing increased number of matured oligodendrocytes in corpus callosum of the Wwox null after treatment with AAV-mWwox compared to Wwox null injected with AAV-GFP virus.

Graph represents the quantification of CC1 and PDGFRα positive cells in corpus callosum (area 0.5 mm2) of WT (n=3), KO (n=3) and KO+A-mWwox (n=3) counted from three similar brain sagittal sections per mouse in each genotype.

Data information: Error bars represent ±SD. (*P <0.05, **P <0.01, *** P <0.001, Student's t-test). Scale bars A) 2 mm (top panel), 250 µm (middle and lower panels), B) 250 µm (top panel), 50 µm (middle and lower panels)
Neonatal neuronal WWOX gene therapy rescues Wwox null phenotypes (2021)
Figure 5

Figure 5. Electron microscopy analysis showing improved myelination in corpus callosum upon WWOX restoration.

Electron micrograph (EM) images from mid sagittal section of corpus callosum showing increased number of myelinated axons in AAV9-hSynI-mWwox-injected mice compared to Wwox-null mice (KO) at P17.

Graph showing average number of myelinated axons in corpus callosum from WT (n=3), KO (n=3) or KO mice injected with AAV9-hSynI-mWwox (n=3) per field of view (FOV).

g-ratio analysis showing reduced myelin thickness of axons in corpus callosum KO (n=3) compared to the KO+AAV-Wwox (n=3). Axon diameter and myelin thickness was calculated from the electron micrograph images of corpus callosum (total no of axons n=350 per each group).

EM images from mid sagittal section of corpus callosum showing similar number of myelinated axons in rescued mice (AAV9-hSynI-mWwox, n=3) compared to wild type (n=3) at the age of 6 months. Unmyelinated axons are noted with the red star symbols in EM images.

Graph represents the number of unmyelinated axons (corpus callosum) per FOV counted from representative EM images shown in D.

Graph represents the measured g-ratio of myelinated axons (n=300 per each group) in WT and rescued mice at 6 months (P value 0.011334).

EM images showing increased number of myelinated axons in optic nerve in AAV9-hSynI-mWwox injected mice compared to KO mice at P17.

Graph showing the average number of myelinated axons (counted per FOV) in optic nerve from WT (n=3), KO (n=3) and KO+AAV-mWwox(n=3).

g-ratio's of myelinated axons from optic nerves showing improved myelin thickness (less g-ratio) after WWOX restoration (KO+AAV-mWwox) compared to KO alone (n=3 per genotype, total no of axons n=200 per each group).

Data information: Error bars represent ±SD. (**P <0.01, *** P <0.001, Student's t-test). Scale bars A) 10 µm (top panel), D) 5 µm (inset 1 µm), G) 5 µm (inset 2 µm).
Neonatal neuronal WWOX gene therapy rescues Wwox null phenotypes (2021)
Figure 6

Figure 6. WWOX restoration in neurons reverses the abnormal behavioral phenotypes of Wwox null mice.

Representative images of open field test showing tracking pattern (open arena) of WT (females, n=7, males n=6) and AAV-mWwox-injected KO mice (females n=7, males n=5) at 8-10 weeks.

B, C, D and E. Dot plot graphs represent the velocity (B), total distance travelled (C), the frequency to enter into the center (D) and periphery (E) in an open arena from the open field tracking as shown in A.

F. Mice tracking images of elevated plus maze test from WT and KO+AAV-mWwox mice (age 8-10 weeks). O. A. (open arm) C.A. (closed arm) are shown with dotted white boxes on the left mice tracking image.

G-I. Dot plot graphs represent the duration (sec) in closed arm (G), open arm (H) and movement velocity (I) from WT (females, n=7, males n=6) and KO+AAV-mWwox (females n=7, males n=5).

J, K. Graphs represent the latency (sec) of the mice (WT and rescued mice at 8-10 weeks) to fall from the rota rod. WT (females, n=7, males n=6) and KO+AAV-Wwox (females n=7, males n=5).

L. Representative images of open field test showing tracking pattern (open arena) of WT (females, n=6) and KO with AAV injected (females mWwox n=3, hWWOX n=3) at 8-9 months.

M-O. Dot plot graphs represent the represent the velocity (M), total distance travelled (N), the frequency to enter into the center and periphery (O) in an open arena from the open field tracking of mice shown in L.

Data information: Error bars represent ±SD. n s (no significance), (*P <0.05). Statistical analysis was done using Two-way ANOVA with Benforroni post hoc comparisons.
BH3-in-groove dimerization initiates and helix 9 dimerization expands Bax pore assembly in membranes (2015)
Figure 1

Figure 1. Activated Bax proteins integrate into the MOM and dimerize via a BH3-in-groove interface.

A. The crystal structure of the BH3-in-groove Bax homodimer (PDB entry 4BDU) is shown with one monomer colored gray and other colored green, magenta, blue and red for its α2, α3, α4 and α helices, respectively, as indicated. The residue pairs that were replaced with cysteine pairs in (B) are presented in stick form, and their β carbon atoms linked by dashed lines with the distances ranging from 5.0 to 6.0 Å.

B. The in vitro synthesized [35S]Met-labeled single-cysteine Bax proteins were activated and targeted to the mitochondria that were pretreated with NEM to block the sulfhydryls of mitochondrial proteins. The resulting mitochondria were isolated and oxidized by CuPhe for 30 min. NEM and EDTA were then added to stop the oxidation. For the “0 min” controls, NEM and EDTA were added before the addition of CuPhe. The resulting samples were analyzed by phosphor-imaging after non-reducing or reducing SDS-PAGE (see Appendix Figure S2A).

C. Oxidized mitochondria with the radioactive single-cysteine Bax protein pair or double-cysteine Bax protein were prepared and analyzed as in (B).

D. Oxidized mitochondria with the radioactive single-cysteine Bax protein pair or double-cysteine Bax protein were prepared as in (B). After an aliquot was withdrawn as input, another aliquot was extracted by Na2CO3 (pH 11.5) and centrifuged through a sucrose cushion to separate the integral proteins in the membrane pellet from the soluble and peripheral proteins in the supernatant. The input, pellet and supernatant were analyzed by non-reducing SDS-PAGE and phosphor-imaging. In a parallel control experiment, the pellet and supernatant were analyzed by reducing SDS-PAGE and immunoblotting with an antibody specific to PDHE1α, a soluble mitochondrial matrix protein.

In (B-D), protein standards are indicated on the side of phosphor-images or immunoblots by their molecular mass (Mr). Open circles indicate Bax monomers. Upward arrows indicate disulfide-linked dimers of the same single-cysteine Bax mutant (e.g., M79C), and downward arrows disulfide-linked dimers of two different single-cysteine Bax mutants (e.g., L59C+M79C) or of the same double-cysteine Bax mutant (L59C,M79C). Closed circles indicate the disulfide-linked heterodimer of Bax M79C and the BH3 peptide (with a cysteine at position 62). The number of independent replicates done for each Bax mutant in (B-C), n = 2 for C62+M79C, T56C, and R94C; 3 for L63C, L63C+A112C, A112C, K64C, K64C+M99C, and M99C; 4 for L59C+M79C, M79C, C62, C62+L76C, and T56C+R94C; 6 for L59C, and L76C; 8 for L59C,M79C. For each Bax mutant and PDHE1α in (D), n = 2.