EMBO/soda-nel huggingface dataset:
A pathogenic role for cystic fibrosis transmembrane conductance regulator in celiac disease (2018)
Figure 1

Figure 1. CFTR malfunction favors gliadin responsiveness in vivo

(A) Immunoblot with anti-TG2 or anti-β-actin as loading control in in whole lysates from small intestine homogenates of CftrF508del/F508del and wild-type (CftrWT) mice (n=5 per group) and densitometric analysis of immunoblots. Mean ± SD of triplicates of independent pooled samples. **p<0.01 (Student's t test).

(B) Detection of NLRP3 expression (top) and caspase-1 cleavage (bottom) by immunoblot of whole lysates from small intestine homogenates of CftrWT and CftrF508del/F508del mice (n= 5) and densitometric analysis of immunoblots. Mean ± SD of triplicates of independent pooled samples. **p<0.01 CftrWT vs CftrF508del/F508del (Student's t test).

(C) Protein levels of IL-17A and IFN-γ from small intestine homogenates of CftrWT and CftrF508del/F508del (n= 10). Mean ± SD of triplicates of independent pooled samples. ***p<0.001 CftrWT vs CftrF508del/F508del (Student's t test).

(D) IL-15 mRNA (left) and protein (right) levels in small intestine homogenates from CftrF508del/F508del, Cftr-/- or their CftrWT littermates (n= 10 per group). Mean ± SD of triplicates of independent pooled samples. **p<0.01 CftrF508del/F508del vs CftrWT(FVB/129), or °°p<0.01 Cftr-/- vs CftrWT(B6.129P2) (ANOVA, Bonferroni post hoc test).

(E) IL-15 mRNA levels in small intestine homogenates from CftrWT mice or CftrF508del/F508del or TG-/-/CftrF508del/F508del or TG-/- mice (n= 10 per group). Mean ± SD of triplicates of independent pooled samples. ***p<0.001 vs CftrWT, °°p<0.01, ##p<0.01 vs CftrF508del/F508del (ANOVA, Bonferroni post hoc test).

(F) Effects of 4 weeks of oral administration of gliadin on IL-15, IL-17A and IFN-γ protein levels in small intestine homogenates from CftrF508del/F508del and CftrWT mice (n =10 mice per group of treatment). Mean ± SD of triplicates of independent pooled samples. **p<0.01, ***p<0.001 (CftrF508del/F508del vs CftrWT mice prior gliadin challenge), °°°p<0.001 (CftrF508del/F508del mice vs CftrF508del/F508del mice after gliadin challenge), (ANOVA, Bonferroni post hoc test).

(G-I) BALB/c mice (G) fed with a gluten-free diet for at least 3 generations, or (H) NOD or (I) NOD-DQ8 mice orally challenged with vehicle or gliadin for 4 weeks (5 mg/daily for one week and then 5 mg/daily thrice a week for 3 weeks). Representative traces of CFTR-dependent Cl- secretion measured by forskolin (Fsk)-induced increase of chloride current (Isc (μA/cm2)) in small intestines mounted in Ussing chambers; quantification of the peak CFTR inhibitor 172 (CFTRinh172)-sensitive Isc (∆Isc) in tissue samples (n=3 independent experiments). Mean±SD of samples assayed; **p<0.01, ***p<0.001 vs challenged with gliadin (Student's t test).

Data information: The blots are representative of one experiment for group of treatment.
A pathogenic role for cystic fibrosis transmembrane conductance regulator in celiac disease (2018)
Figure 2

Figure 2. P31-43 binds NBD1 and inhibits CFTR channel function

(A) Representative traces of CFTR-dependent Cl- secretion measured by forskolin (Fsk)-inducible chloride current (Isc (μA/cm2)) in Caco-2 cells mounted in Ussing chambers after 3 h of incubation with P57-68 or P33-mer or P31-43 peptides (20 µg/ml) and of P31-43 in the presence of up to 30 minutes of pre-treatment of VX-770 (10 µM); quantification of the peak CFTR Inhibitor 172 (CFTRinh172)-sensitive Isc (∆Isc) in Caco-2 cells (n= 3 independent experiments). Mean ± SD of samples assayed. ***p<0.001 vs P31-43 challenge, ##p<0.01 vs Caco-2 challenged with P31-43 in the presence of VX-770 (ANOVA, Bonferroni post hoc test).

(B) Incubation of Caco-2 cells with P31-43 or control peptides (p57-68 or PGAV) for 1 h that were preceded or not by 20 min of pre-treatment with VX-770. Immunoprecipitation in non-reducing and non-denaturing conditions of CFTR protein and immunblot with streptavidin-HRP or CFTR antibody.

(C) Protein-Protein docking and Molecular Dynamics of P31-43 (violet) bound to NBD1 (orange). Left side: general view of P31-43 and NBD1 interaction. Upper right: detailed interaction pattern, highlighting the most important amino acids. Lower right: in silico NBD1/P31-43 complex compared to the original crystallographic positions of Trp401 (red) and ATP (yellow).

(D) Graphical view of the in silico sampling percentage of P31-43 against NBD1/NBD2.

(E) Surface plasmon resonance (SPR) analysis of increasing concentrations of P31-43 and P57-68 peptides on rhNBD1 covalently bound to the CM5 sensor chip.

(F) Surface plasmon resonance (SPR) analysis of rhNBD1 binding to P31-43 and P57-68 biotinylated peptides immobilized on SA sensor chip.

(G-H) Blue native polyacrylamide gel electrophoresis (PAGE) western blotting of P31-43 and P57-68 biotinylated peptides in the presence of rhNBD1 (G) and of WT and double NBD1 mutants in the presence of biotinylated P31-43 (H). All the recombinant proteins and the indicated peptides were pre-incubated in an appropriate buffer at 4°C for 30 min and then resolved in native conditions to preserve the formation of peptide/protein complexes.

(I) P31-43 induced modifications on NBD1 ATP binding site using the intrinsic W401 fluorescence.

(J) P31-43 effect on NBD1 ATPase activity. For further details, see Material and Methods.

(K) P31-43 effect on NBD1 ATPase activity, applied at its IC50, compared to that obtained with 5µM of the non-hydrolyzable ATP analogue (P-ATP) as a positive control of inhibition. Mean ± SD of triplicates of independent experiments.

Data information: The blots are representative of one experiment for group of treatment.
A pathogenic role for cystic fibrosis transmembrane conductance regulator in celiac disease (2018)
Figure 3

Figure 3. Mutual relationship between gliadin, TG2 and CFTR in intestinal epithelial cells

(A) Graphical view of the in silico sampling percentage of P31-43 and of P31-43/4QA (4QA) against NBD1.

(B) SPR analysis of 4QA peptide on rhNBD1 covalently bound to CM5 sensor chip. For further details, see Material and Methods.

(C and D) Incubation of Caco-2 cells with P31-43 or with modified P31-43 (P31-43-2QA 4-5th or P31-43-2QA 10-11th or P31-43/4QA mutant (4QA) peptides) for 1 h. (C) Immunoprecipitation in non-reducing and non-denaturing conditions of CFTR protein and immunblot with streptavidin-HRP or CFTR antibody (n=3 independent experiments). (D) Representative traces of CFTR-dependent Cl- secretion measured by forskolin (Fsk)-induced increase of chloride current (Isc (μA/cm2)) in Caco-2 cells mounted in Ussing chambers; quantification of the peak CFTR inhibitor 172 (CFTRinh172)-sensitive Isc (∆Isc) in Caco-2 cells (n= 3 independent experiments). Mean ± SD of samples assayed. °°p<0.01 vs P31-43 challenge, ***p<0.001 vs Caco-2 challenged with 4QA (ANOVA, Bonferroni post hoc test).

(E) In vitro Blue native PAGE western blotting of enhanced binding of P31-43 to rhNBD1 in the presence of activated rTG2 that in turn promotes the formation of higher molecular weight complexes. As described in the text, only P31-43 (lanes 1, 3, 5), and not P57-68 (lanes 2, 4, 6), has the ability to form stable complexes with NBD1 and TG2 (n=3 independent experiments).

(F-L) Incubation of Caco-2 cells with P57-68 or P31-43, in the presence or absence of pre-treatment with TG2 inhibitor Z-DON, TG2-siRNA, BAPTA-AM or VX-770. (F) Immunoprecipitation in non-reducing and non-denaturing conditions of CFTR protein and immunoblot with anti-isopeptide glutamime-lysine and CFTR or (G) immunoprecipitation of CFTR and then immunoblot with anti-TG2 or anti-CFTR antibodies (n=3 independent experiments). (H and I) Effects of treatment with VX-770 and Z-DON on TG2 transamidating activity in situ. (H) In situ detection of TG2 activity in Caco-2 cells pulsed with Ca2+ by immunoblotting of the TG-catalyzed incorporation of 5-biotinamidopentylamine (BAP) and blotting with anti-biotin antibody (n=3 independent experiments). (I) Assay of TG2 activity by immunostaining of the TG-catalysed incorporation of monodansylcadaverin in Caco-2 cells pre-treated with VX-770 or Z-DON and then pulsed with P31-43. Scale bar, 50µm. (J) Immunoprecipitation of CFTR and immunoblot with anti-CFTR and anti-TG2 antibodies. (K) Immunoprecipitation in non-reducing and non-denaturing conditions of PKAr2α protein and immunoblot with isopeptide glutamine-lysine and PKAr2α antibodies (n=3 independent experiments). (L) Immunoblot of phospho-PKA (phPKA) protein; densitometric analysis of protein levels relative to β-actin. Mean±SD of triplicates of independent experiments. **p<0.01 vs P31-43, °°p<0.001 vs VX-770 (ANOVA, Bonferroni post hoc test).

Data information: The blots are representative of one experiment for group of treatment.
A pathogenic role for cystic fibrosis transmembrane conductance regulator in celiac disease (2018)
Figure 4

Figure 4. CFTR malfunction drives P31-43 induced epithelial stress response

Caco-2 cells incubated with P57-68 or PGAV or with P31-43 in the presence or absence of VX-770 or Vrx-532.

(A) Immunoblot of PPARγ or phospho-ERK1/2 (phERK1/2) and densitometric analysis of protein levels relative to β-actin. Mean ± SD of triplicates of independent experiments. **p<0.01 vs P31-43, °°p<0.001 vs VX-770 (ANOVA, Bonferroni post hoc test).

(B-D) Immunoblotting with specific antibodies in Caco-2 cells challenged for 2 or 4 h in the presence or absence of VX-770. NF-κB p65 in cytoplasmic and nuclear extracts (B), NLRP3 expression (C) and caspase-1 cleavage (D). Densitometric analysis of protein levels relative to loading control. Mean ± SD of triplicates of independent experiments. **p<0.01 and °°p<0.001 vs 2h or 4h of culture with P31-43 in the presence of VX-770 (ANOVA, Bonferroni post hoc test).

(E) Immunoblot of membrane protein fractions with anti-NHERF-1, anti-ezrin Abs and anti-flotillin as a control. Densitometric analysis of immunoblot (mean ± SD of triplicates of independent experiments); *p<0.01 vs treatment with VX-770 or °°p<0.01 vs treatment with Vrx-532 (ANOVA, Bonferroni post hoc test).

(F) Confocal image staining with anti-F-actin. DAPI (blue), nuclear counterstaining. Scale bar, 50 µm.

(G) IL-15 production (quantified by specific ELISA) in CFTR-WT Caco-2 cells treated or not with P31-43 (left) or in Caco-2CFTR-KO cells (right), in the presence or absence of VX-770. Mean ± SD of triplicates of independent experiments. ***p<0.001 vs untreated and °°°p<0.001 vs CFTR-WT Caco-2 cells treated with P31-43+VX-770 (ANOVA, Bonferroni post hoc test). **p<0.01 vs Caco-2 CFTR WT (Student's t-test).

(H) Caco-2 or Caco-2CFTR-KO cells in the presence or absence of pre-treatment with VX-770. Immunoblot of phERK1\2 or β-actin (left); densitometric analysis of protein levels (right). Mean ± SD of triplicates of independent experiments. *p<0.05 (Student's t-test).

Data information: The blots are representative of one experiment for group of treatment.
Increasing Host Cellular Receptor—Angiotensin-Converting Enzyme 2 (ACE2) Expression by Coronavirus may Facilitate 2019-nCoV Infection (2020)
Figure 1.

Transfection of poly (I:C) induces ACE2 expression.HEK293 cells were cultured in six-well plates and transfected with poly (I:C) as indicated above using Lipofectamine 2000. HEK293 cells were then collected for RNA isolation and RT-qPCR analysis at 9 hrs post-transfection. Results in each panel are representative of three independent experiments.
Increasing Host Cellular Receptor—Angiotensin-Converting Enzyme 2 (ACE2) Expression by Coronavirus may Facilitate 2019-nCoV Infection (2020)
Figure 2.

VSV-eGFP infection induces ACE2 expression.HEK293 cells were cultured in six-well plates and infected with VSV-eGFP as indicated above. 6 hrs later, HEK293 cells were collected for RNA isolation and RT-qPCR analysis. Results in each panel are representative of three independent experiments.
Increasing Host Cellular Receptor—Angiotensin-Converting Enzyme 2 (ACE2) Expression by Coronavirus may Facilitate 2019-nCoV Infection (2020)
Figure 3.

Overexpression of TBK1 or TRIF induces ACE2 expression.TBK1 is an essential activator of RNA-sensing pathway. HEK293 cells were cultured in 24-well plates. 24 hrs later, plasmids as shown above were transfected into HEK293 cells. 36 hrs after transfection, total RNA was isolation for subsequent RT-qPCR analysis. Results in each panel are representative of three independent experiments.
Increasing Host Cellular Receptor—Angiotensin-Converting Enzyme 2 (ACE2) Expression by Coronavirus may Facilitate 2019-nCoV Infection (2020)
Figure 4.

ACE2 expression is upregulated in the lungs from SARS-CoV MA15 infected C57Bl/6 mouse.Intranasal instillation of 102, 103 PFU of SARS-CoV MA15 in 50 µl of PBS or mock-infected with PBS alone, lungs were harvested 24 hrs later and total RNA was isolated and subjected to microarray analysis. ACE2 expression data was retrieved from Gene Expression Omnibus (GEO) microarray database (NCBI Accession: PRJNA149057; GEO: GSE33266).
Increasing Host Cellular Receptor—Angiotensin-Converting Enzyme 2 (ACE2) Expression by Coronavirus may Facilitate 2019-nCoV Infection (2020)
Figure 5.

ACE2 expression is upregulated in primary human airway epithelial cells in response to wild type MERS-CoV (icMERS-CoV EMC2012).Human airway epithelial cells were infected with a multiplicity of infection of 5 PFU per cell. Infected samples were collected as indicated above. ACE2 expression data was retrieved from GEO microarray database (NCBI Accession: PRJNA391962; GEO: GSE100504).
Increasing Host Cellular Receptor—Angiotensin-Converting Enzyme 2 (ACE2) Expression by Coronavirus may Facilitate 2019-nCoV Infection (2020)
Figure 6.

ACE2 expression is upregulated in human airway epithelial cells in response to rhinovirus infection.Primary epithelial cells from 4 donors were exposed to rhinovirus or medium only (used as control). 24 hrs post-infection, gene expression was assessed using Affymetrix U133 plus 2.0 human GeneChips. ACE2 expression data was retrieved from GEO microarray database (NCBI Accession: PRJNA137767; GEO: GSE27973).
Increasing Host Cellular Receptor—Angiotensin-Converting Enzyme 2 (ACE2) Expression by Coronavirus may Facilitate 2019-nCoV Infection (2020)
Figure 7.

ACE2 expression is upregulated in human bronchial epithelial cells (HBECs) after IFNβ and H1N1 influenza stimulation.HBECs were treated with IFNβ (1000U/ml) or wild type H1N1 influenza (A/PR/8/34). Control samples were incubated with fresh medium under the same conditions. HBECs were harvested at indicated time points (0.25, 0.5, 1, 1.5, 2, 4, 6, 8, 12, and 18 hours post-treatment). Total RNA was extracted and analyzed using the Affymetrix High Throughput Array. ACE2 expression data was retrieved from GEO microarray database (NCBI Accession: PRJNA121751; GEO: GSE19392).
Increasing Host Cellular Receptor—Angiotensin-Converting Enzyme 2 (ACE2) Expression by Coronavirus may Facilitate 2019-nCoV Infection (2020)
Figure 8.

ACE2 expression is upregulated in human primary keratinocytes exposed to IFNα and IFNγ.Primary keratinocytes from 3 donors were either untreated (control) or exposed to cytokines IFNα, IFNγ. 24 hrs after treatment, total RNA was extracted for subsequent microarray analysis. ACE2 expression data was retrieved from GEO microarray database (NCBI Accession: PRJNA153023; GEO: GSE36287).
Nanoscale organization and dynamics of the siglec CD22 coorporate with the cytoskeleton in restraining BCR signalling (2015)
Figure 1

Figure 1. CD22 restrains BCR and CD19 cytoskeleton-dependent signalling

(A) Wild-type primary B cells were treated with 5 µg/mL of anti-kappa antibody (a-k chain, top) or 1 µM LatrunculinA (LatA, bottom). Intracellular calcium flux was measured by flow cytometry in the presence of Src-inhibitor (PP2), Syk-inhibitor (BAY 613606) or vehicle control.

(B) Wild-type primary B cells were treated with vehicle control (-), 5 µg/mL anti-kappa antibody or 1 µM LatA for the indicated time. Cells were lysed and analysed by SDS-PAGE followed by immunoblotting with phospho-Lyn (Y507) and actin as loading control. The intensity of phosphorylated proteins, normalized to actin, was referred to the respective unstimulated sample, set as 1.

(C and D) Wild-type primary B cells were treated with vehicle control (-), 5 µg/mL anti-kappa antibody or 1 µM LatA for the indicated time. Cells were lysed and analysed by SDS-PAGE followed by immunoblotting with phospho-Syk (pSyk), phospho-CD19 (pCD19), phospho-Akt (pAkt), phospho-ERK (pERK) and total ERK as loading control. The intensity of phosphorylated proteins, normalized to ERK, was referred to the respective unstimulated sample, set as 1.

(E and F) Wild-type primary B cells were treated with vehicle control (-), 5 µg/mL anti-kappa antibody or 1 µM LatA for the indicated time. Cells were lysed and analysed by SDS-PAGE followed by immunoblotting with phospho-PLCγ2 (pPLCγ2) and total ERK as loading control. The intensity of phosphorylated proteins, normalized to ERK, was referred to the respective unstimulated sample, set as 1.

(G and H) Wild-type primary B cells were treated with vehicle control (-), 5 µg/mL anti-kappa antibody or 1 µM LatA for the indicated time. Cells were lysed and analysed by SDS-PAGE followed by immunoblotting with phospho-CD22 (pCD22), phospho-SHP1 (pSHP1), and CD22 or total ERK as loading controls. The intensity of phosphorylated proteins, normalized to CD22 or ERK, was referred to the respective unstimulated sample, set as 1.

(I) Wild-type and Cd22-/- primary B cells were treated with 5 µg/mL anti-kappa antibody or 1 µM LatA and intracellular calcium flux was measured by flow cytometry.

(J) Wild-type and Cd22Y2,5,6F primary B cells were treated with 5 µg/mL anti-kappa antibody or 1 µM LatA and intracellular calcium flux was measured by flow cytometry.

(K and L) Wild-type, Cd22-/- and Cd22Y2,5,6F primary B cells were treated with vehicle control (-) or 1 µM LatA for the indicated time. Cells were lysed and analysed by SDS-PAGE followed by immunoblotting with phospho-CD19 (pCD19), phospho-Akt (pAkt), phospho-ERK (pERK) and actin as loading control. The intensity of phosphorylated proteins, normalized to actin, was referred to the unstimulated sample of the wild type cells, set as 1.

Data are representative of a minimum of three experiments.
Nanoscale organization and dynamics of the siglec CD22 coorporate with the cytoskeleton in restraining BCR signalling (2015)
Figure 2

Figure 2. CD22 density and colocalisation on the surface of primary B cells

(A) Determining the number and density of CD22, CD19, IgM and IgD on the surface of wild-type primary B cells. Cells were stained with PE-conjugated antibody against CD22 (blue profile), CD19 (red profile), IgM (red profile) or IgD (red profile) and their fluorescence profiles compared to those of standard beads (grey profiles), which each possess a well-defined number of binding sites (st0=blank, st1-st4=increasing number of binding sites) against the primary antibody. For IgM, where the range of expression is large, the low and high 25 percentiles were calculated as in Mattila et al. 2013 and shown in brackets.

Data are representative of three independent experiments.

(B-E) Dual-colour SIM analysis of CD22, IgM, IgD and CD19. Wild-type primary B cells were fixed, stained with Atto633 or 488-conjugated antibody against CD22, IgM or IgD and Alexa647-conjugated antibody against CD19 and settled onto nonstimulatory coverslips. Cells were then embedded in agarose, imaged with SIM and analysed.

(B) SIM images of CD22, IgM, IgD and CD19.

(C) Cross-correlation function.

(D) Pearson correlation coefficient.

(E) Cluster size calculated from autocorrelation analysis.

Data are pooled from three independent experiments. *P < 0.0001 (Student’s t-test).
Nanoscale organization and dynamics of the siglec CD22 coorporate with the cytoskeleton in restraining BCR signalling (2015)
Figure 3

Figure 3. CD22 is highly clustered on the surface of resting B cells

(A-C) dSTORM analysis of CD22, CD19, IgM and IgD. Wild-type primary B cells were stained with Alexa647-conjugated antibody against CD22, Cy5-conjugated antibody against CD19 or IgD, or Cy5-conjugated Fab fragments against IgM and settled onto nonstimulatory coverslips. Cells were then fixed, imaged with dSTORM and analysed.

(A) 2D (top and middle) and pseudo 3D (bottom) dSTORM images were reconstructed from single molecule localizations. The white dashed square is shown in magnification (middle) and pseudo 3D (bottom).

(B and C) Quantification of the distribution of CD22, CD19, IgM and IgD with H function and Hopkins index. Error bars (Hopkins index) and thin lines (H function) denote mean ± SEM. Data are pooled from at least three experiments per receptor.

*P < 0.001, **P < 0.0001 (Student’s t-test).
Optimal Sidestepping of Intraflagellar Transport Kinesins Regulates Structure and Function of Sensory Cilia (2020)
Figure 1

Fig. 1. Visualization of IFT at high spatiotemporal resolutions in GFP-knock-in animals

A. Schematic depiction of the Caenorhabditis elegans phasmid cilia (left). Two sensory cilia are aligned into the phasmid channel (the dashed box and enlarged in the middle). Each cilium contains a ciliary base, a middle segment (m.s.), and a distal segment (d.s.). Right shows an inverted fluorescence Hessian-SIM image of GFP-tagged dynein-2 heavy chain CHE-3 in cilia, in which individual puncta were resolved. Scale bar: 1 μm. Also shown in Appendix Figure S4A.

B. Frame series of an anterograde moving IFT-particle from a 7xGFP::CHE-3 worm. Localization precisions are shown under each frame. Time in millisecond (ms); Scale bar, 100 nm. More examples from CHE-2::7xGFP, DYF-11::3xGFP, or 7xGFP::CHE-3; DYF-11::3xGFP animals are in Appendix Figure S3B-C.

C. Localization precisions (mean ± SD) of tracks from multi-GFP-labeled worms. The number of GFP copies was estimated as the following: the dynein-2 heavy chain of CHE-3 forms a dimer, 14 = 2 x 7; IFT-particle subunit CHE-2 is also a dimer, 14 = 2 x 7; IFT-particle subunit DYF-11 can be a tetramer, 12 = 4 x 3; the genetic cross combined 7xGFP::CHE-3 with DYF-11::3XGFP, 26 = 2 x 7 + 4 x 3.

D. The representative diagram shows the displacement of the cargo within the middle ciliary segment. The y-axis grid lines are spaced 16 nm. Protein CHE-3 tagged with 7x GFP was used for tracking experiments. Trace was analyzed using the SIC fitting algorithm (Kalafut & Visscher, 2008).

E. Histogram of 161 displacements detected in 15 tracks extracted from 12 WT worms. The plus and minus displacements are defined as the upward "jumping" or downward "jumping" in Fig. 1D. Protein markers used for tracking experiments are CHE-3 tagged with 7x GFP, CHE-2 tagged with 7x GFP, or CHE-3 tagged with 7x GFP plus DYF-11 tagged with 3x GFP.

F. The proportion of the plus displacement, the minus displacement, and dwell (defined as the segments in Fig. 1D where there was no displacement detected) during imaging time of 15 tracks extracted from 12 WT animals. Proteins used for tracking experiments are the same as in Fig. 1E.