EMBO/SourceData huggingface dataset:
Figure 7(A) TRPV2 RNA expression levels (microarray probe: 219282_s_at) measured in normal skin (n=7 biological replicates), nevi (n=9 biological replicates) , primary (n=31 biological replicates) and metastatic (n=73 biological replicates) melanomas (GEO: GSE46517)(Data ref: Kwong LN, 2013). TRPV2 levels are increased in malignant lesions compared to normal skin and nevi. Data is represented as a whisker-box plot with outliers plotted as individual points (Boxes extend from the 25th to 75th percentiles, whiskers from the 10th to 90th percentiles, the horizontal line in each box is plotted at the median; ns P = 0,1383, *P = 0,0488 and **P = 0,0016, the Kruskal-Wallis test). The horizontal dotted line corresponds to TRPV2 median expression in nevi.(B) Representative melanoma tissue lesions from a tissue microarray (TMA) comparing TRPV2 staining between a benign nevus and a malignant melanoma (See also appendix Fig S10) (Scale bar = 200µm). Top right insets show the same images stained with an anti-melanoma triple cocktail (HMB45+A103+T311). Regions depicted with a black line represent the interface between the diffusely stained tumor (T) and the surrounding normal stroma (S). Right panel shows the scatter plot of the complete quantification of TRPV2 staining in 77 patients with malignant melanoma compared to 16 nevi tissues (dot, single patient sample) with lines for mean ± SEM (*P = 0.0473, the Mann-Whitney test).(F) Analysis of TRPV2 expression by quantitative RT-PCR (left panel) and western-blot (right panel) in the WM115/WM266.4 pair of isogenic melanoma cell lines. The WM115 cell line was derived from an in situ tumor while the WM266.4 was established from a skin metastasis of the same patient. Relative transcript levels are presented as mean ± SEM from n=4-9 biological replicates (**P = 0.0028, the Mann-Whitney test).(G) Kaplan-Meier plot showing the association of TRPV2 expression (10% highest versus 10% lowest expressers in the skin cutaneous melanoma cohort TCGA data set) with melanoma patient survival ((*)P = 0,0144, the Log-rank (Mantel-Cox) test).
Figure 11.SARS-CoV-2 activates delayed innate immune responses in lung epithelial cells Measurements of replication and innate immune induction in Calu-3 lung epithelial cells infected with SARS-CoV-2 at MOIs 0.08, 0.4 and 2 TCID50VERO/cell. (A) Replication of SARS-CoV-2 genomic and subgenomic E RNAs (qRT-PCR). (B) Quantification of N staining from cells in (A) by flow cytometry. Mean percentage of N-positive of all live-gated cells is shown +/- SEM, n=2.(C) Representative example of immunofluorescence staining of N protein (green) after SARS-CoV-2 infection of Calu-3 at MOI 0.4 TCID50VERO/cell, at time points shown. Nuclei (DAPI, blue), cell mask (red). Scale bar represents 50µM.Fold induction of (F) interferons (IFNβ, IFNλ1 and IFNλ3) pro-inflammatory mediators (IL-6 and CCL5) each overlaid with SARS-CoV-2 E (qRT-PCR).Fold induction of IFN stimulated genes (CXCL10 and IFIT2) each overlaid with SARS-CoV-2 E (qRT-PCR).
Figure 2Peak SARS-CoV-2 replication precedes innate immune activation. (A-I) (A,C) Representative images of NF-κB p65 (A) (red) and IRF3 (C) (red) nuclear localisation in mock or SARS-CoV-2 infected (MOI 0.4 TCID50VERO/cell) Calu-3 cells at 24 hpi. SARS-CoV-2 N protein (green. (E and G) Representative images of IL-6 mRNA (E) detected by FISH (red) and N protein (green) , or IFIT1 mRNA (G) (green) with N protein (red), both with nuclei (DAPI, blue) in mock or SARS-CoV-2 infected (MOI 0.4 TCID50VERO/cell) Calu-3 cells at 24 hpi. (B, D, F, H, I) Single cell analysis time course quantifying the Integrated Nuclear Intensity of NF-κB p65 (B), IRF3 (D) , or overall integrated intensity for IL-6 (F) or IFIT1 (H) mRNA over time in N protein positive cells and N protein negative cells (I). n=2. Kruskal-Wallis test with Dunn's multiple comparison. * (p<0.05), **** (p<0.0001). Scale bar represents 50µM.
Figure 3(E-H) Measurement of (E) viral genomic and subgenomic E RNA (F) fold induction of CXCL10, (G) IFIT2 and (H) and IL-6 at 24 hpi, of Calu-3 cells with SARS-CoV-2 (MOI 0.04 TCID50VERO/cell) with Remdesivir treatment (5μM) prior to, at the time of, or 8 h post-infection. Mean +/- SEM, n=3, One way ANOVA with Dunnett's multiple comparisons test to compare to untreated infected condition ('mock'), ** (p<0.01), *** (p<0.001), **** (p<0.0001).(I) Representative example of immunofluorescence staining of dsRNA (red) and N protein (green) after SARS-CoV-2 infection of Calu-3 at MOI 0.4 TCID50VERO/cell, at time points shown. Nuclei (DAPI, blue). Scale bar represents 50µM.(Q-R) (Q) Viral E RNA and (R) released infectious virus (TCID50VERO/cell) at 24 hpi of infected Calu-3 cells depleted for MAVs, RIG-I or MDA5. Mean +/- SEM, n=3. Each group compared to siCtrl by One Way ANOVA with Dunnett's multiple comparisons test, *, p>0.05, ** (p<0.01), n.s : non significant.
Figure 4(G-H) Viral genomic and subgenomic E RNA at 24 hpi (RT-qPCR) with DMSO or TPCA (G) or Rux (H) treatment. Data information: (A-H) Mean +/- SEM, n=3, statistical comparisons are made by unpaired t test comparing inhibitor-treated to mock-treated SARS-CoV-2 infected conditions at each MOI and each timepoint. * (p<0.05), ** (p<0.01), *** (p<0.001), **** (p<0.0001).
Figure 5(B-J) Calu-3 cells were transfected with siRNA targeting MAVS or non-targeting control (siCtrl) (B-D) or treated with DMSO vehicle or inhibitors 10 μM TPCA-1 (E-G) or 2 μM Ruxolitinib (Rux) (H-J) as shown, and were mock-infected or infected with SARS-CoV-2 at MOI 0.04 TCID50VERO/cell. Virus containing conditioned media (CoM) was harvested at 48 hpi. MDM were treated with Calu-3 virus containing CoM for 6 hpi, before washing and measuring MDM gene expression (B, E, H), and MDM activation markers by flowcytometry 48 h later (C,D,F,G,I,J) , plotting relative median fluorescent intensity (MFI) compared to mock-infected siCtrl (C, D) or mock-infected DMSO control (F, G, I, J). Legends in (B), (E) and (H) apply to (C,D), (F,G) and (I,J) respectively. The inhibitors in (E) and (H) were tested side-by-side with the same mock condition. Mean +/- SEM shown, data from 4-6 independent MDM donors is shown. Statistical comparison by two-tailed paired t-test comparing MDM exposed to control infected CoM to siMAVS/inhibitor treated infected CoM. * (p<0.05), ** (p<0.01), *** (p<0.001).
Figure 6Calu-3 cells were infected with SARS-CoV-2 (MOI 0.04 TCID50VERO/cell) in the presence or absence of 10ng/ml IL-1b. (U) Non-adherent cells were collected at 48h post infection and live/dead stained. Cells were acquired by flowcytometry and cell counts determined by time-gating. For statistical comparison, total cell numbers were compared. (V) Viral release into culture supernatants at 24 h was measured by TCID50 on VeroE6 cells. (Q-V) Mock and SARS-CoV-2 infected conditions were compared with or without IL1b-treatment, respectively, by unpaired T test (n=3). *, p<0.05; n.s., non-significant. Mean +/- SEM shown.
Figure 1A Immunoblot analysis of overexpressed and ALFA-tagged CNX in CNX knockout cell lines clones #1 and #2, which were used for mass spectrometry. Endogenous CNX level of wildtype HEK293T cells is shown as a control.C Volcano plots derived from LC-MS/MS analyses of ALFA-tagged CNX, immunoprecipitated in 1% NP-40 buffer from transfected CNX deficient HEK293T cells after DSSO crosslinking and compared to empty vector (EV) control co-IPs. Depicted is an enlarged section of the associated complete volcano plot in the upper right. Among the significantly enriched proteins, annotations are Uniprot gene names. CNX is shown in green. ER proteins with the GO-term annotation "endoplasmic reticulum", which are localized to the ER lumen, are highlighted in blue and among those several known CNX interaction partners are indicated with their names. Membrane proteins are shown in red and those which are non-glycosylated are additionally labeled with their gene names.E Co-immunoprecipitations of endogenous CNX with FLAG-tagged Cx32 WT and its mutants including quantification and statistical analysis. Constructs were expressed in HEK293T cells. One representative immunoblot is shown. Monomers and dimers of Cx32 are indicated with brackets. All samples were normalized to WTF Immunofluorescence images of Cx32 and its mutants. COS-7 cells were transiently transfected with the indicated FLAG-tagged Cx32 constructs and immunofluorescence microscopy was performed using anti PDI (green) as an ER marker. Detection of Cx32 was performed using anti FLAG antibodies and suitable labeled secondary antibodies (red). Nuclei were stained with DAPI (blue). All three channels are overlayed. Images are representative of cells from at least three different biological replicates. GJ denotes gap junctions observed at cell-cell junctions of cells transfected with WT Cx32.
Figure 2B Co-immunoprecipitations of endogenous CNX with the different CL-TMD constructs (NVT glycosylation site mutated to QVT) including quantification and statistical analysis. One representative immunoblot is shown. The asterisk (*) denotes a fraction of cleaved species as observed previously for similar constructs (Behnke et al., 2016). All samples were normalized to the construct containing TMD1C Interaction analysis of Calnexin with the first transmembrane domain of Cx32. Intracellular BMH crosslinking of transiently transfected V5-tagged CNX with the transiently transfected R26C mutant of Cx32-TMD1. In the AA mutant of CNX, the two cysteines within the CNX TMD were replaced by alanines. The species at approx. 250 kDa are possibly CNX dimers.E COS-7 cells were transiently transfected with the indicated constructs and immunofluorescence microscopy was performed using anti PDI (green) as an ER marker. Detection of minCNXTMD and both control constructs was carried out by mScarlet-I fluorescence (red). Nuclei were stained with DAPI (blue). Images are representative of cells from at least three different biological replicates.F Interaction of Cx32-TMD1 with the minCNX system. MinCNXTMD constructs and controls (see (D)) were co-transfected with Cx32-TMD1 into HEK293T cells. Interaction between the proteins was analyzed by co-immunoprecipitation experiments followed by immunoblotting.
Figure 3B HEK293T cells were transiently transfected with plasmids expressing ConMem or the indicated variants, where either the first (N1) or the second (N2) NVT glycosylation motif was ablated (N to Q mutation). Lysates were treated with or without EndoH or PNGaseF as indicated, and analyzed by immunoblotting. N-glycosylation occurs in the ER, complex glycosylation in the Golgi.C COS-7 cells were transfected with the indicated constructs and fluorescence microscopy was performed using Sec61 mCherry (red) as an ER marker. ConMem, detected by sfGFP fluorescence (green), localizes to the plasma membrane. Nuclei were stained with DAPI (blue). Images are representative of cells from at least three different biological replicates.
Figure 4B Representative blots from co-immunoprecipitation experiments from HEK293T cells transfected with the indicated ConMem constructs with or without ER-lumenal glycosylation site (N1) (mean ± SEM, N = 3, *P value < 0.05, two-tailed Student's t tests). Individual values were normalized to ConMem N1Q (Val) values that were set to 1.C Interaction of ConMem N1Q variants as described in (A) with endogenous CNX (mean ± SEM, N ≥ 5, *P value < 0.05, two-tailed Student's t tests ). Individual values were normalized to ConMem N1Q (Val) values that were set to 1. D Same as in (C) only that selected amino acids (proline and arginine) where shifted through the ConMem N1Q TMD segment to five different positions as shown in the illustration, revealing positional binding dependencies (mean ± SEM, N ≥ 4, *P value < 0.05, two-tailed Student's t tests). Individual values were normalized to ConMem N1Q (Val) values that were set to 1.
Figure 5D Interaction of non-glycosylated ConMem N1Q constructs with V5-tagged CNX. In CNX variants, amino acids important for interaction as revealed in (A) were replaced by valine residues. HEK293T cells were transiently transfected with the indicated constructs and cell lysates and HA-immunoprecipitates were analyzed for HA-tagged ConMem N1Q and co-immunoprecipitating CNX mutants. E The same as in (D) for Cx32-TMD1 F Analysis of mutations in the CNX TMD region and their effect on binding glycosylated ConMem. G The same as in (D) for Cx32 full-lengthH Interaction of Cx32-TMD1 with CNX mutants, where amino acids not predicted to be important for interaction as shown in (A) were exchanged by valine residues. HEK293T cells were transiently transfected with the indicated constructs.I Shifting of the residues Y, T and L, most important vor CNX substrate interaction, and replacing these against valine residues, impacts interactions with Cx32-TMD1. HEK293T cells were transiently transfected with the indicated constructs.
Figure 6A Analysis of the interaction of the binding deficient CNX YTL TMD variant with Rhodopsin. CNX knockout HEK293T cells were transiently transfected with either N-terminal ALFA-tagged Rhodopsin alone or in combination with the indicated V5-tagged CNX constructs. In the absence of any CNX, Rho is hardly detectable (long exposure shown). Cell lysates and ALFA-immunoprecipitates were analyzed for Rhodopsin and co-immunoprecipitating CNX.B Analysis of Rhodopsin degradation and its dependency on CNX. CNX knockout HEK293T cells that were transfected with the indicated constructs were incubated with cycloheximide and lysates were collected at different timepoints. CNX WT and CNX YTL immunoblots are from the same blot (dashed line), showing similar expression levels. Hsc70 was used as a loading control (also from the same blot, dashed line). Quantifications are shown below representative immunoblots
Figure 1(C, D) Quantification of SCOT and BDH1 expression levels relative to VCP in various murine organs. Data are presented as mean ± SD. n=3; *, p<0.05; ***, p<0.001 unpaired Student's t-test.(G) Immunoblot of SCOT, BDH1, CD31 and VCP in cardiac endothelial cells (EC) and non-endothelial cell fraction (non-EC) isolated from adult C57Bl/6J mice.
Figure 2(A, B) Murine cardiac endothelial cells (MCEC) were treated with 2 mM D-β-hydroxybutyrate (β-OHB) or 2 mM acetoacetate (AcAc) for 24 hours. Quantification of concentrations of TCA cycle intermediates compared to control treatment. Data are presented as mean ± SD. n≥4; ns, not significant; *, p<0.05; **, p<0.01; ***, p<0.001 unpaired Student's t-test.(D) Quantifications of [13C]-labeled fractions of amino acids (asparagine, L-aspartate, L-glutamate, L-proline), putative lipid species (nervonic acid, 1-palmitoylglycerol, ceramide (d18:1/16:0)) and uridine diphosphate (UDP) species (uridine monophosphate, uridine 5'-diphosphate, UDP-N-acetylglucosamine) in MCEC treated with 1 mM 13C4-β-hydroxybutyrate or 1 mM 13C4-acetoacetate for 24 hours or the same concentration of unlabeled ketone bodies ([12C] isotope). Data are presented as mean ± SD. n≥4; ns, not significant; *, p<0.05; **, p<0.01; ***, p<0.001 unpaired Student's t-test.
Figure 3(A, B) Murine cardiac endothelial cells (MCEC) were treated with different concentrations of D-β-hydroxybutyrate (βOHB) or acetoacetate (AcAc) for 24 hours. Relative BrdU absorbance was quantified compared to control treatment (H2O for βOHB and EtOH for AcAc). Data are presented as mean ± SD. n≥3; *, p<0.05; **, p<0.01; ***, p<0.001 unpaired Student's t-test.(I-M) Angiogenic capacity of MCEC in response to ketone body treatment was analyzed using a spheroid-based sprouting assay. Spheroids were treated with media containing 30 mM D-β-hydroxybutyrate (βOHB), 30 mM acetoacetate (AcAc), 10 mM acetate, 1 mM octanoate, 1 mM butyrate, 30 mM L-β-hydroxybutyrate (L- βOHB), 1 mM niacin or the respective controls (final concentration of reagents is diluted to approximately 10 %) and analyzed after 48 hours. The (I, L) average number of sprouts per spheroid, the (J, M) accumulated total sprout length and (K) average sprout length were quantified. Data are presented as mean ± SD. n≥3; *, p<0.05; **, p<0.01; ***, p<0.001 unpaired Student's t-test.
Figure 4(B, Angiogenic capacity of Oxct1 knockout cells in response to ketone body treatment was analyzed using a spheroid-based sprouting assay. Spheroids were treated with media containing 30 mM D-β-hydroxybutyrate (βOHB), 30 mM acetoacetate (AcAc) or the respective controls (final concentration of reagents is diluted to approximately 10 %) for 48 hours. The average number of sprouts per spheroid was quantified. Data are presented as mean ± SD. n=4; *, p<0.05; **, p<0.01; unpaired Student's t-test.
Figure 5(C, D) Heatmaps showing expression levels of genes involved in KEGG pathways (C) fatty acid degradation and (D) ketone body metabolism in cardiac endothelial cells isolated from male C57Bl/6J mice that were fed a ketogenic diet or control diet for two weeks.
Figure 6(A, B) Representative images of heart sections of animals kept on a ketogenic diet or control diet for two weeks stained against ERG and Ki67 or EdU, double positive cells are indicated by arrowheads, and quantification of double positive cells in heart sections per high power field (HPF). Scale bar: 50 μm. Data are presented as mean ± SD. n≥3; *, p<0.05; **, p<0.01; ***, p<0.001 unpaired Student's t-test.(F) Quantification of Ki67+ endothelial cells in several organs of mice kept on a ketogenic diet or control diet for four weeks (gastrocnemius and soleus muscle) or two weeks (brain, lungs, subcutaneous adipose tissue, liver). Data are presented as mean ± SD. n≥3; *, p<0.05; **, p<0.01; ***, p<0.001 unpaired Student's t-test.
Figure 7(B) Representative images of heart sections of mice kept on a control or ketogenic diet eight weeks after TAC surgery stained against Ki67 and ERG. Scale bar: 50 µm.
Figure 3(A-C) Tissue lysates from (A) spleen, (B) liver and (C) kidney from 6- to 9-day old indicated mice were immunoblotted to determine cleavage of caspase-1. Each lane represents a lysate from a different mouse. Positive control lanes (+) are BMDMs stimulated with LPS/ATP; negative control lanes (-) are respective organ homogenates from caspase-1-/- mice.(D-G) Tissue lysates from (D, F) spleen and (E, G) liver from 6- to 9-day old indicated mice were immunoblotted to determine pro-IL-1β cleavage to IL-1β (D-E) or GSDMD cleavage to its p30 and p20 fragments (F-G). Each lane represents a lysate from a different mouse. Positive control lanes (+) are BMDMs stimulated with LPS/ATP; negative control lanes (-) are respective organ homogenates from (D-E) caspase-1-/- or (F-G) Gsdmd-/- mice.
Figure 2Localization of CASPR in semi-thin sections of retinas from either MOG/CFA-injected mice (EAE mice) or CFA-injected control mice (9 days after injection). Semi-thin sections were double-immunolabelled with antibodies against CASPR1 and RIBEYE (A-B OPL, outer plexiform layer; INL, nuclear layer; IPL, inner plexiform layer. Scale bars: 20μmLocalization o visual-arrestin (v-arrestin) in semi-thin sections of retinas from either MOG/CFA-injected mice (EAE mice) or CFA-injected control mice (9 days after injection). Semi-thin sections were double-immunolabelle with antibodies against v-arrestin and RIBEYE (C-D) V-arrestin, visual arrestin; OPL, outer plexiform layer; INL, nuclear layer; IPL, inner plexiform layer. Scale bars: 20μm
Figure 3Localization of CNTN1 in semi-thin (0.5μm-thin) sections of retinas from either MOG/CFA-injected mice (EAE mice) or CFA-injected control mice (9 days after injection). (A,B) Sections were double-immunolabelled with antibodies against CNTN1 and RIBEYE proteins in the OPL and IPL. ONL, outer nuclear layer; OPL, outer plexiform layer; INL, nuclear layer; IPL, inner plexiform layerQuantification of the immunosignals of the indicated proteins in the OPL and IPL. ONL, outer nuclear layer; OPL, outer plexiform layer; INL, nuclear layer; IPL, inner plexiform layer; n.s., non-significant; ***, p<0.001 (Mann-Whitney U test, actual p-values are given in C) (N=6 independent set of experiments; n=295 images (CFA); n=274 images (MOG/CFA) for quantification of RIBEYE immunosignals; n=95 (CFA); n=92 (MOG/CFA) for quantification of CASPR1 immunosignals; n=91 (CFA), n=82 (MOG/CFA) for CNTN1 quantification; n=109 (CFA); n=100 (MOG/CFA) for quantification of v-arrestin. Scale bars: 20μm
Figure 4MOG/CFA- but not CFA-injected mice contain reactive antibodies for retinal proteins in blood and show altered spatial visual behavior. (A) 7-9 days after injection, blood from MOG/CFA-injected (red) but not from CFA-injected mice (black), elicited strong antibody responses in ELISA. Antibodies for RIBEYE and SV2 were used as positive controls (green), secondary antibody only as a negative control (white). Normalized data are given as mean ± S.E.M. (N=4 (CFA); N=5 (MOG/CFA). ** p<0.01 (significance was assessed by Mann-Whitney test, precise p-values given in the figure)Control Western blot of HEK293 protein lysates obtained from cells transfected with mCherry (lane 1) or CASPR1-mCherry cDNA (lane 2) and probed with anti-mCherry antibody(C,D) Western blot of protein lysates as in B) probed by blood samples from mice before and 7 to 9 days after MOG/CFA injection. Only blood samples obtained from MOG/CFA-injected mice revealed immunoreactivity against CASPR1 (lanes 15,16; top panels) whereas samples obtained before immunization (lanes 7,8; top panels) did not. The same filters were re-probed with anti-actin antibodies as loading control(C,D) Western blot of protein lysates as in B) probed by blood samples from mice before and 7 to 9 days after MOG/CFA injection. Only blood samples obtained from MOG/CFA-injected mice revealed immunoreactivity against CASPR1 (lanes 15,16; top panels) whereas samples obtained before immunization (lanes 7,8; top panels) did not. The same filters were re-probed with anti-actin antibodies as loading control. (E) Summary of data from (C,D) shown as mean ± S.E.M. (N=4, before injection; N=5, MOG/CFA-injected). ***, p<0.001; n.s., non-significant (unpaired two-tailed Student's t-test, precise p-values given in the figure)Spatially guided visual behavior, measured as frequency threshold with a virtual optokinetic system 9 days after immunization, is significantly decreased in MOG/CFA-injected mice compared to CFA-injected control litters. Data are shown as median; boxes and whiskers illustrate 25-75 and 5-95 percentiles of values, respectively. Points below and above whiskers are drawn as individual points. At each trial, mice were measured three times. Home-made and commercial MOG/CFA suspensions were compared as well as PLP-injected mice with control-injected mice. Statistical analysis was done by ANOVA (0,05 significance level), mean comparison probabilities were Bonferroni corrected. N=32, n=96 (MOG/CFA); N=24, n=72 (CFA). ***, p<0.001 (Mann-Whitney test). Results from mice injected with home-made and commercial MOG/CFA suspensions as well as results obtained from PLP/CFA-injected mice (and respective control-injected mice) are displayed
Figure 5Optic nerve parameters are unaltered in early EAE (9 days after injection). (A,B) Cryostat sections of the optic nerve from MOG/CFA-injected mice (own lab-made suspension; for comparison with commercial MOG/CFA suspensions, see Appendix Fig. S12) and CFA-injected control mice were stained with anti-Nav antibody (1:50 dilution) to label the node of Ranvier and with anti-CASPR1 antibody (1:500 dilution) to stain the paranodal region. Length of the node of Ranvier and length of the paranodal region were quantified in C) D)(E1, E2) Richardson-Blue stained semi-thin sections from the indicated optic nerve samples. (E2, E3, F2, F3) Transmission electron micrographs of the optic nerves from MOG/CFA- and CFA-injected animals. Abbreviations: ax, axon; my, myelin sheath. Axon number and myelin thickness were quantified in G) and H). Scale bars: 1μm (A,B,E,F)