EMBO/SourceData huggingface dataset:
Figure 7The amount of CASPR1 protein in the synapse is also altered in the PLP mouse model of multiple sclerosis. Semi-thin sections of PLP/CFA and CFA-injected mice were probed with the indicated antibodies (A, B; see also Appendix Fig. S13)The synaptic amount of CASPR1 is strongly reduced both in the OPL and IPL. RIBEYE is only reduced in the OPL but not significantly in the IPL. CNTN1 is strongly reduced in both OPL and IPL (C, see also Appendix Fig. S13). N=4 PLP/CFA-injected mice; N=4 CFA-injected mice; n=140 slices (CFA, RIBEYE); n=143 slices (PLP/CFA, RIBEYE); n=47 slices (CFA, CASPR1); n=53 slices (PLP/CFA, CASPR1); n=49 slices (CFA, CNTN1); n=46 slices (PLP/CFA, CNTN1); n=44 slices (CFA, v-arrestin); n=44 slices (PLP/CFA, v-arrestin)(D,E) Cryostat sections of the optic nerve from PLP and control mice were stained with anti-Nav-channel antibody to label the node of Ranvier and with anti-CASPR1 antibody to stain the paranodal region. Length of the node of Ranvier and length of the paranodal region were quantified in G) H). (F) transmission electron micrographs of the optic nerves from PLP/CFA- and CFA-injected animals (F1-F4). Axon numbers and myelin thickness were quantified in I) J)
Figure 1Confocal micrographs show CK7 (green) and vimentin (red) in patient-derived HGSC cells cultured in 3D collagen for 7 d. Scale bars: 50 μm.Confocal micrographs of EphA2 (red) in HGSC cells cultured in 3D collagen for 7 d and treated for 72 h with 10 μM cisplatin (5 μM for OCKI_p01). The intensity of EphA2 is comparable only between mock and treatment conditions for each patient. Scale bars: 20 μm.Chart illustrates EphA2 fold change after treatment. Mock is set to one. N = 3.
Figure 2EphA2 (total and phosphorylated at S897 or Y588) in OVCAR3, OVCAR4 and OVCAR8 after treatment with 0-20 μM cisplatin for 72 h was assessed by immunoblotting (A) and quantified for pS897/pY588 ratio (B). N = 4.EphA2 (total and phosphorylated) in corresponding untreated cells.Cytotoxicity assay results after cell treatment with 0-20 μM cisplatin for 72 h. N = 6.Quantitative assessment of EphA2 (total and phosphorylated) and pS897/pY588 ratio in early passage patient-derived HGSC cultures treated with 0-20 μM cisplatin for 72 h (see immunoblots in Appendix Fig S2A). N = 6 patients, pooled.Corresponding EphA2 pS897/pY588 ratios for individual patient cells.EphA2 (total and phosphorylated) in OVCAR4 and OVCAR8 after treatment with 0-80 μM carboplatin for 72 h (G) along with pS897/pY588 quantification (H). N = 3.
Figure 3Bioluminescence images visualize mock and carboplatin treated OVCAR4 xenograft tumors (day 53; after 13 d i.p. treatment). Color scale unit: p/sec/cm2/sr. Charts illustrate the tumor growth (B) and ascites volumes at the end of the follow-up (C). Carboplatin treatment started on day 35. Bioluminescent unit: p/sec/cm2/sr. N = 5 mice/group.Representative micrographs of hematoxylin-eosin staining in the xenografts tumors. Scale bars: 100 μmEphA2-pS897 immunofluorescence in the xenografts tumors. Scale bars: 10 μmQuantitative assessment of tumor EphA2 (F) and EphA2-pS897 (G). N = 5 mice/group.Confocal micrographs of tumor EphA2-pS897 (red) and cleaved caspase-3 (H; clCasp3, green) and corresponding clCasp3 quantification (I). Scale bars: 20 μm. N = 5 mice/group.
Figure 4Immunoblot images show EphA2 (A; total, pS897 and pY588) and corresponding pS897/pY588 ratios (B) of TYK-nu and TYK-nu.R after treatment with 0-10 μM cisplatin for 72 h . N = 5.EphA2 (total and phosphorylated) in the cells treated with 0-80 μM carboplatin for 72 h.EphA2 (total and phosphorylated) in the cellsEphA2 (total and pS897) in TYK-nu after treatment with inhibitors against MEK (UO126, 10 μM), PKA (H89.2HCl, 10 μM), PI3K (LY294002, 10 μM) or its alpha subunit (BYL-719, 5 μM) and RSK (BI-D1870, 10 μM) in combination with 0-10 μM cisplatin for 72 h (E). Corresponding EphA2-pS897 quantification for BI-D1870 treatment (F) N = 5.EphA2 (total and pS897) in TYK-nu.R treated with 0-20 μM cisplatin without or with 10 μM BI-D1870 for 72 h.EphA2, RSK and ERK1/2 (total and phosphorylated) in TYK-nu (H) and OVCAR4 (I) treated with 0-30 μM carboplatin without or with 50 μM LJH685 for 72 h.
Figure 5Charts show OC cell viability after 72 h treatment with 0-20 μM cisplatin and 10 μM BI-D1870Charts show OC cell viability after 72 h treatment with 0-20 μM cisplatin and 50 μM LJH685RSK and EphA2 (total and phosphorylated) in OC cells treated with 10 µM cisplatin and 25 µM LJH685 as indicated for 72 h.Charts illustrate the corresponding quantified inhibition of EphA2-pS897 and increase of EphA2-pY588 shown in (C).
Figure 6Immunoblot images (A) and quantification (B) show GPRC5A 46/41 kDa ratios in OC cells treated with 0-10 µM cisplatin and with 25 µM LJH685 alone or as a combination for 72 h. The ratio in mock cells was set to 1. N = 4.Confocal micrographs of EphA2 (red) and GPRC5A (green) in TYK-nu and TYK-nu.R treated with 5 µM cisplatin and 10 μM LJH685 as indicated for 72 h. Arrows point co-localization of the receptors. Scale bars: 20 μm.EphA2 (total and phosphorylated), RSK1/2 and GPRC5A in TYK-nu were assessed by immunoblotting after RSK1/2 knockdown for 2 d and following treatment with 0-5 μM cisplatin for 72 h. Asterisks indicate unspecific bands.EphA2 (total and phosphorylated), RSK1/2 and GPRC5A and these proteins coupled with PARP and PCNA in TYK-nu.R were assessed by immunoblotting after RSK1/2 knockdown for 2 d and following treatment with 0-5 μM cisplatin for 72 h. Asterisks indicate unspecific bands.Chart illustrates the viability of the RSK1/2 depleted TYK-nu.R after cisplatin treatment. N = 3.
Figure 7Confocal micrographs show cleaved caspase-3 (clCasp3, green), mRFP (orange) and phalloidin (F-actin, red; only shown for CAF) in 3D OVCAR8-RFP and OCKI_p22 CAF mono- and co-cultures treated without or with 20 µM cisplatin or 25 µM LJH685 alone or in combination for 20 h. Scale bars: 100 μm.Quantitative assessment of clCasp3+ cells in the 3D cultures. N = 4.Confocal micrographs show cytokeratin 7 (CK7, green) and phalloidin (F-actin, red) in 3D patient OCKI_p13 mono- and co-culture with OCKI_p22 CAF treated without or with 20 µM cisplatin or 25 µM LJH685 alone or in combination for 20 h. Scale bars: 50 μm. Quantitative assessment of clCasp3+ OCKI_p13 cells in 3D co-cultures of patient-derived cells. N = 4.Immunohistochemistry images and quantifications of TUNEL in OVCAR4 xenografts in mock (N = 4), carboplatin (N = 4) and carboplatin+BI-D1870 (N = 5) treated mice. Scale bars: 100 μm.Immunohistochemistry images and quantifications of Ki67 in OVCAR4 xenografts in mock (N = 4), carboplatin (N = 4) and carboplatin+BI-D1870 (N = 5) treated mice. Scale bars: 100 μm.
Figure 8Representative confocal micrographs show EphA2 (red), RSK (green: top) and GPRC5A (green: bottom) in frozen sections of HGSC patient tumors. S indicates the stroma. Scale bars: 50 μm.Indicated proteins and EphA2 phosphorylation were assessed by immunoblotting in patient-derived HGSC cells and CAFs.GPRC5A in earlier (top) and later passage (bottom) HGSC cells. See normalized GPRC5A relative to OCKI_p01 below the immunoblot images.Charts illustrate EphA2-pS897 inhibition (circles) and EphA2-pY588 increase (squares) by 10 µM BI-D1870 alone (unbroken line) or in combination with 0-10 µM cisplatin (dotted line) in the GPRC5Ahigh HGSC cells. See Appendix S7B for immunoblots.Cell viability of the GPRC5Ahigh OCKI_p02 and OCKI_p06 after treatment with a combination of 0-20 µM cisplatin and 10 µM BI-D1870 for 72 h. N = 3.Cell viability upon 72 h combinatorial treatment with 0-20 µM cisplatin and 25 µM LJH685 in patient-derived CAFsCell viability upon 72 h combinatorial treatment with 0-20 µM cisplatin and 10 µM BI-D1870 in mesothelial cells (G; N = 3).Chart illustrates the changes of EphA2-pS897 and EphA2-pY588 by BI-D1870 alone and with cisplatin in mesothelial cells. Upon combination with 10 µM cisplatin for 72 h, 10 µM BI-D1870 decreased EphA2-pS897 and EphA2-pY588. See Appendix Fig S7J for immunoblot.Confocal micrographs of EphA2 (red) and GPRC5A (green) in primary OCKI_p06 patient cells cultured in 3D collagen for 4 d before 72 h treatment with or without 10 µM cisplatin. Scale bars: 20 μm.
Figure 9Representative immunostainings for GPRC5A scoring in HGSC primary and metastatic tumors. For maximum intensity scores an optical 4-point scale (0: negative, 1: mild, 2: moderate, 3: high) was used. For survival analyses a 2-point scale (low: scores 0-2, high: score 3) was used. Scale bars: 200 μm.Kaplan Meier survival curves illustrate the overall (OS) and progression-free (PFS) survival of patients with high or low GPRC5A. In primary tumors, mean OS for GPRC5Ahigh was 39 months vs 56 months for GPRC5Alow (C). In patients with metastatic tumors, mean OS for GPRC5Ahigh was 37 months vs 60 months for GPRC5Alow. Mean PFS in metastatic tumors was 20 months for GPRC5Ahigh vs 35 months for GPRC5Alow (D). No significant difference in mean PFS in primary tumor.Kaplan Meier survival estimates for the PFS of patients with high or low (top 25% vs bottom 25%) GPRC5A (F), EPHA2 (G), GPRC5A+EPHA2 (H) and GPRC5A+EPHA2+RSK1(RPS6KA1)+RSK2(RPS6KA3) combined (I) mRNA expression. Mean PFS for GPRC5A+EPHA2high was 15 months vs 26 months for GPRC5A+EPHA2low (H). Mean PFS for GPRC5A+EPHA2+RPS6KA1/3 high was 14 months vs 31 months for GPRC5A+EPHA2+RPS6KA1/3low (I).
Figure 3Assays were performed in HEK293 cells for A) F508del-CFTR For the F508del-CFTR measurements since this mutant does not respond well to Fsk alone (Fsk + VX-770 potentiator is required to obtain a detectable signal) we compared the CFTR inh172 (inhibitor) response (F1/'F-CFTRinh172'; where F1 represents peak RFU immediately before inhibitor addition and 'F-CFTRinh172' is the minimum RFU reached at the end of the CFTR inh172 measurement cycle). For each of F508del-CFTR data for three proteins not detected as interactors (first three bars following CFTR only) are shown as negative controls. Fluorescence signals were normalized to the CFTR-only control and the statistical significance of differences assed by unpaired t-test (*p<0.05, **p<0.01 and ***p<0.001). Samples run in n=3 replicates, and all values are mean ± s.d.Assays were performed in HEK293 cells for B) wt-CFTR. For the wildtype measurements, we compared the maximum Fsk response (Maximum F /F0; where F0 represents baseline RFU right before the stimulation). For each of wt-CFTR data for three proteins not detected as interactors (first three bars following CFTR only) are shown as negative controls. Fluorescence signals were normalized to the CFTR-only control and the statistical significance of differences assed by unpaired t-test (*p<0.05, **p<0.01 and ***p<0.001). Samples run in n=3 replicates, and all values are mean ± s.d.
Figure 5A) Blot images showing bands B and C as well as the calnexin internal control.B) Blot quantification showing relative fold change of band B and band C versus treatment with a non-targeting siRNA (Neg1). One-way Anova followed by Dunnett's post hoc test was performed to determine significance versus baseline (siNeg1 + DMSO treatment, *p<0.05) and 5 µM VX-661 (#p<0.05). Measurements were obtained from biological triplicates (n=3) and all values are mean ± s.d.
Figure 6B) qPCR Validation of lentivirus-mediated shRNA knockdown of FGL2 and CFTR in intestinal organoids. Significance of sample differences was calculated using the one-tailed student t-test (n=2 biological replicates). All values are mean ± s.d. EV = empty vector.C) Representative confocal microscopy images of calcein green labelled FIS organoids of a healthy individual and a CF patient, before and 60 min after stimulation with Fsk (5 uM). Scale bar, 200 μm.D) Quantification of FIS response in wt-CFTR organoids (ORG-01(HC)) shown in (C) as area under the curve at 60 min. Significance of sample differences was calculated using two-way ANOVA (n=2 biological replicates). All values are mean ± s.d. E) Quantification of FIS response in F508del-CFTR organoids (ORG-BX-002 (dF/dF)) shown in (C) as area under the curve at 60 min. Significance of sample differences was calculated using two-way ANOVA (n=2 biological replicates). All values are mean ± s.d. F
Figure 1 Wild type (WT) and TRIM21 knockout (KO) mice were infected with 10^5 FFU LCMV clone 13 (6 mice per group) and 2 mice were uninfected (UI). Weights were compared over 15 days post infection (pi).  Wild type (WT) and TRIM21 knockout (KO) mice were infected with 10^5 FFU LCMV clone 13 (6 mice per group) and 2 mice were uninfected (UI). Kaplan-Meier survival were compared over 15 days post infection (pi).  C) Sera from WT mice was analysed for N protein (N) and glycoprotein (GP)-specific antibodies by ELISA, each data point corresponds to one mouse from the same experiment.  D) N-specific antibody ELISA was used to compare antibody responses in sera of WT and KO mice.  E-F) In vitro neutralisation experiments were performed with sera collected day 8pi from WT, KO and UI mice. E) Sera was pre-incubated with LCMV, then the virus-serum mix was added to MEF cells and LCMV infection titre after 16 hours was measured by FFA. F) Sera was electroporated into MEFs, then cells were plated in triplicate and LCMV was added 4 hours later. LCMV infection titre after 16 hours was measured by FFA (three replicates).  G) Anti-N mAb KL53 was electroporated into WT and KO MEFs and subsequent LCMV infection titres were measured by FFA.  H) Anti-N mAb KL53 was co-electroporated with recombinant N protein into WT and KO MEFs, and immunoblotting for N was performed after 3 hours. Electroporation of recombinant KL53 expressing the TRIM21 non-binding mutation H433A was unable to mediate N protein degradation.
Figure 2 Wild type (WT) and TRIM21 knockout (KO) mice were infected with 0.5 x 10^5 FFU LCMV clone 13 (6 mice per group), and either received anti-N mAb KL53 (+) or control (-) intraperitoneally on days 1 and 3pi. Two mice were uninfected (UI). A) Weights were monitored throughout infection, with final day 8 weights of individual mice presented separately.  Wild type (WT) and TRIM21 knockout (KO) mice were infected with 0.5 x 10^5 FFU LCMV clone 13 (6 mice per group), and either received anti-N mAb KL53 (+) or control (-) intraperitoneally on days 1 and 3pi. Two mice were uninfected (UI). Viral titres in the spleen, liver, lung and kidney of all mice were determined by FFA day 8pi. Each data point represents one mouse, with results from two repeat experiments combined.
Figure 3 A) CD8 T cells in WT mice were depleted by administration of anti-CD8 mAb 1 day prior to infection with 10^5 FFU LCMV. Anti-N mAb KL53 or PBS control was passively transferred IP on days 1 and 3pi. Viral titres in the spleen, lung, liver and kidney of all mice were determined by FFA day 8pi.  B) Spleens from WT and KO mice +/- mAb KL53 day 8pi with 0.5 x 10^5 FFU LCMV were analysed for the presence of LCMV N-specific CTLs by staining with the class I N396-404 tetramer.  C) Spleens from naïve WT and KO mice day 10pi with 0.5 x 10^5 FFU LCMV were stained with N tetramer.  D) Macrophages were depleted in WT mice by administration of clodronate liposomes 1 day prior to infection with 0.5 x 10^5 FFU LCMV. KL53 was administered on days 1 and 3pi, and N396-specific CTLs in the spleen were measured day 8pi by tetramer staining.
Figure 4 A) Splenocytes from uninfected CD45.1 mice, either pulsed with 3 concentrations of N peptide and cell trace violet (CTV) or unlabelled control cells, were transfused intravenously into WT and KO mice (CD45.2) that had been infected with 0.5 x 10^5 FFU LCMV 8 days earlier. After 3 hours, spleens from recipient mice were harvested and the proportion of CTV-labelled CD45.1 cells was analysed by flow cytometry. Histograms from single representative uninfected (UI), WT and KO mice are presented, showing the proportion of CD45.1 cells remaining for each of the labelled fractions normalised to mode. Summary data from all individual mice in the same experiment is presented in associated scatter plot, showing the mean ± standard error. B) Labelled splenocytes as for (A) were transfused into WT and KO mice that had been infected with LCMV 8 days earlier, and received mAb KL53 on days 1 and 3pi. Flow cytometry histograms from single representative mice of each genotype. Summary data from all mice in the experiment is presented, showing the mean ± standard error.
Figure 3Genes with higher dominant PAS usage (DPU) (A) or with lower APA Shannon index (B) express at higher levels (liver shown as example). The number of genes in each group is indicated on top. Mann-Whitney U test significance levels are indicated for neighboring groups: *P < 0.05; **P < 0.01; ***P < 0.001. The boxplots show the Q1 to Q3 quartile values (the box limits), the median (the horizontal green lines) and values within the 1.5 * IQR (the whiskers).Spearman correlations between gene expression level and dominant PAS usage (DPU) in each tissue for all genes (blue), genes with DPU equal to or above 90% and genes with DPU less than 90%. Genes with DPU less than 90% exhibit weaker Spearman correlations between gene expression level and dominant PAS usage in each tissue. An empty square indicates that the P value of Spearman's rho is above 0.01. (ESC: embryonic stem cells, CER: cerebellum, COR: cerebral cortex, HEA: heart, KID: kidney, LIV: liver, LUN: lung, MUS: muscle, SPL: spleen.)
Figure 4Fractions of orthologous genes in different groups exhibiting different subcellular localizations of mRNA isoforms in rat neurons. Same: isoforms enriched in same cellular location. Diff: isoforms enriched in different cellular locations, i.e. one isoform in neuropil and another one in soma. (Fisher exact test: *P < 0.05; **P < 0.01; ***P < 0.001).
Figure 72D clustering of Pearson correlation coefficients between all pairwise sample combinations of C57BL/6J (BL6) or SPRET/EiJ (SPR) allelic PAS usages from 5,264 genes (see Methods). 2D clustering of Pearson correlation coefficients between all pairwise combinations of BL6 or SPR allelic gene expression levels from 5,264 genes. All clustering was performed hierarchically using Pearson correlations between samples.
Figure 1B-K. Transmission electron microscopy (TEM) images of 5 week old adult retinas showing the overall structure of groups of ommatidia in (B) wild type; (C) ADAM17-/- mutant; (D) ADAM17-/Deficiency; (E, F) RNAi against ADAM17 and control (lacZ), expressed throughout the retina; (G, H) RNAi against ADAM17 and control (lacZ), expressed in the neurons; (I,J) RNAi against ADAM17 and control (lacZ), expressed in the pigmented glial cells; and (K) a kuz-/- mutant.L. Quantitation of the percentage of normal, abnormal and missing rhabdomeres from TEM images for the genotypes above, expressed as a stacked bar chart; n=180 ommatidia from 3 different fly retinas for each.M. Whole-mount retina stained with anti-ADAM17 (green) and phalloidin to mark the photoreceptor membranes (red).N. Line intensity profiles delineating expression of ADAM17 and phalloidin across individual ommatidia, n=10 fly retinas. Scale bars: 10μm.
Figure 2A-C. TEM images of 1day old adult retinas showing the overall structure of clusters of ommatidia (A1, B1, C1), or a single ommatidium (A2, B2, C2) in wild type (A1, A2), ADAM17-/- mutant (B1, B2) or ADAM17-/Df (C1, C2). Red arrows show lipid droplets in PGCs, green shading highlights PGCs. Scale bars for A2, B2 and C2: 2μm and 10μm for all other panels.D-J. Fluorescent images of 1 day old fly retinas stained with BODIPY (green) and FM-dye (red) to mark lipid droplets and the photoreceptor membranes respectively; (D) wild type; (E) ADAM17-/- mutant; (F) ADAM17-/ Deficiency; (G) knockdown of ADAM17 throughout the retina; (H) knockdown in glial cells; (I) knockdown in neurons; and (J) kuz-/-; n=10 fly retinas. Scale bars 10μmQuantitation of the BODIPY signal shown as integrated total lipid for the genotypes The box end points are the upper (75%) and lower (25%) quartiles, the whiskers define the maximum 95th percentile and minimum 5th percentile values respectively, the central band is the median and the square is the mean. Data were analysed using the Kruskal Wallis test followed by Dunn's test for post hoc analysis for significance due to unequal sample sizes. ***p<0.001, **p<0.01.Quantitation of the BODIPY signal shown as normalised LD size of lipid droplets for the genotypes The box end points are the upper (75%) and lower (25%) quartiles, the whiskers define the maximum 95th percentile and minimum 5th percentile values respectively, the central band is the median and the square is the mean. Data were analysed using the Kruskal Wallis test followed by Dunn's test for post hoc analysis for significance due to unequal sample sizes. ***p<0.001, **p<0.01.M-N. Q-PCR analysis of mRNA transcripts of lipogenic genes -Acetyl CoA carboxylase (ACC) and Fatty Acid Synthase (FASN) from heads of wild type and ADAM17-/- mutants; n=4 and n=3 biological replicates for M and N respectively, with 3 technical replicates for each genotype. The box end points are the upper (75%) and lower (25%) quartiles, the whiskers define the maximum 95th percentile and minimum 5th percentile values respectively, the central band is the median and the square is the mean. Data were quantified for significance using Student's t test. ***p<0.001, **p<0.01.
Figure 3A-D. Fluorescent images of 1 day old retinas, stained with BODIPY (green) and FM-dye (red) to stain lipid droplets and the photoreceptor membranes respectively; (A) wild type; (B) ADAM17-/- mutant; (C) overexpression of lipase in glial cells of ADAM17-/- mutant; (D) overexpression of lipase in neurons of ADAM17-/- mutant. Scale bars: 10μm.Quantitation of the BODIPY signal shown as (E) LD numbers; (F) normalised LD size for the genotypes n=10 for each genotype. The box end points are the upper (75%) and lower (25%) quartiles, the whiskers define the maximum 95th percentile and minimum 5th percentile values respectively, the central band is the median, the square is the mean and the diamond an outlier. Data were quantified for significance using Student's t test. ***p<0.001, **p<0.01.Quantitation of the BODIPY signal shown as (G) integrated total lipid, for the genotypes n=10 for each genotype. The box end points are the upper (75%) and lower (25%) quartiles, the whiskers define the maximum 95th percentile and minimum 5th percentile values respectively, the central band is the median, the square is the mean and the diamond an outlier. Data were quantified for significance using Student's t test. ***p<0.001, **p<0.01.H-K. TEM images of 5 week old adult retinas corresponding to (H) wildtype; (I) ADAM17-/- mutant; (J) glial overexpression of lipase in wild-type glial cells; (K) neuronal overexpression of lipase in the ADAM17-/-mutant. Scale bars: 10μm.L. Quantitation of the percentage of normal, abnormal and missing rhabdomeres observed in the TEM images for the genotypes mentioned above, presented as a stacked graph plot; n=180 ommatidia from 3 different fly retinas for each. Data were quantified for significance using Student's t test. ***p<0.001, **p<0.01.