EMBO/SourceData huggingface dataset:
Figure 7(A) Single Particle Tracking using PALM (SPT-PALM) was performed on α3-NKA-Dendra expressing primary neurons. A representative image shows an epifluorescence image and trajectories (strands of different colors) of minimum 5 points in length. Scale bars: 1 µm in panel (A)(B, C) Extraction of diffusion coefficient using mean squared displacement (MSD) approach using minimum 5-point trajectory length. Spine and shaft were visually identified. An upward shift of MSD curve, indicating reduced confinement was observed following exposure to Fib-Tau (0.36nM, 60 min) (B). As expected, an increased diffusion coefficient (i.e. increased mobility) (C) of α3-NKA is observed in dendritic shaft following exposure to Fib-Tau. Mann-Whitney-test, n=21 cells, obtained from 4 independent experiments; Each dot represents average diffusion coefficient of all trajectories value per cell. Data information: *p<0.05; ns= not significant.Extraction of the number of diffusive states of α3-NKA and the transition rates between these diffusive states using thousands of short (minimum 2-point) trajectories using Hidden Markov Model The occupancy time of α3-NKA in bound-state also decreased whereas that in free-state increased upon neuron exposure to Fib-Tau (E). Box-plot represents: median, interquartile range and entire distribution, Mann-Whitney-test on averaged value per movie from 4 experiments (n=21 cells). Data information: *p<0.05; ns= not significant.Extraction of the number of diffusive states of α3-NKA and the transition rates between these diffusive states using thousands of short (minimum 2-point) trajectories using Hidden Markov Model Dwell-time of α3-NKA is also reduced in bound state indicating destabilization of native α3-NKA-complexes following interaction with Fib-Tau (F). Box-plot represents: median, interquartile range and entire distribution, Mann-Whitney-test on averaged value per movie from 4 experiments (n=21 cells). Data information: *p<0.05; ns= not significant.(G-H) Cell-surface biotinylation on primary neurons exposed or not to Fib-Tau (1.8nM, 60 min). The amount of α-NKA at cell surface decreased upon exposure of neurons to Fib-Tau (G). Quantification was performed by normalizing the western blot band intensity with input. Paired-t-test, 3 independent experiments. Data information: *p<0.05; ns= not significant.(I) Paraformaldehyde fixation α3-NKA-pHluorin (green, top row) reveals endocytic spots otherwise not visible in live neurons (green, bottom row). Many Fib-Tau-ATTO647 (red) clusters overlapped (arrow) with α3-NKA-pHluorin spots (exposure, 0.36nM, 60 min). Scale bars 2µm(J) The majority of the overlapping Fib-Tau-ATTO647 clusters (red) and α3-NKA-pHluorin spots (green) localized on top of Clathrin-mRFP (blue) puncta. Scale bars: 2µm
Figure 3(A) A boxplot showing log2 TPM values of HSPA5 gene expression compared between tumors and matched normal samples across 23 tumor types from TCGA with normal tissue data available. The boxplot indicates the median (central band) and the interquartile range (boxes) of the expression value and whiskers indicates values that outside of the middle 50% (interquartile). The barplot on the top shows the number of normal tissue samples for each corresponding tumor type. Asterisks indicate significant differences by student's t-test after Benjamini-Hochberg multiple testing correction (FDR < 0.05).
Figure 6(B) Analysis and quantification of Xbp1 splicing in DLD1 and SKOV3 cells treated with varying concentrations of Reversine (Rv) for 12, 24, 48, and 72 hours. Thapsigargin (Tg) is shown as positive control.(D) Xbp1 splicing in B16 melanoma cells and eight B16:MEF fused clonal cell lines.(E) Detection of mRNA expression (by RT-PCR) of UPR-associated genes (Ern1, Af4, Ddit3 and Atf6) in tumor cells treated with Reversine (Rv) at 4µM (DLD1 cells) or 0.5µM (SKOV3 cells) for 6 hours. Data points refer to triplicate samples collected at the same time point, run in duplicate, and expressed as means ± SD.(F) Western Blot analysis of the activation of IRE1, PERK and ATF branches of the UPR in DLD1 and SKOV3 cells treated or not with Rv at 4µM for 24H (DLD1 cells) or 0.5µM for 3 days (SKOV3 cells). Thapsigargin (Tg) was used as positive control.(G) Western Blot analysis of phosphorylation of GCN2 and PKR defining the integrated stress response (ISR) in DLD1 and SKOV3 cells treated or not with Rv as in F). Cells starved for 24 hours then treated for 30 min with Calyculin-A (Cal-A) were used as positive control.
Figure 7(A) Xbp1 splicing analysis (left panel) and quantification (right panel) of bone marrow-derived macrophages (BMDM) cultured in conditioned medium (CM) of Rv-treated DLD1 and SKOV3 cells or respective control medium (culture medium of cancer cells not treated with Rv), and CM of fused B16 melanoma cells or their nonfused parental cells.(B, C) mRNA expression (RT-PCR) of Il6 (B) and Arg1 (C) in BMDM cultured in the CM of Rv-treated SKOV3 cells (0.5µM for 3 days) or control cells, and CM of fused B16 cells or their nonfused parental cells. Data points refer to triplicate experiments expressed as means ± SD.(E) Flow cytometry analysis of IFNγ and Granzyme B production by human T cells activated by anti-CD3/anti-CD28 Dynabeads in the presence of CM from Rv-treated SKOV3 cells or control SKOV3 cells (cultured without Rv). Column graphs represent the percentage expression of IFNγ (upper panel) and Granzyme B (lower panel). Percentages in parenthesis refer to change from control. Data representative triplicate samples at the same time point repeated at least twice. Data are expressed as means ± SD.(F) Flow cytometry analysis of IFNγ and Granzyme B production by human T cells activated by anti-CD3/anti-CD28 Dynabeads in complete medium spiked with Rv (0.5µM). Column graphs represent the percentage expression of IFNγ (upper panel) and Granzyme B (lower panel). Data representative triplicate samples at the same time point repeated at least twice. Data are expressed as means ± SD.(G) IFNγ (left) and Granzyme B (right) production by human T cells activated by anti-CD3/anti-CD28 Dynabeads cultured in CM from fused B16 melanoma cells or their parental non-fused cells. Percentages in parenthesis refer to change from parental non-fused cells. Data representative triplicate samples at the same time point repeated at least twice. Data are expressed as means ± SD.
Figure 1A Kinetics of intracellular Ca2+ after EDN stimulation. UACC257 and A2058 (WT and ENDRB-KO) were seeded on 96 well plates, loaded with Fluo-4 dye and stimulated with 100 nM EDN or PBS. Fluo-4 fluorescence was monitored over ten minutes (n = 6). Representative example of three independent experiments.B Time course of AKT and CREB phosphorylation. Melanoma cell lines were stimulated with 100 nM EDN for 2-90 min. AKT and CREB phosphorylation and expression was evaluated by Western blot. Representative example of three independent experiments.D High reproducibility in the UACC257 and A2058 datasets. All CV values were grouped according to cell line to represent dataset specific CV distributions. Numbers and lines indicate the median.
Figure 2A Phosphopeptide MS1 intensities in UACC257 samples were analysed by PCA. Colours indicate experimental groups and shading is mapped to time.C Comparison of the kinetics of the 242 shared target phosphopeptides. Log2 FC of EDN/Ctr is plotted from top to bottom for each phosphopeptide for both cell lines with increases shown in green and decreases shown in red.
Figure 4A,B EDN target phosphopeptides for (A) UACC257 and (B) A2058 were segregated into six clusters each, using c-means clustering (MFuzz). Each line represents one phosphopeptide and cluster membership is indicated by colour. Most likely kinases for each phosphosite were predicted using NetworKIN and enrichment of kinase substrates was calculated for each cluster. Enriched kinases are indicated above each cluster. Note that substrates of these kinases only account for a fraction of all phosphosites in a cluster.C Average MS1 intensity z-score kinetics for 217 predicted CaMKII-α sites that are shared between both cell lines. For comparison, average (n = 6) calcium abundance kinetics are shown.
Figure 1F Clonogenic growth assay of indicated HPAF-II cells left untreated or in the presence of 150nM LGK974 for 10 days; representative of three independent replicates.G Immunoblot of cytoplasmic βcatenin expression from lysates of HPAF-II cells from indicated genotype, treated with vehicle or 100nM LGK974 for 48h; representative of three biological replicates.
Figure 3A Immunoblot of CCNL1 and FBXW7 expression in HPAF-II wild-type and FBXW7-/- cells, representative blot of 3 independent replicates.B Immunoblot of CCNL1 expression following a cycloheximide chase in HPAF-II wild-type and FBXW7-/- cells , representative blot of 3 independent replicates. C Quantification of cycloheximide chase in B, mean ± SEM of three independent replicates.D Immunoblot of CCNL1 expression in HPAF-II wild-type and dN-Cul1 expressing cells, representative blot of 3 independent replicates.G Immunoblot of lysates following cycloheximide treatment of HEK293T cells expressing wild-type or degron-mutated CCNL1, representative blot of 3 independent replicates. H Quantification of cycloheximide chase in G, mean ± SEM of three independent replicates.
Figure 4A Immunoblot of immunoprecipitation of FLAG-FBXW7 overexpressed in HEK293T cells, detecting endogenous CCNL1. Representative of three independent replicates.B Immunoblot of immunoprecipitation of FLAG-CCNL1 overexpressed in HEK293T cells, detecting endogenous FBXW7. Representative of three independent replicates.C Immunoblot of cellular ubiquitination assay demonstrating the requirement of the 325-TPALS-329 degron for ubiquitination of CCNL1. Representative of three independent replicates.D Immunoblot of cellular ubiquitination assay demonstrating the requirement of FBXW7 for ubiquitination of CCNL1. Representative of three independent replicates.E Immunoblot of cellular ubiquitination assay showing re-expression of FBXW7 in knockout cells leads to increased CCNL1 ubiquitination. Representative of three independent replicates.
Figure 5B Immunoblot of HPAF-II wild-type and FBXW7-/- released from overnight nocodazole treatment at indicated time points,D Representative images of GFP-tubulin labeled wild-type, FBXW7-/-, and CCNL1OE HPAF-II cells monitoring cellular progression through cytokinesis following arrest in prometaphase using monastrol (150μM for 18 hours). E Quantification of cytokinetic timing, n=20, 25, 26 (wild-type, FBXW7-/, CCNL1OE ) pooled from three independent experiments F Immunoblot of wild-type, FBXW7-/- and CCNL1OE HPAF-II cell lines demonstrating varying levels of CCNL1.
Figure 6A Clonogenic growth assays for HPAF-II wild-type and FBXW7-/- polyclonal, and FBXW7-/- clones in presence of various OTS964 doses for 14 days. Representative images of 4 independent replicates, quantified by crystal violet absorbance at A595 and B Clonogenic growth assays for HPAF-II wild-type and CCNL1OE polyclonal, and CCNL1OE clones in presence of various doses of OTS964 for 14 days. Representative images of 4 independent replicates, quantified by crystal violet absorbance at A595C Immunoblot for the indicated proteins from lysates extracted from the individual FBXW7-/- and CCNL1OE clones.
Figure 7B Immunoblot of lysates from cervical cell lines demonstrating C33A cells (FBXW7R465H) express high levels of CCNL1, representative of three independent replicates.C Clonogenic growth assay of C33A, Caski, and SiHa cells in presence of various doses of OTS964 for 14 days. Representative images of 3 independent replicates, quantified by crystal violet absorbance at A595
Figure 1(A) Detection of VgpA in supernatants from indicated strains by immunoblotting (IB) with anti-VgpA antisera.(B) T3SS2 translocation of VgpA into Caco-2 cells using CyaA-based assay; VopV is a known T3SS2 effector. Error bars represent mean ± standard deviation (n=3 biologically independent experiments).(C) Localization of translocated VgpA (upper panel) and VgpAL10A (lower panel) during infection.
Figure 2(A) Silver-stained PAGE gel of proteins from a nuclear extract that interact with either GST-VgpA or GST glutathione agarose. The protein bound by GST-VgpA (red arrow), but not GST, was identified as EBP2 by mass spectrometry; GST-VgpA is labeled with a blue arrow.(B) Western blot using anti-EBP2 as antibody confirmed that EBP2 binds VgpA.(C) Interaction of VgpA or VgpA mutants (VgpAΔ10 and VgpAL10A) with EBP2. (D) Localization of VgpA (upper panel) or in VgpAL10A (lower panel) in HeLa cells after transfection. EBP2 was detected with anti-EBP2 antibody (red), VgpA and VgpAL10A were detected with anti-VgpA sera (green) and nuclei were stained with DAPI (blue). White arrows indicate co-localization of EBP2 and VgpA (upper panel). VgpAL10A localizes primarily to the nucleoplasm (white arrow, lower panel).
Figure 3 (A) Fluorescence micrographs of uninfected Caco-2 cells or Caco-2 cells infected with the indicated V. parahaemolyticus strains. Cells were stained with DAPI (blue) and anti-BrdU antibody (green).  (B) Fluorescence micrographs of Caco-2 cells transfected with siRNA targeting EBP2 (siEBP2) or control siRNA (siNC) and subsequently infected with vopZ'ΔvscN1. Cells were stained with anti-BrdU antibody (green) and DAPI (blue).  (C) The percentage of BrdU-positive cells (green) among the total cells (green + blue) was analyzed for 100 cells in three experiments. Error bars represent mean ± standard deviation (n=3 biologically independent experiments). One-way analysis of variance (ANOVA) was used for statistical analysis. *P<0.05 (when compared to the uninfected Caco-2 cells).  (D) The percentage of BrdU+ cells was analyzed as above for cells treated with siNC or siEBP2. Error bars represent mean ± standard deviation (n=3 biologically independent experiments). Two-way ANOVA was used for statistical analysis. *P<0.05 (when compared to siNC-transfected cells that are infected with vopZ'ΔvscN1).  (E) Immunoblot of EBP2 and Actin in Caco-2 cells transfected with siEBP2 or siNC.
Figure 4 (A) Localization of c-Myc in uninfected Caco-2 cells (first row), Caco-2 cells infected with V. parahaemolyticus vopZ'ΔvscN1 (wild type VgpA, second row), Caco-2 cells infected with vopZ'ΔvscN1vgpA' (third row), Caco-2 cells infected with vopZ'ΔvscN1vgpA':pvgpA (fourth row) and Caco-2 cells infected with vopZ'ΔvscN1vgpA':pvgpAL10A (fifth row). Fixed cells were stained with DAPI (blue), and anti-EBP2 (red), and anti c-Myc (green) antibodies. White arrows are examples indicating the co-localization of EBP2 and c-Myc.  (B Detection of c-Myc or EBP2 in nuclear extracts from uninfected Caco-2 cells or after infection with indicated strains. Following infection, nuclear extracts were obtained and loaded on protein A/G beads cross-linked with anti-EBP2 (B, IP: anti-EBP2) Input and eluates were blotted with anti-EBP2, anti-c-Myc or anti-VgpA antibodies, respectively. Quantified value (mean ± standard deviation) as measured by band intensity (normalized to uninfected) from three independent experiments are presented under the signals of the blot of IP samples. n.d., not determined.  C) Detection of c-Myc or EBP2 in nuclear extracts from uninfected Caco-2 cells or after infection with indicated strains. Following infection, nuclear extracts were obtained and loaded on protein A/G beads cross-linked anti-C-Myc (C, IP: anti-c-Myc) antibodies, respectively. Input and eluates were blotted with anti-EBP2, anti-c-Myc or anti-VgpA antibodies, respectively. Quantified value (mean ± standard deviation) as measured by band intensity (normalized to uninfected) from three independent experiments are presented under the signals of the blot of IP samples. n.d., not determined.  (D) Purified proteins EBP2, c-Myc and GST-VgpA or GST-VgpAL10A were mixed and then pulled down with protein A/G beads cross-linked with anti-EBP2. Input and eluates were blotted with anti-EBP2, anti-c-Myc or anti-VgpA antibodies, respectively. Quantified value (mean ± standard deviation) as measured by band intensity (normalized to EBP2+c-Myc sample without GST-VgpA and GST-VgpAL10A) from three independent experiments are presented under the signals of the blot of IP samples. n.d., not determined.  (E) Caco-2 cells were infected with the indicated strains and treated with cycloheximide (CHX) for the indicated time. Whole cells lysates were analyzed by western blot using anti-c-Myc and anti-Actin antibody.
Figure 5 (A) Amounts of rRNA were from uninfected Caco-2 cells or cells infected with indicated strains. *P<0.05 (when compared to the uninfected Caco-2 cells).  (B) Amounts of rRNA in uninfected or infected Caco-2 cells transfected with control (siNC) or EBP2-targeting (siEBP2) siRNAs. *P<0.05 (when compared to the siNC-transfected cells that are infected with vopZ'ΔvscN1).  (C) Amounts of RNA polymerase I (POLR1A) were from uninfected Caco-2 cells or cells infected with indicated strains. *P<0.05 (when compared to the uninfected Caco-2 cells).  (D). Amounts of rRNA in intestinal homogenates from infant rabbits infected with indicated strains 18 and 38 h after infection. *P<0.05 (when compared to the uninfected rabbits at 18 hours). ΔP<0.05 (when compared to the uninfected rabbits at 38 hours post infection).
Figure 6 (A) VgpA (upper panel: animals infected with WT) and VgpAL10A (lower panel: animals infected with vgpAL10A) localization in intestinal epithelial cells of infected animals. Arrows in upper panel indicate VgpA localized as punctae in the nucleus; the arrow in the lower panel indicates VgpAL10A localized diffusively in the nucleoplasm.  (B) VgpA and VgpAL10A interactions with EBP2 in intestinal epithelial cells isolated from infant rabbits infected with WT or vgpAL10A V. parahaemolyticus. Inputs represent proteins detected in intestinal epithelial cells of uninfected rabbits or rabbits infected with the indicated strains. Outputs represent eluates from protein A/G beads cross-linked with anti-EBP2 (IP: anti-EBP2) or anti-VgpA (IP: anti-VgpA) antibodies and analyzed with anti-VgpA and anti-EBP2 antibodies.  (C) Frozen sections from uninfected rabbits or rabbits infected with the indicated strains for 18 hours were stained with anti-Ki67 antibodies to detect actively dividing cells (green), phalloidin to visualize F-actin (red) and DAPI to detect nuclei (blue).  (D) Eighteen hours after infant rabbits were infected with WT (middle panel) or vgpAL10A (lower panel) V. parahaemolyticus, fixed sections from the small intestine were stained with antibodies to c-Myc (green), EBP2 (red), and DAPI (blue). Uninfected rabbits were processed similarly and were included as a control (upper panel). White arrows indicate co-localization of c-Myc and EBP2 (middle panel).
Figure 7 (A) CFU in homogenates from the distal small intestines of infant rabbits infected with the indicated strains 18, 28 and 38 h following inoculation. Black horizontal line indicates mean CFU (n=7-10 animals/group). One-way ANOVA was used for statistical analysis. *P<0.05 for the indicated comparison.  (B) H&E stained sections from the distal small intestine from the rabbits infected with the indicated strains 18 hours after infection. Black arrows point to bacterial colonies closely attached to the epithelium. (C) Fluid accumulation in rabbits infected with the indicated strains 38 hours post inoculation. Error bars represent mean ± standard deviation (n=7 animals/group). One-way ANOVA was used for statistical analysis. *P<0.05 (when compared to the rabbits infected with WT).
Figure 2C, AltFUS (arrow) endogenous expression in Human tissues (brain, muscles and kidney - 100 μg), in HEK293 and HeLa cultured cells (100 μg) and using the over-expression of altFUS CDS in HEK293 cells (50 μg) as positive control (representative image from n=3). The asterisk indicates a protein species detected with the anti-altFUS antibody specifically in the brain.AltFUS (arrow) endogenous expression in the motor cortex of three C9orf72 and three sporadic ALS patients (D) (representative image from n=3). The asterisk indicates a protein species detected with the anti-altFUS antibody specifically in the brain.AltFUS (arrow) endogenous expression in iPSC-derived motor neurons of three lines from controls and from ALS patients (E) (representative image from n=3). The asterisk indicates a protein species detected with the anti-altFUS antibody specifically in the brain.I, Endogenous altFUS mitochondrial expression in HEK293 cells following fractionation (representative image from n=3), with Tubulin as a marker of the cytosolic fraction and VDAC as a marker of the mitochondrial fraction. We used siFUS transfected cells as a negative control and altFUS transfected cells as a positive control for altFUS expression (WCL = Whole Cell Lysate, Cyto = Cytosol fraction, Mito = mitochondrial fraction).J, Representative images of the mitochondrial network (Tomm20 in red) in mock and altFUS-Flag (green) transfected HeLa cells (n=3). The white scale bar corresponds to 10μm. K, Proportion of tubules and globules in the mitochondrial network of mock HeLa cells and HeLa cells transfected with altFUS-Flag Quantification was done over a minimum of 100 cells across a technical duplicate per independent experiments (n=3, i.e. a minimum of 300 cells per biological conditions, p-value < 0.001, Mann-Whitney U test). The boxes extend to the 25th and 75th percentiles, with the median marked. The whiskers correspond to the 5th and 95th percentiles.
Figure 3E, Scatter plot of the proteins identified by AP-MS indicating their enrichment (Fold-change over control) and their SAINT probability score. Proteins above the 0.8 threshold (grey line) are indicated in black, others in grey. AltFUS is indicated in red (bait) and preys known to regulate the autophagy or the cellular stress response are indicated in green.
Figure 4B, LC3-II accumulation after bafilomycin treatment from mock, altFUS, FUS, FUS(Ø), FUS-R495x, FUS(Ø)-R495x transfected cells and FUS(Ø)-R495x and altFUS co-transfected cells across 3 independent experiments (n=3, mean ± SD). The quantification corresponds to the treated/untreated ratio of LC3-II abundance Statistical significance is relative to the mock condition unless otherwise indicated (**** = p value < 0.0001, *** = p value < 0.001, ** = p value < 0.01, n.s. = non-significant, two-way ANOVA with Tukey's multiple comparison correction).D-E, Quantification of FUS cytoplasmic granules, number (D) and area (μm2) (E) in cells over-expressing the bicistronic (+) or monocistronic (-) construct for FUS, FUS-G156E, FUS-R495x, FUS-K510E, FUS-Q519x, FUS-Q519I-fs527x, FUS-R521C, and FUS-P525L. Statistical comparisons are made between bicistronic and monocistronic versions of each construct (n=3 - biological replicates, mean ± SD, **** = p value < 0.0001, *** = p value < 0.001, ** = p value < 0.01, * = p value < 0.05, n.s. = non-significant, one-way ANOVA test with Sidak's multiple comparison).
Figure 5C-E, Locomotion assay represented by the percentage of climbing success in control and RU-486-treated transgenic Drosophila expressing mCherry or altFUS (C), the bicistronic FUS or the monocistronic FUS(Ø) (D), and the bicistronic FUS-R495x or the monocistronic FUS(Ø)-R495x (E) at day 1, 10 and 20 post-induction. Statistical comparisons were made between each population (n=4 - biological replicates). Indicated significance is between the monocistronic and the bicistronic transgenic flies of the RU-486-treated population (mean ± SD, n.s. = non-significant, * = p value < 0.05, *** = p value < 0.001, two-way ANOVA with Tukey's multiple comparison correction).