EMBO/SourceData huggingface dataset:
Figure 2CD68 immunoreactivity in white adipose tissue (scale bar = 25 μm, images representative of one animal each from a total of four animals per experimental group). Arrow points to a region stained with CD68 antibody. *P = 0.0109, Student's t-test.Epididymal fat mass was analyzed in mice (n = 6 animals/group) 7 days after i.c.v. injection of vehicle or AβOs. Data are representative of three independent experiments with similar results. *P = 0.0255.Relative expression of leptin (C), TNF-α (D) and IL-6 (E), respectively, in white adipose tissue of mice (n = 7 Veh; 9 AβOs) 7 days after i.c.v. injection of vehicle or AβOs. In (C), *P = 0.0394; in (D), **P = 0.0038; in (E), *P = 0.0305; Student's t-test.p-JNK (F; n = 5 animals/group) and IRS-1pSer312 (G; n = 6 animals/group) levels (normalized by total JNK and total IRS-1, respectively) in skeletal muscle of mice 7 days after i.c.v. injection of vehicle or AβOs. In (F), *P = 0.0464; in (G), *P = 0.0081; Student's t-test.Representative images of GLUT-4 immunofluorescence in insulin-stimulated skeletal muscle from mice that were i.c.v.-injected with vehicle (Veh) or 10 pmol AβOs. Bar graphs show quantification of GLUT-4 surface immunoreactivity in skeletal muscle of mice that received intraperitoneal injections of PBS or insulin (1 IU/kg body weight) 7 days after i.c.v. injection of vehicle or AβOs, as indicated (n = 5 animals/group). Scale bar = 25 μm. *P = 0.0144, one-way ANOVA followed by Bonferroni post hoc test.GLUT-4 mRNA (n = 4 animals/group) and total protein levels (normalized to actin levels; n = 5 Veh; 6 AβOs) were unchanged in skeletal muscle of Swiss mice injected with vehicle (Veh) or 10 pmol AβOs.Plasma levels of insulin (K; n = 12 animals/group), leptin (L; n = 11 Veh; 12 AβOs), cholesterol (M; n = 8 Veh; 6 AβOs), triglycerides (N; n = 8 Veh; 6 AβOs) or noradrenaline (O; n = 7 Veh; 8 AβOs) measured 7 days after i.c.v. injection of vehicle (Veh) or 10 pmol AβOs. In (O), *P = 0.0361, Student's t-test.
Figure 3Representative immunocytochemistry images of mature hypothalamic neurons in culture exposed to vehicle (Veh) or AβOs (500 nM) for 3 h. Binding of AβOs was detected using anti-oligomer monoclonal antibody NU4 (red). Neurons were double-labeled using MAP-2 antibody (green). Images represent typical results from experiments with three independent hypothalamic cultures (three coverslips/experimental condition per independent experiment). Scale bar = 30 and 10 μm for main panels and insets, respectively.Representative immunocytochemistry image of mature hypothalamic culture exposed to AβOs (500 nM) and immunolabeled with anti-GFAP (green) and NU4 (red) antibodies. Insets show AβOs binding to neuronal dendrites, whereas no binding was detected to GFAP-positive cells.Representative DCFfluorescence images from hypothalamic neuronalcultures exposed to vehicle or AβOs (500 nM, 4 h). Insets show optical zoom images of the indicated areas. Scale bars = 100 and 50 μm for main panels and insets, respectively. Graph shows integrated DCFfluorescence intensities (relative units; see Materials and Methods) (n = 3 independent hypothalamic cultures; three wells/experimental condition per experiment; three images acquired per well). Bars represent means ± SEM. #P = 0.0604, one-sample t-test compared with a fixed value of 100 RUs.LDH activity (IU/l) in culture media of hypothalamic cultures exposed to vehicle or AβOs (500 nM, 3 h).Representative immunofluorescence images of eIF2α-P in hypothalamic cultures exposed to vehicle or AβOs (500 nM, 3 h) in the absence or presence of infliximab (1 μg/ml). Scale bar = 30 μm. Graph represents integrated immunofluorescence intensities of eIF2α-P levels from three independent hypothalamic cultures (three coverslips/experimental condition per experiment, 20 images per coverslip). Bars represent means ± SEM. *P = 0.0489, one-way ANOVA followed by Bonferroni post hoc test comparing AβO-treated versus vehicle-treated cultures.Representative images of hypothalamic cultures exposed to AβOs (500 nM, 3 h) and double-labeled with NU4 (oligomer-sensitive) and eIF2α-P antibodies. Arrow points to a neuron presenting high levels of eIF2α-P in the absence of AβO binding. Nuclear staining (DAPI) is shown in blue. Scale bar = 30 μm.Representative images of hypothalamic neurons labeled with NU4 antibody exposed to AβOs (500 nM, 3 h) in the absence or presence of infliximab (1 μg/ml). Similar patterns of AβO binding were observed in both conditions. Scale bar = 20 μm.
Figure 4Western blot analysis of eIF2α-P levels in the hypothalamus of mice 4 h (A; n = 4 animals/group) or 7 days (B; n = 6 Veh; 5 AβOs) after a single i.c.v. injection of vehicle (Veh) or 10 pmol AβOs. Graphs show densitometric data normalized by total eIF2α levels. *P = 0.0213.Western blot analysis of ATF4 levels in the hypothalamus of mice 4 h after i.c.v. injection of vehicle (Veh) or 10 pmol AβOs; graph shows densitometric data normalized by β-actin (n = 7 Veh; 8 AβOs). #P = 0.0731; Student's t-test.Western blot analysis of hypothalamic phospho-IKKβ levels in the hypothalamus of mice 4 h (C; n = 6 animals/group) or 7 days (D; n = 4 Veh; 5 AβOs) after i.c.v. injection of vehicle or 10 pmol AβOs. Graphs show densitometric data normalized by total IKKβ levels. In (D), *P = 0.0437; in (E), *P = 0.0444; Student's t-test.Western blot analysis of IκBα (F; n = 6 animals/group) and cytoplasmic phospho-p65-NF-κB (G; n = 4 Veh; 5 AβOs) in the hypothalamus of mice 4 h after i.c.v. injection of vehicle or 10 pmol AβOs. Graphs show densitometric data normalized by actin (F) or total NF-κB levels (G). *P = 0.0207.Nuclear NF-κB levels in the hypothalamus 6 h after i.c.v. injection of vehicle or 10 pmol AβOs in mice. Graphs show NF-κB levels normalized by nuclear marker, lamin B (n = 6 animals/group). **P = 0.0024; Student's t-test.IRS-1pSer636 (I; n = 4 animals/group) and pTyr465 (J; n = 6 Veh; 4 AβOs) levels in the hypothalamus 7 days after i.c.v. injection of vehicle or AβOs in mice. Graphs show IRS-1pSer or IRS-1pTyr levels normalized by total IRS-1. In (I), *P = 0.0043; in (J), *P = 0.0275; Student's t-test.Twelve-hour food intake after a single i.c.v. infusion of insulin (200 mU) in mice. Experiment was performed 7 days after i.c.v. injection of vehicle or AβOs (n = 5 PBS; 5 Veh + Insulin; 9 AβOs + Insulin), data are representative of two independent experiments with similar results. ***P < 0.0001, one-way ANOVA followed by Bonferroni post hoc test comparing Veh-Insulin versus PBS groups.
Figure 5Representative images of AβO immunoreactivity (detected using anti-oligomer monoclonal antibody NU4) in the hypothalamus of control, sham-operated adult cynomolgus macaques (Sh; n = 3) or macaques that received i.c.v. injections of AβOs (n = 3; see Materials and Methods). Nuclear staining (DAPI) is shown in blue. Insets show optical zoom images of the areas indicated by white dashed rectangles in the main panels. Scale bars = 100 and 20 μm for main panels and insets, respectively.Representative images showing eIF2α-P (B), phospho-IKKβ (C) and IκBα (D) immunoreactivities in the hypothalamus of cynomolgus macaques that received i.c.v. injections of AβOs or control (sham-operated; Sh) macaques (n = 3 animals/group). Graphs show immunolabeling optical density analysis from three images acquired in the hypothalamus of each macaque (three control versus three AβO-injected animals). In (B), #P = 0.0523; in (C) #P = 0.1123; unpaired Student's t-test with Welch's correction for unequal variances; AβO-injected monkeys compared to sham-operated monkeys. Scale bars = 50 μm
Figure 6Accumulated chow intake (normalized by body weight) measured during 7 days following a single i.c.v. injection of vehicle or 10 pmol AβOs in mice (n = 13 Veh; 10 AβOs; data are representative of two independent experiments with similar results). ***P < 0.0001; Student's t-test.Daily body weight measured during 7 days after i.c.v. injection of vehicle or AβOs (n = 7 animals/group; data are representative of two independent experiments with similar results).Adult Swiss mice received a single i.c.v. injection of vehicle or 10 pmol AβOs, and hypothalamic levels of mRNA for AgRP (C; n = 6 Veh; 5 AβOs), NPY (D; n = 6 Veh; 5 AβOs) and POMC (E; n = 7 animals/group) were analyzed 7 days after injection. In (C), *P = 0.0191; in (D), *P = 0.0115; Student's t-test.Swiss mice received a single i.c.v. injection of vehicle (Veh) or 10 pmol AβOs, and their brains were analyzed by Fluorojade staining of degenerating cells 7 days after the injection. Representative images of Fluorojade staining in the hypothalamus of vehicle- or AβO-injected mice (n = 4/group). Scale bar = 100 μm in left panels (top and bottom) and 20 μm in right panels (top and bottom). Positive control (bottom left panel) was the hippocampus of a mouse that received one i.c.v. injection of quinolinic acid (36.8 nmol) and was analyzed 24 h after.
Figure 7Glucose tolerance test (2 g glucose/kg body weight, i.p.) in mice that received i.c.v. injections of vehicle, vehicle + TUDCA, AβOs or AβOs + TUDCA (when used, TUDCA was administered in 5 i.c.v. injections of 5 μg TUDCA each, before and after oligomer injection; see Materials and Methods. Control groups received injections of saline). Glucose tolerance test (GTT) was performed 7 days after i.c.v. injection of vehicle or AβOs. Bar graph represents areas under the curves (AUC) in the time course plots (n = 15 Veh; 15 AβOs; 10 Veh + TUDCA; 16 AβOs + TUDCA). Data are representative of two independent experiments with similar results. Left panel: **P = 0.0048, **P = 0.003, two-way ANOVA followed by Bonferroni post hoc test; right panel: *P = 0.0384, one-way ANOVA followed by Bonferroni post hoc test.Plasma noradrenaline (NA) levels measured 7 days after i.c.v. injection of vehicle, vehicle + TUDCA, AβOs or AβOs + TUDCA in mice (n = 7 animals/group). Data are representative of two independent experiments with similar results. *P = 0.0071, one-way ANOVA followed by Bonferroni post hoc test.Glucose tolerance test (2 g glucose/kg body weight, i.p.) in TNFR1−/− mice or wild-type littermates performed 7 days after i.c.v. injection of vehicle or AβOs. Bar graph represents areas under the curves (AUC) in the time course plots (n = 8 WT + Veh; 7 WT + AβOs; 7 TNFR−/− + Veh; 8 TNFR−/− + AβOs). Left panel: **P = 0.0049, ***P < 0.0001, two-way ANOVA followed by Bonferroni post hoc test; right panel: *P = 0.0001, one-way ANOVA followed by Bonferroni post hoc test.Western blot analysis of phospho-IKKβ (D; n = 6 WT + Veh; 6 WT + AβOs; 4 TNFR−/− + Veh; 6 TNFR−/− + AβOs) and IRS-1pSer636 levels (E; n = 6 WT + Veh; 5 WT + AβOs; 6 TNFR−/− + Veh; 5 TNFR−/− + AβOs) in the hypothalamus of wild-type (WT) or TNFR1−/− mice 10 days after i.c.v. injection of vehicle or AβOs. Representative images from Western blot experiments were always run on the same gels but represent noncontiguous lanes. In (D), *P = 0.0088, Student's t-test; in (E), *P = 0.0428, one-way ANOVA followed by Bonferroni post hoc test.Adult Swiss mice pre-treated with minocycline or PBS received a single i.c.v. injection of vehicle or 10 pmol AβOs, and hypothalamic levels of mRNA for AgRP (F; n = 5 Veh; 6 AβOs; 5 Veh + Mino; 4 AβOs + Mino) and NPY (G; n = 14 Veh; 13 AβOs; 8 Veh + Mino; 9 AβOs + Mino) were analyzed 7 days after injection. In (F), *P = 0.0097, one-way ANOVA followed by Bonferroni post hoc test; in (G), *P = 0.0219, one-way ANOVA followed by Bonferroni post hoc test.Glucose tolerance test (GTT) in APP/PS1 mice before and after i.c.v. injection of infliximab (0.2 μg daily for 4 days). Bar graph represents areas under the curves (AUC) in the time course plots (n = 9 animals/group). Left panel: *P = 0.0177, two-way ANOVA followed by Bonferroni post hoc test; right panel: *P = 0.0327, paired t-test.
Figure 1B: Scatter Plot of mass spectrometry assay. Dots depict protein intensity (IBAQ) and relative significance obtained from mass spectrometry assay. X-axis: Difference of Che-1 intensity versus IP control; on the y-axis are the corresponding −log10 P-values. Significant Che-1 interacting proteins relevant in this study are represented in blue.C: Nuclear extracts from Kms27 MM cells treated or not with RNase A were subjected to immunoprecipitation with the indicated antibodies (abs) and analyzed by western blot (WB) with the indicated abs. Input corresponds to 5% of the total extract used for immunoprecipitation.D: (Top) qRT-PCR approach described in the schematic representation. (Bottom) RIP-qPCR analysis with anti-Che-1 specific antibody or control rabbit IgG of Kms27 MM cells. Data are expressed as percent of Input. Error bars represent the standard error of three different biological experiments performed in technical duplicate (Two Tailed T test ***P<0.005, ****P<0.001, n.s. = not significant).E: Colocalization assays in Kms27 and RPMI8226 MM cell lines by combining In Situ Hybridization (FISH) staining of NEAT1 (Stellaris) with immunofluorescence of Che-1. Scale bar 10 μm.F: (Left) RT-qPCR analysis of NEAT1 expression levels in Kms27 MM cells transiently transfected with LNA GapmeR NEAT1A or GapmeR Control oligonucleotides. Values were normalized to Actin expression. Error bars represent the standard error of three different biological experiments performed in technical duplicate (Two Tailed T test ****P<0.001). (Right) Nuclear extracts from KMS27 MM cells were immunoprecipitated with the indicated abs and analyzed by WB with the indicated abs.
Figure 3A: Quantitative RT-PCR (RT-qPCR) of Kms27 MM cells transiently transfected with two different LNA GapmeR NEAT1 or GapmeR Control oligonucleotides. Values were normalized to Actin expression. Error bars represent the standard error of three different experiments performed in duplicate (Two Tailed T test ****P<0.001).B: WB analysis with indicated abs of total extracts from Kms27 MM cells transiently transfected as in A.C: Representative confocal images of Kms27 MM cells transiently transfected with LNA GapmeR NEAT1A, GapmeR NEAT1B or GapmeR Control oligonucleotides and immunostained with anti-Che-1 antibody. Scale bar 10 μm.D: Isolated cell compartments from Kms27 MM cells transfected with LNA GapmeR NEAT1A or GapmeR Control oligonucleotides, were subjected to WB with the indicated abs.E: Che-1 ChIP-seq signal intensity of LNA GapmeR NEAT1A (2 replicates on right) and GapmeR Control (2 replicates on left) and at co-localizing Che-1/NEAT1 sites (N=14654 sites). Signal intensity from 1 (weak) to 12 (strongest).F: FISH images of Kms27 MM cells depleted or not for Che-1 expression (siChe-1A) labeled with Hoechst (cell nuclei) and Quasar 670 (NEAT1 RNA). Scale bar 10 μm.G: NEAT1 ChIRP-seq signal intensity of Kms27 MM cells transiently transfected with siChe-1A or siControl at co-localizing Che-1/NEAT1 sites (N=14654 sites). Signal intensity from 1 (weak) to 14 (strongest).
Figure 4B: Chromatin extracts from Kms27 and RPMI8226 MM cells treated or not with RNase H, were subjected to immunoprecipitation with S9.6 antibody and analyzed by WB with the indicated abs. Input corresponds to 5% of the total extract used for immunoprecipitation.C: Representative confocal images showing the interaction between Che-1 and S9.6 revealed by PLA in Kms27 MM cells. Negative controls were performed by omitting Che-1 or S9.6 primary antibody. Scale bar 10 μm.D: Chromatin extracts from Kms27 MM cells transfected with LNA GapmeR NEAT1A or GapmeR Control oligonucleotides were immunoprecipitated with S9.6 antibody and analyzed by WB with the indicated antibodies. Input corresponds to 5% of the total extract used for immunoprecipitation.E: (Left) Representative confocal images of Kms27 MM cells treated or not with RNase H, transiently transfected with siChe-1A or siControl and immunostained with S9.6 antibody and. Scale bar 10 μm. (Right) Box plot showing S9.6 signal nuclear intensity per cell (Arbitrary Unit-AU). siChe-1A cells are described in green color, while siControl in grey color. 100 cells were counted in each replicate. Each dot represents a cell. Median scores per sample are written at the top of the median red line. P value: <1.02-18. ANOVA test was performed to evaluate statistical significance. The red center line denotes the median value (50th percentile) while the black box contains the 25th to 75th percentiles of data. The whiskers mark the 5th and 95th percentiles.F: (Top) S9.6 antibody was used for dot blot analysis to evaluate RNA:DNA hybrids formation in Kms27 MM cells by serial dilutions of genomic DNA starting at 1.5 micrograms. (Bottom) Quantification of S9.6 levels by densitometric analysis. Values were normalized to a Methylene blue loading control. Error bars represent the standard error of three different biological experiments (Two Tailed T test ***P<0.005, ****P<0.001).G: Enrichment density of DRIP-seq signals at NEAT1/Che1 co-localizing sites in Kms27 MM cells identified in siRNA control (siControl) (grey) and siRNA Che-1 (siChe-1A) (dark green) samples. Enrichment density was calculated on the relative size of each peak (start to end of each fragment) extended 2000 bp upstream and downstream. Right frame represents the signal obtained from 14654 randomly selected sites from the same experiments.
Figure 5D: (Top) Representative confocal images of Kms27 MM cells transfected and treated as in C and immunostained with S9.6 antibody. Scale bar 10 μm. (Bottom) Box plot showing S9.6 signal nuclear intensity per cell (Arbitrary Unit-AU). siChe-1A cells are described in dark grey, siChe1A plus CX5461 in green color, while siControl in grey color. Median scores per sample are written at the top of the median red line. P value: <1.30-54. ANOVA test was performed to evaluate statistical significance. The red center line denotes the median value (50th percentile) while the black box contains the 25th to 75th percentiles of data. The whiskers mark the 5th and 95th percentiles.E: (Top) Dot blot analysis of decreasing amounts of genomic DNA extracts from Kms27 MM cells depleted or not for Che-1 expression (siChe-1A) and treated with CX5461, were probed using abs directed against RNA:DNA hybrids. (Bottom) Quantification of S9.6 levels by densitometric analysis. Values were normalized to a Methylene blue loading control. Error bars represent the standard error of three different biological experiments (Two Tailed T test ****P<0.001).
Figure 6A: Differential analysis of siControl vs. siChe1A transcriptome in Kms27 MM cells. Volcano plot shows 135 significantly upregulated (dark green) and 392 downregulated genes (dark grey). x-axis reports the base-2 logarithm of fold change which is approximated to the B value coming from the statistical Wald test. y-axis reports the base-10 logarithm of Q value of significant genes.D: WB analysis of total extracts of Kms27 MM cells depleted or not for Che-1 (siChe-1A) and probed for the indicated abs.E: (Left) Luciferase activity of human NF-kB promoter was measured in Kms27 MM cells transiently transfected with siChe-1A or siControl oligonucleotides or with empty vector or p65/RelA protein expressing vector. Error bars represent the standard error of three different biological experiments performed in duplicate (Two Tailed T test ***P<0.005, ****P<0.001). (Right) WB analysis of total extracts of Kms27 MM cells transiently transfected as described in E and incubated with the indicated antibodies.RT-qPCR analysis of the levels of the indicated genes in Kms27 MM cells transiently transfected with siChe-1A or siControl. Values were normalized to Actin expression. Error bars represent the standard error of three different experiments in duplicate (Two Tailed T test ***P<0.001, ****P<0.001).G: RT-qPCR analysis of the levels of the indicated genes in Kms27 MM cells transiently transfected with siChe-1A or siControl. Values were normalized to Actin expression. Error bars represent the standard error of three different experiments in duplicate (Two Tailed T test ***P<0.001, ****P<0.001).H: WB analysis for the indicated antibodies of total extracts from Kms27 MM cells with siChe-1A or siControl and where indicated with GFP-tagged RNaseH expression vectors.I: RT-qPCR analysis of IFNB and IFNG levels in Kms27 MM cells transiently transfected as in H. Values were normalized to Actin expression. Error bars represent the standard error of three different biological experiments performed in duplicate (Two Tailed T test **P<0.01, ***P<0.005.)
Figure 7A: Representative S9.6 immunofluorescence levels in healthy and tumor CD138+ plasma cells purified from patient bone marrow. Nuclei were visualized by staining with Hoechst dye. Scale bar 10 μm.B: Dot blot performed to evaluate the enrichment of RNA:DNA hybrids formation using the S9.6 antibody by serial dilutions of genomic DNA starting at 1.0 micrograms. Methylene blue normalization of S9.6 signal image acquired by using Alliance Mini HD6 system by UVITEC Ltd.C: Boxplot showing relative S9.6 nuclear mean intensity in CD138+ plasma cells from 15 MM patients (red) and 5 healthy (light blue). Dot plot representing each cell considered overlay the boxplot. Y-axis: S9.6 Nuclear Mean Intensity (AU). The center line denotes the median value (50th percentile) while the boxes contain the 25th to 75th percentiles of data. The whiskers mark the 5th and 95th percentiles.D: Representative WB analysis with the indicated antibodies of healthy and neoplastic CD138+ purified plasma cells from patients.F: Unsupervised clustering heatmap showing enrichment profiles of 9 IFN signature genes. From the top: ISS stages 1,2,3 of each patient (N=687). IFN signature High (red, N=71) and Low (white, N=614) assigned to each patient. Color scheme representing up- (red) and down- (blue) regulation of the selected genes across the patient dataset. Colors are proportional to normalized (cpm) read enrichment - the relative read enrichment median of each gene within the full patient cohort.
Figure 8A: WB analysis of total extracts from Kms27 and U266 human MM cells with the indicated abs.B: (Left) Kms27 and U266 MM cells were subjected to immunostaining with S9.6 antibody Scale bar 10 μm. (Right) Dot plot showing relative S9.6 nuclear mean intensity cell in the nucleus. 100 cells were counted in each replicate. Median scores per sample are written at the top of the median red line. P value: 3.3-13. T-test was performed to evaluate statistical significance.C: Different expression of IFNG and IFNB in Kms27 and U266 MM cells was assessed by RT-qPCR. Values were normalized to Actin expression. Error bars represent the standard error of three different biological experiments performed in duplicate (Two Tailed T test ***P<0.005).D: WB analysis with the indicated abs of total extracts from U266 MM cells treated or not with Thapsigargin (THAP) 0.5 μg/ml for 16 hours. G: WB with indicated abs of total extracts from Kms27 MM cells treated or not with ISRIB 1μM for 6 hours.E: (Left) U266 MM cells treated or not with THAP as in D and immunostained with S9.6 antibody Scale bar 10 μm. (Right) Dot plot showing relative S9.6 nuclear mean intensity per cell. 100 cells were counted in each replicate. Median scores per sample are written at the top of the median red line. P value: 1.1-20. T-test was performed to evaluate statistical significance. H: (Left) Immunostaining with S9.6 antibody of Kms27 MM cells treated or not with ISRIB as in G Scale bar 10 μm. (Right) Dot plot showing relative S9.6 nuclear mean intensity per cell. 100 cells were counted in each replicate Median scores per sample are written at the top of the median red line. P value: 4.7-23. T-test was performed to evaluate statistical significance.F: RT-qPCR analysis of IFNG and IFNB in U266 MM cells treated or not with THAP. Values were normalized to Actin expression. Error bars represent the standard error of three different biological experiments performed in duplicate (Two Tailed T test ***P<0.005, ****P<0.001) I: RT-qPCR analysis of IFNG and IFNB levels in Kms27 MM cells treated as in G. Values were normalized to Actin expression. Error bars represent the standard error of three different biological experiments performed in duplicate (Two Tailed T test ****P<0.001).