EMBO/SourceData huggingface dataset:
Figure 4A. Quantification of cortical thickness in the somatosensory (SS), somatomotor (SM) and visual (V) area, in untreated Ppt1-/- or Ppt1+/+ mice and in mice transplanted (tx) with hPPT1-LV transduced HSPCs IV, ICV or ICV+IV or mock-transplanted, analyzed at study termination. * = p < 0.05; ** = p < 0.01; *** = p < 0.001; **** = p < 0.0001; Kruskal Wallis followed by Dunn's post-hoc test.B. Quantification of CD68 immunoreactivity in the cortex, hippocampus, thalamus and cerebellum of untreated (UT) or gene therapy treated Ppt1-/- mice. * = p < 0.05; ** = p < 0.01; *** = p < 0.001; **** = p < 0.00001; Kruskal Wallis followed by Dunn's post-hoc test.
Figure 5A. Significant increase of the survival in Ppt1-/- mice transplanted at the symptomatic stage with hPPT1-LV transduced HSPCs administered IV+ICV.E. Representative fluorescence microscope photomicrographs showing autofluorescent storage material (AF, green) in different brain regions of Ppt1-/- mice transplanted with hPPT1-LV transduced HSPCs at 18-20 weeks of age, analyzed at study termination. Ppt1 -/- mice mock transplanted at 18-20 weeks of age and analyzed at about 250 days, i.e. humane end point, are shown as reference. Neurotrace fluorescent Nissl staining (NT, red) is used to highlight neurons in different brain regions. Scale bar = 100 μm. F. Top panel: quantification of autofluorescent (AF) storage material in different brain regions of untreated or transplanted mice analyzed at study termination. ** = p<0.01; *** = p< 0.001; **** = p< 0.0001; 2-way ANOVA followed by Tukey's post-hoc test. Bottom panel: histograms showing the quantification of cortical thickness. SM = somatomotor; SS = somatosensory; V = visual cortex. * = p <0.05; ** = p<0.01; **** = p<0.0001; Kruskal Wallis followed by Dunn's post-hoc test (for SM and V area) or ANOVA followed by Tukey's post-hoc test (for SS area). S
Figure 1Immunofluorescence microscopy detection of Tmem117 in the mouse hypothalamus and pituitary: (A, B) Positive immunostaining was observed in the PVN, the SON and the posterior pituitary. (C, D) No immunostaining was detected in the hypothalamus and pituitary of mice with constitutive inactivation of Tmem117 in AVP neurons. Data information: 3V: 3rd ventricle, AP: Anterior pituitary, MZ: Medial zone, opt: optic tract, PP: Posterior pituitary, PVN: Paraventricular nucleus, SCh: Suprachiasmatic nucleus, SON: Supraoptic nucleus. Scale bar=100μm for panelsImmunofluorescence microscopy detection of Tmem117 in the mouse pituitary: (E) Co-staining for Tmem117 and AVP in the PVN. (F) Co-staining for Tmem117 and AVP in the SON. (G) Co-staining for Tmem117 and AVP in the posterior pituitary. (H, I) Quantification of AVP+/Tmem117+ cells in the PVN and SON, respectively. Data information: 3V: 3rd ventricle, opt: optic tract, PP: Posterior pituitary, PVN: Paraventricular nucleus, SON: Supraoptic nucleus. Scale bar=100μm for panels(J) Higher resolution depiction of an AVP+/Tmem117+ neuron in the PVN captured with structured illumination microscopy. The white arrows highlight representative positions of signal co-localization. Data information: 3V: 3rd ventricle, PVN: Paraventricular nucleus, Scale bar= 10μm for panel J.
Figure 2(B) Glycemia one hour after saline (black) or insulin (red) i.p. injection [n=15-16 mice per group]. (C) CPP plasma levels one hour after saline or insulin injection [n=13 mice per group; mean±SEM for INS: WT 89±5 vs KO 121±13 pg/ml]. (D) GCG plasma levels one hour after saline or insulin injection [n=15-16 mice per group; mean±SEM for INS: WT 124±19 vs KO 186±21 pg/ml]. (E) Glycemia one hour after saline (black) or insulin (red) i.p. injection [n=18-22 mice per group]. (F) CPP plasma levels one hour after saline or insulin injection [n=18-19 mice per group; mean±SEM for INS: WT 78±6 vs KO 92±8 pg/ml]. (G) GCG plasma levels one hour after saline or insulin injection [n=17-20 mice per group; mean±SEM for INS: WT 242±14 vs KO 277±18 pg/ml]. (H-J) Analysis of glycemic levels and of CPP and GCG secretion following insulin injection in female mice at each stage of the estrus cycle [n=3-7 mice per group]. (H) Glycemic levels. (I) CPP plasma levels. (J) GCG plasma levels. Data information: d: day, di: diestrus, est: estrus, INS: insulin, met: metestrus, pro: proestrus, For panels B-G, bars correspond to the mean value per group. For panels H-J, lines correspond to the mean value per group and error bars represent ± SEM. B-G: 2-way ANOVA RM with Bonferroni post-hoc test; H-J: unpaired t-test; *p<0.05, **p<0.01, ***p<0.001.
Figure 3C57BL6/N male mice were injected i.p. with saline or insulin and c-Fos expression (green) in AVP (magenta) neurons was quantitated by immunofluorescence microscopy 2 hours later. (A) C-Fos expression in the PVN after saline injection. (B) C-Fos expression in the PVN after insulin injection (C) Quantitation of c-Fos positive cells in the PVN after saline and insulin injections [n=4 mice per group]. Data information: 3V: 3rd ventricle, AVP: Vasopressin, INS: insulin, PVN: Paraventricular nucleus, SAL: saline, Scale bar=100μm. Lines correspond to the mean value per group and error bars represent ± SEM. Unpaired t-test; ns: p>0.05, *p<0.05, **p<0.01.C57BL6/N male mice were injected i.p. with saline or insulin and c-Fos expression (green) in AVP (magenta) neurons was quantitated by immunofluorescence microscopy 2 hours later. (D) C-Fos expression in the SON after saline injection. (E) C-Fos expression in the SON after insulin injection (F) Quantitation of c-Fos positive cells in the SON after saline and insulin injections [n=4 mice per group]. Data information: AVP: Vasopressin, INS: insulin, opt: optic tract, SAL: saline, SON: Supraoptic nucleus. Scale bar=100μm. Lines correspond to the mean value per group and error bars represent ± SEM. Unpaired t-test; ns: p>0.05, *p<0.05, **p<0.01.(H) Tmem117 mRNA levels in the PVN [n=4 mice per group]. (I)Tmem117 mRNA levels in the SON [n=4 mice per group Data information: INS: insulin, PVN: Paraventricular nucleus, SAL: saline, SON: Supraoptic nucleus. Lines correspond to the mean value per group and error bars represent ± SEM. Unpaired t-test; ns: p>0.05, *p<0.05, **p<0.01.
Figure 4(B) C-Fos expression in the SON Data information: AVP: Vasopressin INS: insulin opt: optic tract, SAL: saline, Scale bar=100μm.(G) Membrane potential was increased comparably in response to 0.1 mM glucose in both groups [n=7-9 cells per group]. (H) No change in membrane potential in NR neurons of both genotypes [n=9 cells per group]. (I) Membrane resistance was decreased in 6 out of 9 AVPTM117WT and 6 out of 7 AVPTM117KO cells in response to 0.1 mM glucose [n=7-9 cells per group]. (J) Membrane resistance in NR neurons was stable for both genotypes [n=9 cells per group]. Data information: AVP: Vasopressin, d: day, GI: glucose inhibited, NR: non-responding, Lines correspond to the mean value per group and error bars represent ± SEM. G-J: paired t-test for all graphs except Δ membrane potential (unpaired); nsp>0.05, *p<0.05, ***p>0.001.
Figure 5(A) Quantification of BiP mRNA per cell in AVP-positive cells of the SON [n=483-704 cells per group; each group consisted of 4 mice]. (B) Quantification of BiP mRNA per cell in AVP-negative cells of the SON [n=347-584 cells per group; each group consisted of 4 mice]. Data information: AVP: Vasopressin For the violin plots dashed lines correspond to the median value and dotted lines to the quartile values. unpaired t-test ; *p<0.05, ***p>0.001.(C) Fluorescence microscopy detection of AVP mRNA (green) and BiP mRNA (magenta) in the SON. Data information: AVP: Vasopressin opt: optic tract Scale bar=20μm.(F) Fluorescence microscopy detection of DHE-derived fluorescence (red) in the SON. (G) Quantification of DHE-derived fluorescence in the SON [n=8 SON per group; each group consisted of 4 mice]. Data information: AVP: Vasopressin, d: day, DHE: dihydroethidium , opt: optic tract, SON: Supraoptic nucleus,. Scale bar=20μm. For panels G data are represented as mean ± SEM. G: unpaired t-test; ; *p<0.05, ***p>0.001.(J) Calcium recordings in AVP magnocellular terminals 10 minutes before and one hour after the i.p injection of insulin (red and orange) or saline (black). The vertical arrow corresponds to the i.p. injection (time=0). (K) Quantification of mean signal intensity in 10-minute time bins. [n=6-8 mice per group]. Data information: AUC: area under the curve, AVP: Vasopressin INS: insulin, SAL: saline For panels K, data are represented as mean ± SEM. K: 1-way ANOVA with Tukey's post-hoc test; *p<0.05, ***p>0.001.(M) Fluorescence microscopy detection of GCaMP (green) in posterior pituitary. Immunofluorescent labelling was used to localize the AVP terminals (magenta). Data information: AVP: Vasopressin ,. Scale bar=20μm.
Figure 6(B) Immunofluorescence microscopy detection of AVP in the PVN and the SON. Data information: 3V: 3rd ventricle, AVP: Vasopressin, KO: AVPTM117KO opt: optic tract, PVN: paraventricular nucleus, SON: Supraoptic nucleus, WT: AVPTM117WT. Scale bar=100μm.(C) Quantitation of AVP positive cells in the PVN [n=3-4 mice per group]. (D) Quantitation of AVP positive cells in the SON [n=3-4 mice per group]. Data information: AVP: Vasopressin, KO: AVPTM117KO WT: AVPTM117WT. For panels C and D lines correspond to the mean value per group and error bars represent ± SEM. For panels C,D: unpaired t-test; ; *p<0.05, **p<0.01, ***p<0.001.(E) Glycemia one hour after saline (black) or insulin (red) i.p. injections in AVPTM117WT and AVPTM117KO male mice [n=8-9 mice per group]. (F) CPP plasma levels one hour after saline or insulin injection in AVPTM117WT and AVPTM117KO male mice [n=8 mice per group]. (G) GCG plasma levels one hour after saline or insulin injection in AVPTM117WT and AVPTM117KO male mice [n=8-9 mice per group]. Data information: AVP: Vasopressin, d: day, INS: insulin, KO: AVPTM117KO, SAL: saline, WT: AVPTM117WT. For panels E-G bars correspond to the mean value per group and error bars represent ± SEM. E-G: 2-way ANOVA for each timepoint in comparison to SAL baseline with Bonferroni post-hoc test; *p<0.05, **p<0.01, ***p<0.001.
Figure 2(A) Volcano plot of phosphorylation changes in iKras PDAC cells in response to KrasG12D induction. Phospho- to total proteomic changes were quantified from three independent biological replicates of Dox treated vs. untreated cells, via TMT (Dataset EV8). 872 phosphorylations were found to be significantly increased, while 82 phosphorylations were significantly decreased (FDR < 0.05). Significantly changing phosphorylations on RBPs whose RNA-binding activity was significantly increased (red) or decreased (blue) as per Fig 1C are highlighted on the plot.(E) KrasG12D induction enhances phosphorylation of CK2α (T360/S362), in an Erk1/2-dependent manner. IKras PDAC cells were grown in the absence of Dox for 48 hrs, before its addition to the indicated cells, with or without Trametinib (10 nM), for a further 24 hrs. Cells were then lysed and analyzed by immunoblotting (IB) with the indicated antibodies. The red triangle marks the main phospho band which overlaps with the total CK2α.(G) Analysis of the RNA-binding activity of WT, Phospho-defective (S4A), and phospho-mimicking (S4D) mutants of Ncl by OOPS. IKras PDAC cells were transiently transfected with myc-tagged WT, S4A, and S4D Ncl constructs. Cells were subsequently grown in the absence of Dox for 48 hrs, followed by Dox addition for 24 hrs to induce KrasG12D expression. Cells were then subjected to OOPS to isolate the interface (RNA-bound proteins), or whole cell lysis (total lysate), followed by immunoblotting with anti-Ncl antibody. For comparison, Gapdh, which also binds RNA but does not show a change in its RNA-binding activity (Dataset EV1), was also blotted for. A fraction of each interface or total lysate sample was also subjected to RNA extraction, which was resolved and quantified by capillary electrophoresis (CE) as loading control.
Figure 3(B) Immunofluorescence analysis of WT, S4A, and S4D Myc-Ncl subcellular localization. IKras PDAC cells ectopically expressing either WT, S4A, or S4D Myc-Ncl were fixed and immunostained with anti-Myc-tag antibody, along with anti-Fibrillarin (Fbl) antibody as a Nucleolar marker, and Hoechst, followed by confocal microscopy analysis.(C) Distribution of WT, S4A, and S4D Myc-Ncl crosslink sites across the annotated 47S pre-rRNA genomic region. Peak heights represent mean crosslink intensities from three independent iCLIP replicate experiments.(D) Quantification of the percentage of crosslinks in each region of the 47S pre-rRNA for WT, S4A, and S4D Myc-Ncl. Percentage of crosslinks relative to 47S total were quantified from three independent biological replicate iCLIP experiments. (E) Quantification of the proportional density of crosslinks in each region of the 47S pre-rRNA for WT, S4A, and S4D Myc-Ncl. Proportional density was calculated from three independent iCLIP experiments by normalizing the number of crosslinks in each region to the sequence length of that region.
Figure 5(A) Colony formation of control and Ncl depleted iKras PDAC cells, in presence or absence of KrasG12D. Cells were transfected with a non-targeting control siRNA, or two independent siRNAs against Ncl, followed by clonogenic assay for 7 days in presence or absence of Dox. Colonies were visualized by Crystal Violet staining. (B) Quantification of Crystal Violet staining levels from (A). A total of 6 biological replicate experiments were quantified. Error bars depict SD. (****: P < 0.0001; n.s.: not significant - calculated from two way ANOVA with Šídák's multiple comparisons test). ((C) 3D proliferation of control and Ncl depleted iKras PDAC cells, in presence or absence of KrasG12D. Cells were transfected with a non-targeting control siRNA, or two independent siRNAs against Ncl, before being reseeded onto 3D Collagen-I gels, with or without Dox, and allowed to grow for 48 hrs. Cells were subsequently imaged live by phase contrast microscopy. Scale bar = 200 µm. (D) Analysis of the relative percentage of viable cells in 3D collagen-I cultures from (C). Cells were subjected to luminescence-based viability assay by CellTiter-Glo to quantify the percentage of viable cells. A total of 3 biological replicate experiments were quantified. Error bars depict SD. (****: P < 0.0001; n.s.: not significant - calculated from two way ANOVA with Šídák's multiple comparisons test). (
Figure 6(A) Dose response analysis of CX-5461 impact on nascent rRNA expression. IKras PDAC cells were grown in the absence of Dox for 48hrs. Cells were subsequently treated with or without Dox for 24 hrs to induce KrasG12D expression, followed by pre-treatment with the indicated concentrations of CX-5461 for 30 mins prior to pulse labeling with FUrd. Cells were then fixed and immunostained with anti-FUrd antibody to visualize nascent RNA (green), anti-Ncl antibody to reveal the Nucleolus (red), and Hoechst (blue) as the Nuclear stain, followed by confocal microscopy analysis. (B) Quantification of Nucleolar FUrd levels in images from (A). FUrd fluorescence densities in single nucleoli were quantified from 251-479 individual cells per condition, combined from 2 independent biological replicate experiments (****: P < 0.0001; n.s.: not significant - calculated from one-way ANOVA with Šídák's multiple comparisons test).(G) MRI imaging of orthotopic iKras tumors in untreated or CX-5461 treated mice. IKras PDAC cells were engrafted into the pancreas of Dox-fed nude mice and allowed to form tumors for 4 days. Animals were then divided into two groups, with the first group treated by daily oral administration of CX-5461 (50mg/kg) for a further 10 days, whilst the second group was left untreated for the same period. T2 scans were taken on the indicated days, post-engraftment. Green areas mark the tumors.(K) H&E analysis of the extracted pancreas tissues from (I). Representative pancreatic tissue images from the untreated and CX-5461 treated mice, showing typical PDAC histology with malignant ductal structures surrounded by stroma in the untreated, but a largely normal pancreas histology with a small malignant (M) component (marked by the dashed line) in the treated mice.
Figure 1(C) TNFemARE/ARE mice show significantly elevated serum levels of TNF (wild-type n=5, TNFemARE/+ n=5, TNFemARE/ARE n=5). Data information: data are represented as Mean +/- SEM, n=biological replicates. one-way ANOVA test was used with Tukey's multiple comparisons test, ns= p-value > 0.05, * = p-value ≤ 0.05, ** = p-value ≤ 0.01, *** = p-value ≤ 0.001, **** = p-value ≤ 0.0001.(D) Histologic H&E sections of ileum and hind paw indicate severe ileitis and arthritis in 20-30 w/o SPF TNFemARE/ARE mice (Scale bars upper panel: 100 μm, scale bars lower panel: 500 μm).(G) tSNE analysis performed on CD45+ lineage (excluding CD3+, CD19+, NK1.1+ fractions) of lamina propria of ileum samples (n=5/genotype).(H) Ileal flow cytometry data of 25 w/o TNFemARE mice show a highly activated innate immune system (n=5/genotype). Data information: data are represented as Mean +/- SEM, n=biological replicates. one-way ANOVA test was used with Tukey's multiple comparisons test, ns= p-value > 0.05, * = p-value ≤ 0.05, ** = p-value ≤ 0.01, *** = p-value ≤ 0.001, **** = p-value ≤ 0.0001.(J) Ileal flow cytometry data show an increase of CD4+ cells in transgenic mice, which can be designated to the enrichment of the CD4+RORgt+ (Th17) cell population (n=5/genotype). Data information: data are represented as Mean +/- SEM, n=biological replicates. one-way ANOVA test was used with Tukey's multiple comparisons test, ns= p-value > 0.05, * = p-value ≤ 0.05, ** = p-value ≤ 0.01, *** = p-value ≤ 0.001, **** = p-value ≤ 0.0001.
Figure 2(C) Serum levels of TNF are elevated in hetero- and homozygous transgenic mice, in both SPF and GF conditions (n=5 mice/group, except for GF wild-type n=4). Data information: data are represented as Mean +/- SEM, n=biological replicates, two-way ANOVA test used with Tukey's multiple comparisons test, ns= p-value > 0.05, * = p-value ≤ 0.05, ** = p-value ≤ 0.01, *** = p-value ≤ 0.001, **** = p-value ≤ 0.0001.(D) Quantitative real-time PCR reveals increased expression of the tnf gene in the ileum in homozygous SPF and GF mice (SPF wild-type n=10, SPF TNFemARE/+ n=10, SPF TNFemARE/ARE n=13; GF wild-type n=3, GF TNFemARE/+ n=4, GF TNFemARE/ARE n=4). Data information: data are represented as Mean +/- SEM, n=biological replicates, two-way ANOVA test used with Tukey's multiple comparisons test, ns= p-value > 0.05, * = p-value ≤ 0.05, ** = p-value ≤ 0.01, *** = p-value ≤ 0.001, **** = p-value ≤ 0.0001.(F) Comparing SPF versus GF H&E sections of the ileum shows complete rescue of gut inflammation under axenic conditions in 20-30 w/o TNFemARE/ARE mice (Scale bars: 100 μm).
Figure 3(C) 20-30 w/o SPF TNFemARE/ARE mice clearly suffer from inflammation in the synovio-entheseal complex, which is not rescued in age-matched GF mice. (Scale bars: 200 μm)(F) <em>μ</em>CT images of tails of wild-type versus SPF and GF TNFemARE/ARE mouse display fusion of sacral vertebrae in homozygous SPF and GF mice, as indicated by yellow arrows.
Figure 4(B) Gait analysis of TNFemARE mice reveals distinct moving patterns of SPF and GF TNFemARE/ARE mice. Homozygous mice tend to have smaller footprints, place their feet with lower mean intensity and generally have a smaller stride length. (SPF wild-type n=12, SPF TNFemARE/+ n=12, SPF TNFemARE/ARE n=12; GF wild-type n=8, GF TNFemARE/+ n=20, GF TNFemARE/ARE n=12) Data information: For panel (B), boxplots represent the 25- and 75- percentile with median values as central band, whiskers span min to max value range, n=biological replicates with every datapoint representing 1 paw, two-way ANOVA test used with Tukey's multiple comparisons test, ns= p-value > 0.05, * = p-value ≤ 0.05, ** = p-value ≤ 0.01, *** = p-value ≤ 0.001, **** = p-value ≤ 0.0001.
Figure 5(C) H&E sections of the calcaneo-cuboid joint of 5-15 w/o A20myel-KO mice indicate immune cell infiltrates in the synovium and the fat pad and severe bone marrow edema. (Scale bars: 100 μm)(E) Histologic images of hind paws of old (44 w/o) GF wild-type (left) versus A20myel-KO (right) mice. The morphology of the paw is completely lost in the transgenic mouse due to severe inflammation. (Scale bars: 200 μm)(F) Splenomegaly in GF A20myel-KO mice of 15 w/o indicates systemic inflammation in this mouse model (wild-type n=7, A20myel-KO n=8). Data information: For panels F), data are represented as Mean +/- SEM, n=biological replicates. For graph (F), two-tailed unpaired t-test was used. ns= p-value > 0.05, * = p-value ≤ 0.05, ** = p-value ≤ 0.01, *** = p-value ≤ 0.001, **** = p-value ≤ 0.0001.
Figure 7A) Cells treated with nocodazole are affected in DSB-SPB interaction. Cnm67-CFP and Ddc2-RFP were used as SPB and DSB markers respectively. Diagram depicts the genomic background used. D1: inter-SPB distance; D2: DSB distance to proximal SPB; D3: DSB distance to distal SPB. D1, D2 and D3 distances after inducing a DSB in the presence of nocodazole or mock dimethyl sulfoxide (DMSO) were scored and plotted. Graph represents the mean ± SD of D1, D2 and D3 distances of three independent experiments. At least 100 cells per experiment were scored using the maximum projection of three z-planes. P values were calculated using a 2-tailed unpaired Student t test. A representative picture is shown. Scale bar: 3μm.B) Physical analysis of wild-type cells containing the repair pathway choice assay described in Fig 2B. The diagram with the genomic information, the restriction enzymes used and the location of the probe are shown. Cells were synchronized in G1 by using the α-factor pheromone and released into fresh media for 1 hour. Induction of HO expression was attained by adding galactose for 2 hours. After formation of the DSB, glucose was added to repress the HO in the presence of nocodazole or mock DMSO and samples were taken to analyse the kinetics of the repair. FACS analyses are shown.B) Physical analysis of wild-type cells containing the repair pathway choice assay described in Fig 2B. The diagram with the genomic information, the restriction enzymes used and the location of the probe are shown. Cells were synchronized in G1 by using the α-factor pheromone and released into fresh media for 1 hour. Induction of HO expression was attained by adding galactose for 2 hours. After formation of the DSB, glucose was added to repress the HO in the presence of nocodazole or mock DMSO and samples were taken to analyse the kinetics of the repair. DNA was extracted, digested with EcoRV, separated on agarose gels and blotted. Both probes for the MATa-distal sequence and the HIS3 gene (as loading control) were used. Graphs show the quantification of MATa restoration (NHEJ), mating type switching by gene conversion (HR), and DSB kinetics formation. All data were normalized with the HIS3 gene. Graphs show the mean ± S.D. from three independent experiments. P values were calculated using a 2-tailed unpaired Student t test.