EMBO/SourceData huggingface dataset:
Figure 3TPLSM of EAE lesions in the brainstem of adoptive transfer EAE at the onset of the disease. EAE was induced by transfer of in vitro differentiated 2d2Th17 cells (tdRFP-red, IL-17-EGFP-green) into CD11c-DTR/GFPmice (CD11c-GFP - green); imaging area 300 × 300 µm.(A) Intravascular IL-17hi2d2Th17 cell (arrow, double positive -> green: IL-17EGFP and red: 2d2.tdRFP) rolling cell towards a CD11c-GFP+ cell (arrowhead). White dotted lines mark a venous vessel.(B) Time-lapse TPLSM (maximal intensity projections) of the boxed area in (A) shows that the perivascular elongated CD11c-GFP+ cell (asterisk) enters into contact with IL-17-expressing (GFP - green and 2d2.tdRFP - red double-positive; arrow and arrow head) Th17 cells (scale bar: 20 µm).(C) XYZ-resolved TPLSM depiction reveals that the elongated perivascular CD11c-GFP+ cell makes contact with the intravascular 2d2Th17 cells via a filopodium-like dendrite (xy-plane, 300 × 300 µm, z-depth 70 µm).(D) Strong interaction of a perivascular IL-17hi2d2Th17 with a perivascular CD11c-GFP+ cell (insert upper left contact).(E) Time lapse of the insert in (D): Stopping motility of the round-shaped IL-17hi2d2Th17 cell is followed by entry into the CNSparenchyma.(F) Magnified image of interaction of the upper left and (G) lower right perivascular T cell-DC contacts as shown in (D) and (E).(H) Time-lapse imaging of parenchymal CD11c-GFP+ cells (arrowhead), targeted migration of an IL-17hi 2d2 Th17 cell (arrow, double positive -> green: IL-17EGFP and red: 2d2.tdRFP) towards the CD11c-GFP+ cell (arrowhead). Scale bar is 20 µm.(I) Quantification of the motility pattern of IL-17hi 2d2.tdRFP vs. all 2d2.tdRFP in CD11c-DTR/GFP brainstem lesions before and after (2-5 h) intraperitoneal DTX injection. Evaluation of mean track velocity of the single cell tracking (each dot represents a track); pooled data from at least two independent experiments (>3 imaging areas). For the statistical analysis, the Mann-Whitney-U-test was performed (*** p < 0.0001, n.s. = not significant).(K) Percentage of stopping cells (red bar; instantaneous velocity <2 µm/min) of the data set as shown in (I).
Figure 5(A) CD11c+ cells, CD4+ cells and CX3CR1+CD45intIAb+ (microglia) cells were isolated from peak EAE animals from the CNS. Samples from 6-11 animals were pooled in each experiment; at least two experiments were performed for each subset. Depicted are normalized mRNA expression (to expression in spleen CD11c+ cells) levels as determined by qRT-PCR analysis relative to the housekeeping genes Eukaryotic translation elongation factor 1 alpha 1 (Eef1a1) and Peptidylprolyl isomerase A (Ppia). CNS CD11c+ cells show gene expression of the inflammatory chemokines Ccl2, Ccl5, Cxcl9, Cxcl10 but not Il23 (mean ± SEM).(C) Quantitative expression of EAE-relevant factors Il23 and Csf2 were investigated in the CNS samples as described above(B) CD11c+ cells, CD4+ cells and CD11c-CD11b+IAb+ (monocytes/macrophages) were isolated from EAE affected mice as described in (A). Samples from 6-11 animals were pooled in each experiment; at least two experiments were performed for each subset. Depicted are normalized mRNA expression (to expression in spleenCD11c+ cells) levels as determined by qRT-PCR analysis relative to the housekeeping genes Eukaryotic translation elongation factor 1 alpha 1 (Eef1a1) and Peptidylprolyl isomerase A (Ppia). Relevantly regulated chemokines in other cell populations than CNS CD11c+ cells are shown (mean ± SEM).(D) Quantitative expression of EAE-relevant factors Il23 and Csf2 were investigated in the spleen samples.Figure 5Chemokine expression profiling of CD11c+ cells in the spleen and CNS reveal a key role of CNSCD11c+ cells for inflammatory chemokines.(A) CD11c+ cells, CD4+ cells and CX3CR1+CD45intIAb+ (microglia) cells were isolated from peak EAE animals from the CNS. Samples from 6-11 animals were pooled in each experiment; at least two experiments were performed for each subset. Depicted are normalized mRNA expression (to expression in spleenCD11c+ cells) levels as determined by qRT-PCR analysis relative to the housekeeping genes Eukaryotic translation elongation factor 1 alpha 1 (Eef1a1) and Peptidylprolyl isomerase A (Ppia). CNSCD11c+ cells show gene expression of the inflammatory chemokines Ccl2, Ccl5, Cxcl9, Cxcl10 but not Ccl20 (mean ± SEM).(B) CD11c+ cells, CD4+ cells and CD11c-CD11b+IAb+ (monocytes/macrophages) were isolated from EAE affected mice as described in (A). Relevantly regulated chemokines in other cell populations than CNSCD11c+ cells are shown (mean ± SEM).(C) Quantitative expression of EAE-relevant factors Il23 and Csf2 were investigated in the CNS samples as described above and (D) the spleen samples.Statistical significance was determined using Kruskal-Wallis test. P-values < 0.05 were regarded as statistically significant. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.
Figure 6(A) CD11c-GFP+ DC cluster formation near venous vessel with (B) protrusions attracting 2d2.tdRFP Th17 cells.(C) Low-velocity motility of 2d2.tdRFPT cells in DC cluster compared to outside of the cluster. SHG in upper panel on the left (arrow). In addition, intercellular matrix fibers (arrow) are visualized that are only found in inflamed CNSparenchyma and are due to second harmonic generation (SHG) at 1110 nm wavelength excitation and detection at 535 ± 50nm. Tracking of 2d2.tdRFPTh17 in right panel (color-coded tracks, low velocity: red, high velocity: yellow). (D) Quantitative analysis of T cell tracking: higher mean velocity of 2d2.tdRFPTh17 cells in the onset of the disease with differential distribution at the peak of the disease. Cluster associated tracks have significantly lower mean velocities than Th17 cells outside the CD11c-GFP clusters. (E) Analogously for track straightness; Kruskal-Wallis Test and Dunn's Multiple Comparison Test (*** p < 0.0001) were performed; pooled data from at least three independent experiments per group.
Figure 2A. The unfertilized oocytes were injected with spermatozoa with a variant in IQCN at 15-18 h post-injection. The white arrowheads represent the polar body (PB). PN represents pronucleus.B. The expression and localization of PLCζ in sperm from the affected individuals.C. H&E staining of spermatozoa from affected individuals. The black arrow represents normal spermatozoa. The black arrowheads represent tapered-head spermatozoa with increased major axis length and normal minor axis length. The white arrowheads represent thin spermatozoa with decreased major axis length and minor axis length.D. The ultrastructure of spermatozoa from normal and affected individual. The white squares represent the perinuclear ring. Ac represents acrosome. N represents nucleus.E. The range of acrosomes in sperm heads from normal and affected individual (n=7).
Figure 3A. Fertility assessment experiments in WT and Iqcn-/- male mice after mating with WT female mice (n = 18).B. Size of the testes and epididymides in WT and Iqcn-/- male mice.C. Quantification ratio of the testis weight/body weight in WT and Iqcn-/- male mice (n = 3).D. Sperm counts in the caudal epididymis from WT and Iqcn-/- male mice (n = 3).E. H&E staining of sperm from WT and Iqcn-/- male mice. Red arrowheads represent sperm with no sickle shape heads.F. Percentage of sperm without sickle shape heads from WT and Iqcn-/- male mice (n = 3).G. Ultrastructure of spermatozoa from WT and Iqcn-/- male mice. Ac represents the acrosome.H. Immunostaining of lectin (green) in spermatozoa from WT and Iqcn-/- male mice.I. Percentage of abnormal acrosomes in spermatozoa from WT and Iqcn-/- male mice (n = 3).J. Percentage of spontaneous and induced acrosome reactions in spermatozoa from WT and Iqcn-/- male mice (n = 3).
Figure 4A. Representative two pronucleus (2PN) zygotes, two-cell embryos, and blastocysts after ICSI in WT and Iqcn-/- male mice.B. Percentage of 2PN zygotes, two-cell embryos and blastocysts after ICSI in WT and Iqcn-/- male mice (n = 3).C. Profile of Ca2+ responses induced by oocyte injection with spermatozoa from WT and Iqcn-/- male mice (n = 5). Spermatozoa from WT male mice induced 8-9 Ca2+ spikes over a 3-h period (blue line), while spermatozoa from Iqcn-/- male mice failed to induce spikes (red line).D. Immunostaining of oocytes injected with spermatozoa from WT and Iqcn-/- male mice. The expression of PLCζ (red) and α-tubulin (green) was detected in oocytes. M represents male nucleus and F represents the female nucleus.E. Expression and localization of PLCζ in sperm from WT and Iqcn-/- male mice.F. Expression and localization of PLCζ during acrosomal biogenesis from WT and Iqcn-/- male mice. PLCζ was localized in the acroplaxome in WT spermatids. PLCζ was only localized in the tip of the head in the cap and acrosome phases (white arrowheads) and was lost during the maturation phase in Iqcn-/- mice.
Figure 5A. Expression and localization of IQCN during spermiogenesis in the testes of WT male mice. IQCN (red) was co-localized with α-tubulin (green) from steps 8-14. Sb represents late spermatids. Scale bars, 20 μm.B. Ultrastructure of the testes from WT and Iqcn-/- male mice from steps 8-16. MA represents manchette. PR represents perinuclear ring. CD represents cytoplasmic droplets. Scale bars, 2 μm (steps 8-12; 15-16) and 5 μm (steps 13-14).C. Immunostaining of α-tubulin (green) in spermatids at steps 13-14. MA represents manchette. PR represents perinuclear ring.D. Length of the manchette structure in spermatids at steps 13-14 from WT and Iqcn-/- male mice (n = 10).
Figure 6A. Expression and localization of calmodulin during spermiogenesis in the testes of WT and Iqcn-/- male mice. Calmodulin (red) was co-localized with α-tubulin (green) from steps 8-14 in WT mice, but failed to co-localize in steps 13-14 in Iqcn-/- male mice.B. Interaction between IQCN and calmodulin (CaM) following co-IP by IQCN antibody in testes from WT and Iqcn-/- male mice (n = 3). IQCN interacts with calmodulin in WT mice, the interaction of which is completely lost in Iqcn-/- male mice. GAPDH was used as the internal control.C. Immunostaining of calmodulin (red) and α-tubulin (green) in HeLa cells. The normal (WT) and mutant (c.910C>T and c.2453_2454del) plasmids of IQCN were transfected into HeLa cells. HeLa cells were treated with an antagonist of calmodulin W-7. Multipolar spindles were observed in HeLa cells transfected with mutant plasmids and W-7 treatment.D. Percentage of multipolar spindles in WT and mutant IQCN transfected HeLa cells. (n = 3 biological replicates)
Figure 1C), the percentage of pups gathered in the nest location is significantly reduced in Shank2-/- mice. Mann-Whitney test, ***P<0.001. Data information: All data are presented as mean ± SEM.
Figure 2(H) Shank2+/+ and Shank2-/- mice displayed a significant preference for the novel object versus the familiar one in the test session, two-way mixed ANOVA, effect of object: ***P<0.001, effect of genotype: P=0.167, object x genotype interaction: P=0.667, Shank2+/+ n = 9, Shank2-/- n = 9. Data information: All graphs are presented as mean ± SEM, NS: not significant.
Figure 3(J) Left panel: Electron micrographs of the posterior pituitary gland from Shank2+/+ and Shank2-/- mice in lower (left panel) and higher magnification. Scale bar (500 nm). Right diagram: No significant difference was detected in the number of vesicles in the posterior pituitary gland between Shank2+/+ and Shank2-/- mice, Mann-Whitney test, P=0.507, Shank2+/+ n = 3, Shank2-/- n = 3. (K-M) Additionally, Shank2-/- mice display no significant difference in: (K) Estradiol-, Mann-Whitney test, P=0.475, Shank2+/+ n = 6, Shank2-/- n = 7, (L) Progesterone, unpaired, two-tailed Student's t-test, P=0.570, Shank2+/+ n = 5, Shank2-/- n = 5 or (M) Prolactin- plasma concentrations, unpaired, two-tailed Student's t-test, P=0.980; Shank2+/+ n = 3, Shank2-/- n = 3. Data information: All data are presented as mean ± SEM, NS: not significant. E, endothelium; CL, capillary lumen; M, mitochondria; NG, neurosecretory granule; AE, axonal endings, HYP, Hypothalamus; PIT, Pituitary gland.
Figure 4(A-G) qRT-PCR analysis of mRNA expression levels of hormonal genes and hormonal receptors normalized to house-keeping gene HMBS of 9-10 week old naive and postnatal day 1 Shank2+/+ and Shank2-/- mice: (A) Oxytocin gene expression did not differ between Shank2+/+ and Shank2-/- mice, one-way ANOVA, P=0.335. (B) Vasopressin gene expression did not differ between Shank2+/+ and Shank2-/- mice, one-way ANOVA, P=0.945. (C) Prolactin gene expression did not differ between Shank2+/+ and Shank2-/- mice, one-way ANOVA, P=0.202. Additionally, no significant difference was evident in (D) oxytocin receptor, one-way ANOVA, P=0.675, (E) prolactin receptor, one-way ANOVA, P=0.912, (F) vasopressin receptor, one-way ANOVA, P=0.418 and (G) estrogen receptor alpha expression, one-way ANOVA, P=0.890 in comparison to Shank2+/+ mice. All data are presented as mean ± SEM, NS: not significant. Pituitary gland, (n=3 pituitary glands pooled per value, n=3 hypothalamus (Oxytocin, Vasopressin, Oxytocin receptor, Vasopressin receptor, estrogen alpha) n=2-3 hypothalamus (Prolactin receptor)). HYP, hypothalamus; PIT, pituitary gland.
Figure 5(E-H) Repeated pup exposure does not induce major components of social attachment behavior in Shank2-/- females: (E) pup grooming, unpaired, two-tailed Student's t-test, ***P<0.001 (F) crouching, Mann-Whitney test, ***P<0.001 and (G) nest building, Mann-Whitney test, ***P<0.001, (H) maternal interaction, unpaired, two-tailed Student's t-test, ***P<0.001 (trial 5), Shank2+/+ n = 9, Shank2-/- n = 9. Data information: All data are presented as mean ± SEM, NS: not significant.(L) Levels of synaptic proteins in the crude synaptosomes fraction (P2) from the hypothalamus of Shank2+/+ and Shank2-/- mice. Shank2+/+ n = 5 (black bars), Shank2-/- n = 5 (blue bars). Unpaired, two-tailed Student's t-test.
Figure 6(D-G) DREADD activation by CNO injection selectively rescues major components of social attachment behavior in Shank2-/- mice: (D) grooming, two-way ANOVA, effect of genotype: *P=0.018, effect of treatment: **P=0.002, genotype x treatment interaction: ***P=0.001. (E) Crouching, two-way ANOVA, effect of genotype: P=0.091, effect of treatment: P=0.869, genotype x treatment interaction: P=0.663, (F) nest building, two-way ANOVA, effect of genotype: P=0.415, effect of treatment: P=0.341 genotype x treatment interaction: P=0.093, (G) Maternal interaction, two-way ANOVA, effect of genotype: *P=0.034, effect of treatment: P=0.149, genotype x treatment interaction: *P=0.045. Shank2+/+ vehicle injected n = 6, Shank2-/- vehicle injected n = 6, Shank2+/+ CNO injected n = 6, Shank2-/- CNO injected n = 7. Data information: All data are presented as mean ± SEM, NS: not significant.
Figure 1D H&E (hematoxylin-eosin) staining (left) and oil red O staining (right) of livers. Scale bar, 50μm.E Serum cholesterol concentrations of each group. Liver cholesterol concentrations (F) of each group were examined.H and I Cholesterol levels in p53+/+ and p53-/- HepG2 cells (H) or HCT116 cells (I) were examined. Protein expression was shown by western blotting (bottom panel).
Figure 2C mRNA and protein levels of SQLE in different tissues of p53+/+ or p53-/- mice were determined by qRT-PCR and western blot respectively.F and G p53+/+ and p53-/- HepG2 cells were cultured for 48 hours in medium containing fetal bovine serum (Serum) or lipoprotein-depleted FBS (LPDS). Protein expression was shown by western blotting (F). mRNA levels of SQLE were examined by qRT-PCR (G). P: premature SREBP2; M: mature SREBP2.Protein expression and mRNA levels of p53+/+ and p53-/- HepG2 cells treated with control siRNA or three separated sets of SREBP2 siRNAs for 72 hours as indicated.
Figure 3F Luciferase reporter constructs containing RE1, RE2, RE1mut or RE2mut were transfected into p53-/- HCT116 cells together with control (PRK5-flag vector) or p53 expression vector (PRK5-flag p53) for 48 hours. Renilla vector pRL-CMV was used as a transfection internal control. Relative levels of luciferase are shown. Data represent three independent experiments. Protein expression is shown G Luciferase reporter constructs containing RE1, RE2, RE1mut or RE2mut were transfected into HEK293T cells together with control (PRK5-flag vector) or p53 expression vector (PRK5-flag p53) for 48 hours. Renilla vector pRL-CMV was used as a transfection internal control. Relative levels of luciferase are shown. Protein expression is shown.
Figure 4A-C Squalene (A), lanosterol (B) and cholesterol (C) abundance in p53+/+ and p53-/- HepG2 cells transfected with control or SQLE siRNA for 48 hours were determined via ultra-high pressure liquid chromatography coupled to mass spectrometry (UHPLC-MS). Protein levels were analyzed by western blotting with specific antibodies (A bottom panel).
Figure 5A Protein expression of SQLE in human liver tumors (C) and non-cancerous adjacent tissue (N) were analyzed by western blotting.B SQLE gene expression in HCC and adjacent normal tissue was analyzed in TCGA Liver Hepatocellular Carcinoma (normal [n=50] versus tumors [n=371]). C SQLE gene expression in COAD and adjacent normal tissue was analyzed in TCGA Colon Cancer (normal [n=41] versus tumors [n=286]). J - M p53+/+ HCT116 cells (J and K) and p53+/+ HepG2 cells (L and M) were transfected with SQLE siRNA or control siRNA in the presence or absence of exogenous SQLE cDNA for 48 hours. Cholesterol concentration (upper panel) and protein expression (bottom panel) were determined (J and L). Cell proliferation is shown (K and M).
Figure 6C Growth of p53+/+ and p53-/- HepG2 cells transfected with and without exogenous SQLE (HA-SQLE) in LPDS medium. Protein expression is assayed after transfection 24 hours.G and H Cholesterol levels, protein expression (G) and cell growth (H) of SK-HEP-1 cells stably expressing control shRNA or p53 shRNA in LPDS medium in presence or absence of 5μg cholesterol.L Percentage of SA-β-gal-positive cells of p53+/+ and p53-/- HepG2 cells expressing SQLE siRNA or control siRNA (upper panel). Protein expression (middle panel) and representative images (lower panel) are shown . Sacle bar, 100 μm.
Figure 7p53-/- C57BL/6N male mice (n=7) were injected with a single dose of diethylnitrosamine (DEN) (25mg/kg) at two weeks and then fed with HFD diet at 4 weeks. Oral administration of terbinafine were given at 13 weeks old as indicated. Shown are liver tumor incidence (L) and tumor numbers (M).(N) H&E (left), Ki-67 (middle) and Oil Red O (right) staining of livers of mice.(O) Total liver cholesterol concentrations of mice were examined.
Figure 8representative liver photos (C, left), liver tumor numbers (C, right), representative images (D, left) and quantification (D, right) of Ki-67 staining are shown. Red arrows indicate the tumors in the liver (C).E H&E (left), Ki-67 (middle) and Oil Red O (right) staining of livers of mice
Figure 1A: Serial dilutions of stationary phase cultures of the indicated strains were spotted on YEPD and incubated at the indicated temperatures.Wild type, mps1-3 and mps1-1 mutant cells were synchronised in G1 with α-factor at permissive temperature (25°C) and then released at restrictive temperature (34°C, t=0). Cells were collected at the indicated time points for FACS analysis of DNA contents (C)Wild type, mps1-3 and mps1-1 mutant cells were synchronised in G1 with α-factor at permissive temperature (25°C) and then released at restrictive temperature (34°C, t=0). Cells were collected at the indicated time points for immunofluorescence using anti-tubulin antibodies in order to score metaphase and anaphase spindles (D). Budding and nuclear division were scored on the FACS samples.F: Wild type and mps1 mutant cells bearing the TetO/TetR-GFP system to mark the centromere of chromosome V [43] and expressing the SPBs marker Spc97-mCherry were synchronised as in (C-D) and arrested in anaphase through the temperature-sensitive cdc15-2 allele. At 120 and 150 minutes, cells were fixed for scoring chromosome V segregation (n≥172). Arrowheads indicate the sister chromatids of chromosome V. Error bars: SD. N=3. Representative images of cells for each genotype are shown on the right. Scale bar: 5 μm.G: Wild type and mps1-3 mutant cells carrying the TetO/TetR-GFP markers for CEN5 labelling and expressing mCherry-Tub1 were grown at 25°C and then shifted to 34°C for 1 hour before filming. Cells were filmed at 34°C every 2 minutes by time lapse fluorescence microscopy. DIC: differential interference contrast. Scale bar: 5 μm.
Figure 2A: Wild type and mps1 mutant cells were synchronised in G1 with α-factor at 25°C and then released at 34°C in the presence of nocodazole (t=0). Cells were collected at the indicated time points for FACS analysis of DNA contents.B: Cells were treated as in (A) and after 2 hours from the release α-factor was re-added to prevent cells from undergoing a second cell cycle. Cell samples were collected at the indicated time points for western blot analysis of the indicated proteins. Pgk1 was used as loading control. Cyc: cycling cells.C: Top: in vitro kinase assays with recombinant Mps1 and Mps1-3 purified from E. coli and incubated at 34°C for the indicated times in the presence of radioactive ATP and a recombinant Spc105 N-terminal fragment (aa 1-320) as substrate. Bottom left: kinase activity was quantified on the autoradiographs by Image J and normalized to the levels of the full length protein on the Coomassie Blue-stained gel (right). Bottom right: 1μg of the recombinant Mps1 kinases used for kinase assays was loaded on SDS page and stained with Coomassie Blue for normalisation of kinase assays.D: Wild type and mps1-3 cells were synchronised in G1 with α-factor at 25°C and then released at 34°C in the presence of nocodazole. Cells were collected after 90 minutes and fixed with formaldehyde for ChIP-seq analysis. ChIP sequence reads were normalised against sequence reads from corresponding input samples, and relative enrichment is plotted for chromosome III around the centromere (see the centromeric regions of all 16 yeast chromosomes in Fig. S2). Y‐axis shows enrichment values (linear scale, range is 0-10). Values below 1.5 are shown in grey, and values above 1.5 (i.e. sequences enriched in ChIP samples) are red coloured.E: Cells with the indicated genotypes were synchronised in G1 with α-factor at 25°C and then released in fresh medium at 34°C (t=0). Cells were collected at the indicated time points for FACS analysis of DNA contents.