EMBO/sd-character-level-ner huggingface dataset:
PI3Kα inhibition regulates selective PI-3,4,5-P3 species.Figure 3A-B A, Murine pancreatic tumour cells R211 were treated for 15 min with the vehicle, α-specific A66 or pan-PI3K inhibitor BKM120 at 0.01, 0.05, 0.1, 0.5, 1 or 5 µM in the presence of 10% FBS and protein levels of P-Akt (Ser473), total Akt and β Actin were observed by western blot. B, P-Akt on Ser473 was quantified and normalised with β Actin. n = 3 in each group. R211 cells were treated with the vehicle, α-specific inhibitor A66 or pan-PI3K inhibitor BKM120 at 1 µM and C, living cells were quantified after 3 days with a MTT colorimetric assay R211 cells were treated with the vehicle, α-specific inhibitor A66 or pan-PI3K inhibitor BKM120 at 1 µM D, migrating cells were quantified after 24h in a Boyden chamber assay and compared to vehicle (DMSO). n = 3 in each group. E, Murine pancreatic tumour cells R211 were treated for 15 min with the vehicle (0.01% DMSO) or α-specific A66 or pan-PI3K inhibitor BKM120 at 1µM. Phospholipids were extracted, total PIP, PIP2 and PIP3 were quantified and compared to the vehicle (DMSO). n = 3 in each group. F, the proportion of each PIP3 sub-type was represented. Sub-types whose proportion was modified compared to DMSO were underlined. n = 3 in each group.
Figure 8A-D, Exemplary virtual sections through EM tomograms for stimulatory condition at 36°C and 39oC. Scale bar 100nm. E, Illustration depicting the tomogram analysis (not drawn to scale). Ribbon-associated synaptic vesicles (SVs, green) in the first row ≤80 nm from ribbon (R) and membrane-proximal vesicles (yellow) in ≤50 nm membrane-to-membrane distance from the plasma membrane (blue) and ≤100nm from each side of the presynaptic density. F-I, 3D reconstruction of the ribbon-associated pool of vesicles (front view). Active zone membrane is depicted in blue, presynaptic density in pink, and ribbons in red, scale bars 100nm. F-I, 3D reconstruction of the membrane-proximal pool (top view) and J-M, the ribbon-associated pool of vesicles (front view). Active zone membrane is depicted in blue, presynaptic density in pink, and ribbons in red, scale bars 100nm.N-Q, Cumulative probability distribution of the diameter of round vesicles as well as the longest axis of flattened vesicles in the membrane-proximal pool (N-O) and ribbon-associated pool (P-Q). N-P, one way ANOVA followed by Tukey's test; Q, Kruskal-Wallis test. Round vesicles tested in both the pools appeared larger in OtofI515T/I515T IHC synapses but the long axis of flattened vesicles were comparable for both the genotypes (see also Table EV1).
Figure 6Comparison of changes in gene expression induced by Bhlhe40/Bhlhe41-deficiency in steady-state knockout mice (horizontal axis) and in mixed BM chimeras (vertical axis) as shown in Fig. 5 A, but filtered for the indicated signatures (no TPM filter applied). Closed symbols - genes with associated Bhlhe40 binding (as in Fig. 5 A) revealed by ChIP-seq. Open symbols - genes without Bhlhe40 binding. Inserts show Bhlhe40 ChIP-seq track for selected dysregulated genes. Flow cytometric comparison of expression of the indicated cell-surface markers (identified in panel A) on WT and DKO AMs. A narrow forward/side scatter gate was applied to correct for possible differences in autofluorescence. Representative results of two independent experiments with three and four mice per group.
Figure 7A-D Effects of LAMTOR1 KD and ML-SI1 treatment on TBS-induced LTP (A) or LFS-induced LTD (C) in CA1. B, D Means ± SEM of fEPSPs measured 40 min after TBS (B, n = 4-5 slices from 4-5 mice) or LFS (D, n = 4-8 slices from 4-8 mice) in different groups. Data information: Slopes of fEPSPs were normalized to the average values recorded during the first 10 min baseline Statistical significance was assessed by two-way ANOVA with Tukey's post-test *P < 0.05, **P < 0.01, ***P < 0.001 compared with shSc P < 0.05, ##P < 0.01, ###P < 0.001 compared with shLAMTOR1. Insets show representative traces of evoked fEPSPs before and 40 min after TBS/LFS. Scale bar 0.5 mV/10 ms. E Effects of ML-SI1 treatment on LFS-induced LTD in CA1 from 2-3 weeks old mice. Data information: Slopes of fEPSPs were normalized to the average values recorded during the first 10 min baseline Insets show representative traces of evoked fEPSPs before and 40 min after LFS. Scale bar 0.5 mV/10 ms. F Means ± SEM of fEPSPs measured 45 min after LFS in different groups. N = 6-8 slices from 6-8 mice. Data information: Statistical significance was assessed by Student's t-test *P < 0.05, **P < 0.01, ***P < 0.001 compared with shSc, Vehicle, or TAT, #P < 0.05, ##P < 0.01, ###P < 0.001 compared with shLAMTOR1. G-J Effects of TAT-2031 treatment on TBS-induced LTP (G) or LFS-induced LTD (I) in CA1. H, J Means ± SEM of fEPSPs measured 40 min after TBS (H, n = 6 slices from 6 mice) or LFS (J, n = 3-4 slices from 3-4 mice) in different groups. Data information: Slopes of fEPSPs were normalized to the average values recorded during the first 10 min baseline Statistical significance was assessed by Student's t-test *P < 0.05, **P < 0.01, ***P < 0.001 compared with shSc, Vehicle, or TAT Insets show representative traces of evoked fEPSPs before and 40 min after TBS/LFS. Scale bar 0.5 mV/10 ms. K Effects of LAMTOR1 KD and treatment with a calcineurin inhibitor, FK506, on LFS-induced LTD in CA1. L Means ± SEM of fEPSPs measured 45 min after LFS in different groups (n = 3-4 slices from 3-4 mice). Data information: Slopes of fEPSPs were normalized to the average values recorded during the first 10 min baseline Statistical significance was assessed by two-way ANOVA with Tukey's post-test *P < 0.05, **P < 0.01, ***P < 0.001 compared with shSc, Vehicle, or TAT, #P < 0.05, ##P < 0.01, ###P < 0.001 compared with shLAMTOR1. Insets show representative traces of evoked fEPSPs before and 40 min after LFS. Scale bar 0.5 mV/10 ms.
Figure 3(A) Representative colonies formed by Sirt7+/+, Sirt7-/- and Sirt7-TG bulge SCs that were puriﬁed from telogen-phase HFs and allowed to grow for 2 weeks. (B-C) Quantiﬁcations of colony number and size in (A); n = 3 mice per genotype. Immunoblot analysis of the indicated proteins in HFSC colonies from Sirt7+/+, Sirt7-/- and Sirt7-TG mice (D) proteins in HFSC colonies from Sirt7+/+, Sirt7-/- and Sirt7-TG mice Nfatc1 protein levels were quantified using Image J Plus 6.0 (E, n = 3 mice for each group). (F) Immunoblot analysis of the NFATc1 expression level in human keratinocyte HaCaT cells treated with SIRT7 siRNAs. (G) Immunoblot analysis of the NFATc1 expression in the presence of ectopic SIRT7 expression in HaCaT cells. (H) NFATc1 luciferase activity was monitored in HEK293T cells after the indicated treatments (n=3). The relative luciferase activity was determined by dual luciferase reporter assay. (I-J) IHC analyses of Nfatc1 expression in hair follicles of Sirt7f/f, Sirt7f/f ;K15-Cre and Sirt7-TG mice at the indicted time points. The black dashed line indicates the bulge (Bu) of hair follicle. (K-L) qPCR analysis of Nfatc1-targeted genes in HFSCs colonies cultured from Sirt7+/+, Sirt7-/-and Sirt7-TG mice, n=3 for each genotypes.
Figure 8A) Control and irradiated murine small intestines analysed with RNAscope in situ hybridisation for Myc mRNA (black arrows point to mRNA granules in the control) and by immunostaining for Sox9 protein reveals an expansion in the expression domain of both genes. Nuclear localisation of beta-catenin is readily detectable after irradiation. n = 3 samples per condition, quantification shown in the insets, error bars = 1 SD. Statistical analysis was performed with an unpaired student's t-test. Similar results were obtained at 3dpi and 5dpi. B) Q-PCR analysis of YAP, TEADs and their target genes Ctgf and Cyr61 over a 4 days post irradiation time course at 12Gy. Note the strong upregulation of TEAD4, Ctgf, and Cyr61. In contrast, Axin2, TEAD2 and Sox9 mRNA expression levels are only moderately altered, confirming that Wnt signalling remains active upon irradiation-induced YAP-TEAD activation. n = 4 biological replicates per time point. Statistical analysis was performed with unpaired t-test compared to the control 0Gy condition, * p<0.05, ** p<0.01, ***p<0.001. C) Control and irradiated murine small intestines analysed by immunostaining for Sox9 protein reveals that treatment with Porcupine inhibitor LGK974 strongly reduces the Sox9 expression domain induced by 3 days after 12Gy irradiation. Phosphorylated Src immunostaining reveals an upregulation by 3 days after irradiation which is also lost upon treatment with Porcupine inhibitor LGK974 (n= 4 animals in each condition). D) Q-PCR analysis of Wnt and YAP target gene expression in intestinal crypts isolated at 1 day after 12Gy irradiation. Note the inhibition of Axin2, Sox9, YAP, Ctgf, Cyr61, and TEAD family expression upon treatment with Porcupine inhibitor LGK974. The mild reduction of Sox9 mRNA expression by LGK974 at 1 day after irradiation (1dpi) becomes more pronounced at 3 days after irradiation (3dpi). n = 4 biological replicates per condition, statistical analysis was performed with Student's 2-tailed t-test, error bars = 1 SD, * p<0.05, ** p<0.01, ***p<0.001.
Schematic illustration for (A) the synthesis of PC polymer, and (B) the preparation of pcMNP.Scheme1 Schematic illustration for (A) the synthesis of PC polymer, and (B) the preparation of pcMNP.
Brain metastases are positive for HSP90.Figure 3A-C Immunofluorescence against HSP90 in mouse brains with established metastases. (A) HSP90 positive structures in areas not affected by the metastasis includes the medial habenula, where neurons co-localize with the chaperone. Scale bars: 100 µm (low magnification), 50 µm (medial habenula nucleus), 12 µm (high magnification neurons). (B) Established metastases from different primary origins and oncogenomic profiles stained with HSP90. Dotted lines delineate the metastasis (cc: cancer cells). Scale bars: 75 µm. (C) Iba1 colocalizes with HSP90 within areas affected by metastases. BB: bisbenzamide. Scale bar: 75 µm (low magnification), 12 µm (high magnification). D Immunohistochemistry against HSP90 was performed in human brain metastases (n=60) from lung (40 cases) and breast cancer (20 cases). E Representative human brain metastases showing different intensities or scores for HSP90. Scale bar: 50 µm. F Quantification of HSP90 in human brain metastases. 59 out of 60 (98%) showed positive staining of HSP90 in the tumor, 15 (25%) scored with 3 (strong), 36 (60%) with 2 (moderate) and 8 (13%) with 1 (weak) according to the signal intensity of HSP90 in the cytoplasm of cancer cells. G Human brain metastases (n=30) and their matched primary tumors (n=28 lung and n=2 breast) were evaluated and compared for HSP90 expression by immunohistochemistry. H Quantification of HSP90 in human primary tumors. 29 out of 30 (97%) showed positive staining of HSP90 in the tumor, 6 (20%) scored with 3 (strong), 10 (34%) with 2 (moderate) and 13 (43%) with 1 (weak) according to the signal intensity of HSP90 in the cytoplasm of cancer cells. I Schema showing HSP90 scores in matched pairs of primary tumor and brain metastasis. Red: increase of HSP90 score from primary to brain metastasis; green: decrease of HSP90 score; gray: no changes in HSP90 score. Representative human brain metastases showing different percentages of nuclear HSP90. Scale bars: (J) 50 µm Black arrows point to cancer cells positive for HSP90 in the nucleus. Representative human brain metastases showing different percentages of nuclear HSP90. Scale bars: (K) low magnification: 100 µm; high magnification: 10 µm. L Quantification of nuclear HSP90 in human brain metastases. 54 out of 60 samples (90%) showed positive nuclear HSP90 in the tumor. 27 (45%) showed 1-5% (moderate) and 27 (45%) showed >5% (high) of nuclear HSP90. M Quantification of nuclear HSP90 in human primary tumors. 19 out of 30 (63%) showed positive nuclear HSP90 in the tumor. 9 (30%) showed 1-5% (moderate) and 10 (33%) showed >5% (high) of nuclear HSP90.
Figure 6(A) Overlay of right and left wings of a yki2-only-A female showing fluctuating asymmetry. (B) Quantification of fluctuating asymmetry index FA10 in female flies of the indicated genotypes (F-tests: * p-value<0.05; ** p-value<0.01; *** p-value<0.001; **** p-value<0.0001). (C) Distribution of wing size and wing asymmetry among the flies analyzed in panel B. Each circle represents the area of a single wing. Each wing was measured in duplicate. The difference between Right (orange) and Left (blue) sides of a single fly is schematized by a black line.
Figure 5(A) Schematic diagrams showing the NLRP3 constructs used in 7B. (B) 293T cells were co-transfected with expression plasmids encoding for the indicated V5-tagged domains of NLRP3 for 24h. Whole cell lysate (left blot) and flag specific IP (right blot) were analyzed by immunoblotting. Equal loading was confirmed by blotting against tubulin. Representative blots of two independent experiments are shown. (C) 293T cells were co‑transfected with expression plasmids encoding Flag-NLRP3, V5‑PB1-F2 and V5‑ASC for 24h. Whole cell lysate and flag specific IP were analyzed by immunoblotting for indicated antigens. Equal loading of whole cell lysate was assured by blotting against beta actin. Representative blot of two independent repeats is shown. (D) 293T cells were co-transfected with expression plasmids encoding for Flag-NLRP3, PB1-F2 or NEK7 or combinations thereof as indicated. Whole cell lysate and Flag IP were analyzed by immunoblotting. Equal loading was confirmed by blotting against beta actin. Representative blots of three independent experiments are shown. Right panel: Quantification of IP bands' intensity relative to their input. The mean ± standard deviation of three independent experiments is shown. Statistical analysis was performed by paired two‑tailed Student's t test (*p< 0.05). (E) As in D: 293T cells were co-transfected with expression plasmids encoding for Flag-NLRP3-LRR, PB1-F2 or NEK7 or combinations thereof as indicated. Whole cell lysate and NLRP3-LRR IP were analyzed by immunoblotting. Equal loading was confirmed by blotting against beta actin. Representative blots of three independent experiments are shown. (F) Quantification of IP bands' intensity relative to their input. The mean ± standard deviation of three independent experiments is shown. Statistical analysis was performed by paired two‑tailed Student's t test (*p< 0.05). G) As in D and E: 293T cells were co-transfected with expression plasmids encoding for Flag-NLRP3-LRR, PB1-F2 or V5-NLRP3-Pyrin or combinations thereof as indicated. Whole cell lysate and NLRP3-LRR IP were analyzed by immunoblotting. Equal loading was confirmed by blotting against tubulin. Representative blots of three independent experiments are shown.
Brain region-specific mRNA profiles indicate multicellular PD-related processes (A) Brain region origin of tissues: SN (blue), MTG (green) and AMG (red).Figure 1(A) Brain region origin of tissues: SN (blue), MTG (green) and AMG (red). (B) Non-supervised clustering heatmap plot indicates substantial clustering of transcripts from each tissue but not of transcripts from patients compared to controls. PD samples indicated in red. (C) PCA clustering of all RNA molecules in the SN control (green) and PD samples (blue). (D) qPCR validation of TH levels in SN samples of PD and controls, normalized to beta-actin mRNA. T-test p*=0.025, Data presented as mean ± SD. n=18 for CT and 24 for PD. (E) Top enriched GO pathways of DE genes in the SN of PD vs controls and the corresponding logP values, Wald test (DEseq2 analysis). (F,G) Venn diagrams demonstrating cell type-specific DE genes in the SN of PD vs control tissues, up/down arrows indicate gene groups that were up/down regulated. (H) Module-trait relationship of WGCNA analysis showing correlation and corrected P values (in brackets) for each gene module (indicated by colors) as related to the different external traits selected (disease condition, brain region, age and sex). Correlation test p-value calculated by WGCNA package. (I) Module membership vs. gene significance in selected modules as reflecting external sample traits: disease condition (reflected in the brown and red modules), brain region (the blue module) and sex (the cyan module), regression-based p-value calculated by WGCNA analysis. n=8 for Amygdala control, 15 for Amygdala PD, 8 for MTG control and 13 for MTG PD, 10 for SN control and 15 for SN PD. (J) Significant GO terms that emerged as associated with the blue, red and brown modules and the corresponding logP values, Fisher Exact P-Value.
Fibroblastic FAK inactivation prevents tumour metastasis without impacting tumour growth.Figure 3Tumour cells were syngeneically and orthotopically co-grafted with mouse FAK-WT or FAK-KD fibroblasts. Thirty-eight days after injection, mice were euthanized. (A) Primary tumour mass quantification. Values are means ± SEM from at least 7 mice per group. (B) Representative images from primary tumour stained for H&E, cytokeratin 19 (CK19), α-SMA, Sirius Red and polarized Sirius Red. Scale bar, 100 μm. (C) Quantification of fibrillar collagen I (Yellow-Orange) and III (Green) based on polarized Sirius Red images from B. Each dot represents a mouse (n=11 per group). IHC representative images (left) and quantification (right) the expression of CD31 (D) from primary tumour; scale bar, 100 μm. IHC representative images (left) and quantification (right) the expression of two hypoxia markers CaIX and HIF1α (E) from primary tumour; scale bar, 100 μm. (F) Representative images of H&E, CK19 and α-SMA IHC staining performed on lung sections. Arrow identified lung metastasis. (G) Quantification of lung metastasis size and number based on CK19 positive staining using NanoZoomer software. Values are means ± SEM of 6 mice per group.
Figure 4A. Bright-field microscopy images of WT, TP53KO and onco-RNF43/TP53KO human colon organoid lines grown in medium with high Wnt/Rspo (20% conditioned medium (CM)) or without Wnt/Rspo (20, 2 or 0.2% CM). Images were taken 6 days after splitting. Scale bars represent 1000 μm. Non-cystic, non-proliferative organoids are indicated with red asterisks. B. Bar plot showing the enrichment scores of significantly enriched MSigDB hallmark gene sets in onco-RNF43/TP53KO compared to TP53KO organoids (FDR < 0.05). C. Bright-field microscopy and fluorescence microscopy pictures of WT, TP53KO and onco-RNF43/TP53KO human colon organoid lines grown in two different media and transduced with the TOP-GFP reporter. Images were taken 6 days after splitting. Scale bars represent 1000 μm. D. Gene Set Enrichment Analysis of onco-RNF43/P53KO compared to TP53KO organoids in medium without Wnt/low Rspo (0.2%). Significantly changed intestinal cell-type gene sets from Haber et al., 2017() are shown (FDR < 0.05).
Figure 5Representative immunofluorescence images of HeLa cells transfected with siControl or siCyclin B2. After 36 h of transfection, cells were treated with nocodazole for 2 h. Then cells were fixed and co-stained for Mad2 (green) in D, ACA (red), and DNA (blue) Representative immunofluorescence images of HeLa cells co-transfected with siCyclin B2 plus GFP-tagged Cyclin B2-WT or Cyclin B2-4A. At 36 h post-transfection, cells were treated with nocodazole for 2 h. Then cells were fixed and co-stained for Mad2 (red) in DNA (blue) Scatter graphs illustrating kinetochore intensity of Mad2 ( cells treated as in A and B Bars represent the mean kinetochore intensity (±SD) normalized to the values of the siControl group Representative immunofluorescence images of HeLa cells transfected with siControl or siCyclin B2. After 36 h of transfection, cells were treated with nocodazole for 2 h. Then cells were fixed and co-stained fo Cdc20 (green) in D, ACA (red), and DNA (blue) E) Representative immunofluorescence images of HeLa cells co-transfected with siCyclin B2 plus GFP-tagged Cyclin B2-WT or Cyclin B2-4A. At 36 h post-transfection, cells were treated with nocodazole for 2 h. Then cells were fixed and co-stained fo Cdc20 (red) in E, and DNA (blue) Scatter graphs illustrating kinetochore intensity o Cdc20 (F, cells treated as in D and E) Bars represent the mean kinetochore intensity (±SD) normalized to the values of the siControl group (G) Bar graph illustrating the mitotic index in cells treated with low concentrations of nocodazole for 12 h (H) Immunoprecipitation assay performed with Cdc20 antibody. HeLa cells were transfected with siControl or siCyclin B2. At 36 h after transfection, cells were arrested in mitosis, collected, and lysed. Cell lysates were incubated with Protein A/G beads bound with Cdc20 antibody. Following extensive washes, immunoprecipitates were resolved by SAS-PAGE and analyzed by Western blotting using anti-Cdc20, anti-BubR1, anti-Bub3, and anti-Mad2 antibodies
Figure 4Representative immunostaining of Arl13b (red) and γ-tubulin (green) in the P14 mouse cortical layers. Nuclei were stained with DAPI (blue). Scale bar 10 µm. Primary cilium length was measured in the cortex of Mecp2 null mice Average cilium length was measured in the cortex (mean ± S.E., n=4 WT and n=3 Mecp2 null) (B; *p<0.05, by Mann-Whitney test) and for each cortical layer (C; ***p<0.001, **p<0.01, *p<0.05, by two-way ANOVA followed by Bonferroni post hoc test). In C, cilium length is analysed in separate cortical layers while in B, the values indicate the average of cilium lengths for each layer. Representative immunostaining of Arl13b (green) in the internal (IGL) and external (EGL) granular layers of Mecp2 null (n=3) and heterozygous (het) cerebella (n=3) at P7, and the corresponding WT (n=3 males and n=3 females). Nuclei were stained with DAPI (blue). Scale bar 5 µm. Primary cilium length was measured in the cerebellum of Mecp2 null and heterozygous mice. Histograms show the mean ± S.E. of cilium length (n=3 males WT, n=3 females WT, n=3 Mecp2 null and Mecp2 het; **p<0.01, ***p<0.001, by Mann-Whitney test) in Mecp2 null (E) and heterozygous (F) mice, compared to the corresponding WT. Gli1 protein levels were measured by Western blot in WT and Mecp2 null cerebellum (n=7 WT and n=6 Mecp2 null). Representative bands of Gli1 and α-tubulin are depicted beside the histogram, which shows the mean ± S.E of the percentage of Gli1 expression levels, compared to WT animals (*p<0.05, by Student's t test). The mRNA expression levels of Gli1 and its downstream genes (Cyclin D1 and D2) were analyzed in WT and Mecp2 null cerebella (n=8 WT and n=7 Mecp2 null). The graph shows the fold change of transcripts compared to WT (mean ± S.E., *p<0.05, by Student's t test).
Figure 8A. Ablation of OPA1 using CRISPR-Cas9 gene editing in Drp1-/- 293T cells was confirmed by Western blotting analysis. B. Confocal images of mitochondrial morphology in Drp1-/- (left panel) and Drp1/OPA1DKO 293T (right panel) cells. (Aggr/frag: aggregation/fragmentation of mitochondria; Bulb/tubular: bulb-like/tubular mitochondria). C. Confocal images of mitochondrial morphology in Drp1/OPA1DKO 293T cells treated with the indicated siRNAs, combinations of siRNAs or with Myc-hFis1 plasmid (lower three panels) compared to mitochondrial morphology in Drp1-/- 293T cells overexpressing Myc-hFis1 or not (upper panel). D. Percentages (mean ± s.e.m.) of cells with indicated mitochondrial morphologies in Drp1-/- 293T cells transfected with empty vector or Myc-hFis1 plasmid, and in Drp1/OPA1DKO 293T cells treated with the indicated siRNA, combinations of siRNA or Myc-hFis1 plasmid. The data were collected from three independent experiments. Total cell numbers (n) used for statistical analysis are indicated for each condition. E. Knockdown of the indicated proteins by siRNA in Drp1/OPA1DKO 293T cells was confirmed by Western blotting analysis.