EMBO/sd-character-level-ner huggingface dataset:
Figure 6PRKRIR, PCBP4 and TSC22D1 regulate expression of distinct set of genes in mesenchymal breast cancer cells. (A) Volcano plots showing significantly changing genes upon siRNA-mediated depletion of PRKRIR, PCBP4 and TSC22D1 as compared to NTC. The x-axis and y-axis show fold expression changes (log2 scale) and associated P-value (-log10 scale) respectively. The blue horizontal line marks the cut-off for genes considered significantly changing upon factor depletion. Red dots indicate genes previously implicated in a similar context (Supplementary Table 1). (B) Circle plots of genes highlighted in (A) (red) and all significantly changing genes (grey). (C) mRNA levels for representative genes highlighted in (A) for cells treated like in (Fig 5D) were measured by qRT-PCR relative to Ctcf and plotted on the y-axis. Mean and SEM is plotted from three independent biological replicates. (D-E) Venn diagrams showing the overlap of the genes differentially upregulated and down regulated following knockdown of the three factors mentioned in (A). F) Representative immunofluorescence images showing localization of Tsc22d1 in NMuMG cells following transient expression of Flag-HA tagged Tsc22d1 and detection using an anti-Flag antibody, Bar plot shows quantification of Tsc22d1 localization with respect to DAPI density within the nucleus. Y-axis shows the percentage of total fluorescence intensity for Tsc22d1 in DAPI dense and light regions. SEM is derived from independent biological replicates. (G) ChIP assay using anti-Flag antibody following expression of Flag-HA tagged TSC22D1 in MDA-MB-231 cells. Quantitative PCRs were performed for indicated gene promoters and enrichments are plotted on the y-axis as ratio of precipitated DNA (bound) to total input DNA and then further divided by the same obtained in the empty vector transfected cells. SEM is derived from independent biological replicates. (H) mRNA levels for PRKRIR, PCBP4, TSC22D1 in non-invasive (DCIS) (n=20) and invasive tumor (n=20) samples were measured by qRT PCRs relative to TBP and plotted on the y-axis. ZEB1 was used as an established positive control for invasive tumors. Plotted Mean ± SEM of three biological replicates. * p<0.05, ** p<0.01, *** p< 0.001, Student's t-test.
Figure 1Domain architecture of PTPIP51 and overall structure of PTPIP51_TPR. The transmembrane (TM), coiled-coil (CC), and tetratricopeptide repeat (TPR) domains are presented. Each TPR pair is marked with a different color: red, TPR1 (α1 and α2); orange, TPR2 (α3 and α4); yellow, TPR3 (α5 and α6); green, TPR4 (α7 and α9); blue, TPR5 (α10 and α11). Two α-helices that are not involved in a TPR (α8 and α12) are highlighted in light blue. The lower panel shows helical wheel projections for the PTPIP51_TPR structure showing one of the features typical of a multi-TPR protein: TPR pairs forming a superhelical twist. The diagram was drawn using NetWheels (http://lbqp.unb.br/NetWheels). Detailed views of the hydrophobic interactions of TPRs. There are two kinds of hydrophobic interactions, intra-TPR and inter-TPR. The hydrophobic residues associated with inter-TPR interactions are indicated with asterisks (*). Analytical ultracentrifugation (AUC) of PTPIP51_TPR and PTPIP51_ΔTM. The molecular weights of recombinant PTPIP51 calculated and estimated in AUC experiments are shown. All fusion tags were removed from the respective recombinant proteins for the AUC experiments. Topology diagrams of PTPIP51_TPR and other TPRs: TTC0263_TPR (PDB ID 2PL2), PILF_TPR (PDB ID 2FI7), and CUT9_TPR (PDB ID 2XPI). TPR pairs are presented in different color, which are identical to those in the topology diagrams. Electrostatic surface potentials of TTC0263_TPR, PILF_TPR, CUT9_TPR, and PTPIP51_TPR, showing two different views of the electrostatic surface potential of each TPR. The concave and convex regions of each TPR are shown. The electrostatic surface potentials were calculated using the APBS electrostatics plugin (https://www.poissonboltzmann.org/) at ±3 kT/e.
Both Atg8 subfamilies are required for autophagosome maturation.Figure 4(A) HeLa cells stably expressing YFP‐Atg5 were transfected with non‐targeting siRNA (control siRNA), a pool of LC3 siRNAs, or a pool of GABARAP/GATE‐16 siRNAs using DharmaFect reagent. Seventy‐two hours after transfection, the cells were incubated for 2 h in EBSS medium, fixed, and immunostained with anti‐Atg16 antibodies. Quantification of Atg5 and Atg16‐labelled puncta structures from three independent experiments is presented on the right panel. Scale bar: 20 μm. (B) HeLa cells stably expressing YFP‐Atg5 were treated as in (A) and monitored by live microscopy. Quantification of the lifespan of Atg5‐labelled puncta structures is presented. Mean±s.d. of three independent experiments is presented. *P0.05, **P0.001. (C) YFP‐Atg5 cells were transfected with siRNA pool and starved as in (A). Cryo sections of fixed cells were immunolabelled using anti‐GFP antibodies and analysed by TEM as described in 'Materials and methods'. Quantification of the Atg5‐labelled structures size is presented at the right panel. *P0.05, **P0.001.
a,b) Changes in HEK293T cell cytoplasmic (a) and mitochondrial (b) [Ca2+] in response to ionomycin (2.figf1(a,b) Changes in HEK293T cell cytoplasmic (a) and mitochondrial (b) [Ca2+] in response to ionomycin (2.5 μM) were simultaneously measured by fluo-4 and rhod-2 imaging, respectively. Each bar represents one target gene silenced with pooled siRNA. (c) qrtPCR of MCU, MCUR1 and MICU1 mRNA from mouse tissues (n = 3; mean ± s.e.m.). (d) qrtPCR of MCUR1 mRNA from HEK293T cell clones (n = 3; mean ± s.e.m.). KD, knockdown. Neg shRNA, negative shRNA. (e) qrtPCR of MCUR1 mRNA from HeLa cell clones and of rescued MCUR1 mRNA levels in shHe2 clone cells (n = 3; mean ± s.e.m.). The same lentiviral shRNAs were used to generate shHK4 and shHe1 and shHK5 and shHe2, respectively. (f) MCUR1 protein expression levels (top) and densitometric analysis (bottom left) (n = 3; ± s.e.m.). Bottom right, Flag-tagged MCUR1 protein expression in clone shHe2 cells reconstituted with shRNA-resistant MCUR1 cDNA plasmid. WB, western blot. (g,h) Representative images from movies of HEK293T negative shRNA or shHK5 cells showing cytosolic (green) and mitochondrial (red) [Ca2+] before, during and after ionomycin exposure. Scale bar, 20 μm. (i-p) Cytoplasmic (green) and mitochondrial matrix (red) [Ca2+] responses in HEK293T (i-l) cells challenged with ionomycin or histamine (100 μM), respectively. (n = 3). (i) Wild-type HEK293T cells. (j) Cells expressing negative shRNA. (k) Clone shHK5 (n = 4). (l) Quantification of peak rhod-2 fluorescence. **P0.01 (mean±s.e.m.). (i-p) Cytoplasmic (green) and mitochondrial matrix (red) [Ca2+] responses in HeLa (m-p) cells challenged with ionomycin or histamine (100 μM), respectively. (n = 3). (m) HeLa cells expressing negative shRNA. (n) Clone shHe2. (o) Clone shHe2 re-expressing MCUR1 (n = 3). (p) Quantification of peak rhod-2 fluorescence. *P0.05, **P0.01 (mean±s.e.m.). (q) [Ca2+]c and [Ca2+]m signals evoked by ATP (100 μM) and thapsigargin (Tg, 2 μM) were monitored simultaneously using fura-2/AM and mitopericam (ipcam490), respectively in control (left) and MCUR1-knockdown (right) HeLa cells. [Ca2+]c is calibrated in nanomolar concentrations (black), whereas mitopericam fluorescence is inversely normalized to baseline (F0/F; red). (r) Summary mean [Ca2+]c and [Ca2+]m peaks during ATP stimulation (negative shRNA n = 29; MCUR1 knockdown n = 36 cells). *P0.05 (mean±s.e.m.). (s) Increase in bath [Ca2+](Rfura2) and [Ca2+]m (Ripcam) signals in response to CaCl2 (1 μM) and InsP3 (IP3, 7.5 μM) addition in permeabilized cells. Uncropped images of blots/gels are shown in Supplementary Fig. S6.
Figure 1Family pedigrees for CSF-1R variants ΔA781_N783 and P824R, with Sanger sequencing confirmations for each variant below. Cystic encephalomalacia evident in the right inferior frontal lobe secondary to a historical intraparenchymal haemorrhage. Extensive atrophy evident in frontal lobe. Secondary de-myelination observed with Luxol-Fast Blue stain. Wallerian degeneration observed with spheroids apparent following phosphorylated neurofilament staining. Profound cerebral amyloid angiopathy (CAA), involving meningeal and intraparenchymal blood vessels. Extensive phospho-tau staining displaying a perivascular pattern of deposition.
Figure 3A Relative activity of NF-kB luciferase reporter was measured in A549 cells transfected with plasmids encoding PB1-F2. The mRNA levels of pro-inflammatory cytokines were determined by semi-quantitative RT-PCR. All data are shown as mean (±SEM) from at least three independent experiments (*p < 0.05, **p <0.01, ***p <0.001). Cells were transfected with PB1-F2-expressing plasmids. Relative IFNβ mRNA levels were determined by semi-quantitative RT-PCR (top) and qPCR (bottom). All data are shown as mean (±SEM) from at least three independent experiments (*p < 0.05, **p <0.01, ***p <0.001). C U937 cells were co-transfected with the IFNβ luciferase reporter plasmid and the PB1-F2 or NS1 expressing plasmid and treated with polyI:C for 12 h before harvesting. IFNβ promoter activity was determined by luciferase reporter assay. All data are shown as mean (±SEM) from at least three independent experiments (*p < 0.05, **p <0.01, ***p <0.001). Cells were infected for 24 h with each virus at 1 MOI. The mRNA level of IFNβ was determined by semi-quantitative RT-PCR, and PB1-F2 protein expression was assessed by Western blotting. All data are shown as mean (±SEM) from at least three independent experiments (*p < 0.05, **p <0.01, ***p <0.001). E Cells were transfected with each plasmid encoding PB1-F2. 18 h after transfection, cells were treated with MG132 for 6 h and the level of IFNβ mRNA was determined by semi-quantitative RT-PCR (left) and qPCR (right). PB1-F2 protein expression was assessed by Western blotting. All data are shown as mean (±SEM) from at least three independent experiments (*p < 0.05, **p <0.01, ***p <0.001). F A549 cells were transfected with PB1-F2 clones mutated at positions 68 and 69; 24 h after transfection, the IFNβ mRNA level and PB1-F2 protein expression were determined by semi-quantitative RT-PCR and Western blotting, respectively (left). The relative expression level of IFNβ mRNA was also analyzed by qPCR (right). All data are shown as mean (±SEM) from at least three independent experiments (*p < 0.05, **p <0.01, ***p <0.001).
Molecular phenotyping of murine mesothelioma.Figure 6Wild-type (Wt), KRASG12D, and Trp53f/f mice were intercrossed, and all possible offspring genotypes received 5 x 108 PFU intrapleural or intratracheal Ad-Cre and were sacrificed when moribund. In parallel, C57BL/6 mice received ten consecutive weekly intraperitoneal injections of 1 g/Kg urethane and were sacrificed after six months. Data summary (heatmap) and representative images of immunoreactivity of tissue sections of pleural and pulmonary tissues and tumors from these mice for different markers of human malignant pleural mesothelioma (MPM) and lung adenocarcinoma (LUAD). n = 10 mice/group were analyzed for each marker. Brown color indicates immunoreactivity and blue color nuclear hematoxylin counterstaining. Note the ubiquitous strong expression of Wilms' tumor 1 (WT1), patchy moderate expression of vimentin (VIM), ubiquitous moderate expression of mesothelin (MSLN), ubiquitous strong expression of calretinin (CALB2), podoplanin (PDPN), and osteopontin (SPP1), patchy moderate expression of cytokeratin 5/6 (CK5/6), and the absence of expression of surfactant protein C (SFTPC) in murine KRAS-driven mesotheliomas. Note also the ubiquitous strong expression of WT1, the patchy moderate expression of VIM, the ubiquitous low-level expression of MSLN, the ubiquitous strong expression of CALB2 and SPP1, the ubiquitous low-level expression of PDPN, the variable moderate expression of CK5/6, and the ubiquitous moderate expression of SFTPC in murine KRASG12D-driven and urethane-induced LUAD.
a) p35 interacts with EndoB1 in a GST pulldown assay.figf1(a) p35 interacts with EndoB1 in a GST pulldown assay. (b,c) EndoB1 co-immunoprecipitates (IP) with p35 in COS-7 cell lysates expressing p35 and full-length EndoB1 (b) and adult rat brain lysate (c). (d) Putative Cdk5 phosphorylation site(s) in the EndoB1 sequence. Two proline-directed threonine residues (arrows) are present with a Cdk5 consensus site found at Thr 145 (underlined). (e) Dose-dependent phosphorylation of EndoB1 by Cdk5/p35 in an in vitro kinase assay. (f) Cdk5/p35 phosphorylates EndoB1 at Thr 145 in an in vitro kinase assay. (g) Cdk5/p35 phosphorylates EndoB1 at Thr 145 in COS-7 cells. (h) Cdk5 phosphorylates EndoB1 at Thr 145 in Cdk5+/+, but not Cdk5-/- mouse brains. Quantification of p-Thr145-EndoB1 level is shown in the right panel. Data are means ± s.e.m.; n=3. Uncropped images of blots are shown in Supplementary Fig. S8
Figure 6BMDMs from wild type, Traf3ip3-/- and Mavs-/- mice were treated with or without VSV, SeV, poly (I:C) or HSV-1 respectively as indicated for six hours. Ifnb (A), Ifna4 (B), Il6 induction was measured respectively by qPCR. All data are presented as the mean values based on three independent experiments, and error bars indicate s.d. P values were determined by unpaired two-tailed Student's t-test. *P<0.05 and **P<0.01. NS indicates no statistically significant difference. BMDMs from wild type, Traf3ip3-/- and Mavs-/- mice were treated with or without VSV, SeV, poly (I:C) or HSV-1 respectively as indicated for six hours. Il6 (C), Isg54 (D), and Cxcl10 (E) induction was measured respectively by qPCR. All data are presented as the mean values based on three independent experiments, and error bars indicate s.d. P values were determined by unpaired two-tailed Student's t-test. *P<0.05 and **P<0.01. NS indicates no statistically significant difference. F BMDMs from wild type, Traf3ip3-/- and Mavs-/- mice were infected with or without VSV for six hours. Cells were then collected and following subcellular fractionation, S5 fractions were subjected to native PAGE to examine IRF3 dimer, and P5 fractions contained mitochondria were subjected to SDD-AGE to examine MAVS aggregation.
PAP8 is essential for the PHYB-mediated light induction of photomorphogenesis.Figure 5A Phenotypes of given genotypes subjected to 5 days of illumination at 8 µmol.m-2.s-1 660-nm red light. PBG, pCaMV35S::PHYB-GFP transformed in pap8-1/+ (among 25 lines selected for GFP expression see data source; 2 doubly heterozygous pap8-1/+; PBG/- lines #6 and #7 segregated the photobodies alteration with the albinism). B Hypocotyl length, given as the mean ± SD, of plants grown as in (A) (R8, grey bars) or at 30 µmol.m-2.s-1 660-nm red light (R30, pink bars) showing partial insensitivity of pap8-1 to the PBG overexpression. Measurements are given in source data; in the order of the graph n equals (50, 133, 58, 43, 36, 112, 25, 60) δ-test (comparison of the mean) R8: δPBG/PBGp8-1= 20.38 corresponding to p-value < 10-31 δwt/p8-1= 0.5 < 1.96 not significant at α set to 0.05). Nuclear accumulation of PBG observed under GFP excitation in the given genotypes. (C,D) Epi-fluorescence microscopy. (E,F) Confocal microscopy showing the size of the nuclear bodies. Scale bars equal 5 µm. (G) Box plot (Min, 1st quartile, median as the central band, 3rd quartile, max) on the diameter of the nuclear bodies (NBs); n equals the number of records. H Immuno-blots using a GFP antibody or a PAP8 antibody showing respectively the levels of PHYB-GFP and PAP8 in the given genotypes grown in the dark for 3 days or in light; n.a., not applicable as the pap8-1 mutant can only be visually distinguished from wild type after light exposure; 2 lines (L#06 and L#07) for PBG/pap8-1 were tested. Coomassie blue staining presented as loading; signals were quantified using ImageJ. I RT-qPCR analysis on wild type, pap8-1, PBG and PBG pap8-1. Seedlings were grown in the dark (D) or under white light (L, 30 µmol.m-2.s-1); levels of transcripts are given relative to EF1α; error bars correspond to standard errors on technical triplicates and the dark sample is the wild-type PBG line. δ-test (comparison of the mean) ***, P < 10-72. J Immuno-blots showing the levels of PIF1, PIF3, HY5 in given genotypes grown in the Dark or Light condition as noted: p5/+, mix of an heterozygous pap5-2 siblings progeny undistinguishable from wild type; p5-2, pap5-2 and pifq, quadruple pif1-1 pif3-3 pif4-2 pif5-3 mutant; Histone H3 (H3), RbcL and PAP8 were used as controls; n.a., not applicable.
Figure 4(A) Intravital microscopy of elimination of CDFDA, a fluorescent substrate subject to excretory elimination by hepatocytes via Mrp-2, allows direct visualization of excretory function in sham or septic (PCI) animals treated with vehicle or a hepatocyte-directed inhibitor of PI3Kγ (T-LipoAS). (B) Normalized CDFDA intensities quantified from intravital images depict plasma disappearance curves (A, B) Representative intravital images and analysis (mean ±SD) from 5 (sham), 4 (PCI T-LipoAS) and 3 (PCI Veh) animals. Sham and PCI animals treated with T-LipoAS showed a classical biphasic decay curve, while PCI vehicles showed a relatively linear decay and a higher area under the curve indicative of excretory liver failure. (C) Similar results were obtained when the liver excretory function was tested by indocyanine green clearance. Indocyanine green was detected non-invasively by multispectral optoacoustic tomography. The ability to clear ICG was quantitatively analyzed by comparing areas under the curve (AUC). Groups of 9 (sham vehicle), 8 (sham LipoAS), 12 (PCI vehicle) and 15 (PCI LipoAS) animals were analyzed. Bars depict mean + SD. (D) Representative transmission electron micrographs from bile canaliculi of healthy control (sham) and septic animals (PCI) treated with vehicle or the PI3Kγ inhibitors, free AS, or T-LipoAS. The asterisk marks a bile canaliculus where typical brush borders are lost due to sepsis.
Figure 3A,B FRMD6 expression is strongly induced and CDX2 expression is downregulated both in (A) normal and (B) TA organoid cultures following TGFβ stimulation. C The reduction in CDX2 levels in the TGFβ-treated condition is also observed on the protein level. D,E Gene expression profiles of TGFβ-treated samples show enrichment of genes highly expressed in (D) the mesenchymal CMS4 of CRC (log2FC > 2) and (E) SSA precursor lesions when compared to control samples.
Figure 3Concentrations of IFNβ, measured by ELISA, in the culturing media of MCF7 and T47D cells 48 hours after siRNA transfections. siNC: non-targeting siRNA as a negative control, siHN-1: siRNA sequence 1 for HNRNPC, siHN-2: siRNA sequence 2 for HNRNPC, siLMNA: siRNA for LMNA as another negative control. Each sample has 3 replicates. Error bars represent mean ± SD. The normal MCF7 (left) and T47D (right) cells were cultured in the media collected from the corresponding cells 48 hours after siRNA-mediated gene knock-down. Expressions of the ISGs in these normal cells were measured with qPCR. Each sample has 3 replicates. Error bars represent mean ± SD. Different doses of the IFNβ antibody were added to the media of the siRNA-transfected MCF7 (left) or T47D (right) cells right before the media were transferred to the wild type cells. The wild type MCF7 or T47D cells were then cultured in these media for 48 hours, and the expressions of ISGs were measured by qPCR. Each sample has 3 replicates. Error bars represent mean ± SD. Growth curves of the MCF7 (left) and T47D (right) cells cultured in the media collected from the siRNA-transfected cells. Each sample has 3 replicates. Error bars represent mean ± SD.
Figure 4(A) Jurkat E6.1 T cells were stimulated with 10 μg/ml anti-CD3 and 2 μg/ml anti-CD28 for the indicated times, the cell lysates were immunoprecipitated and immunoblotted as indicated. The relative intensity of the IP bands was quantified by densitometry and is shown below the graph. (B) HEK293T cells transfected with Flag-tagged TAB2 (open arrow) or TAB2-ub (solid arrow) were lysed and immunoprecipitated with anti-Flag, and then immunoblotted with the indicated antibodies. The asterisk indicates a non-specific band. The relative intensity of the IP bands was quantified by densitometry and is presented in the right. (C) HEK293T cells co-transfected with HA-tagged p38IP together with cMyc-tagged TAK1 (open arrow) or ub-TAK1 (solid arrow) were lysed and immunoprecipitated with anti-cMyc, and then immunoblotted with the indicated antibodies. Asterisk indicates non-specific bands. The relative intensity of the IP bands was quantified by densitometry and is presented in the right. (D) HEK293T cells co-transfected with HA-tagged p38IP, Flag-tagged TAB2-ub, together with cMyc-tagged TAK1 (open arrow) or ub-TAK1 (solid arrow), were lysed and immunoprecipitated with anti-cMyc, and then immunoblotted with the indicated antibodies. The relative intensity of the IP bands was quantified by densitometry and is presented in the right. (E) Schematic of dynamic association between p38IP and TAK1 after stimulation.
Activation of htlACT but not InRwt and EgfrACT induces cortex glial overgrowthA-D') Representative images showing that pan-glial overexpression of htlACT, but not InRwt or EgfrACT causes an expansion of cortex glia that enwraps NBs.Figure 4A-D') Representative images showing that pan-glial overexpression of htlACT, but not InRwt or EgfrACT causes an expansion of cortex glia that enwraps NBs. Glial cells are marked with repo-GAL4 >GFP, and NBs are marked with Mira. (A', B', C' and D') are zoomed in images of (A, B, C and D), respectively. Representative images showing that cortex glial overexpression of htlACT but not InRwt or EgfrACT causes an increase in cortex glial membrane size (H) (n=10, 10 brain lobes) NP2222-GAL4>mGFP is used to mark cortex glial membrane in (E-G) Representative images showing that cortex glial overexpression of EgfrACT to mark cortex glial membrane wrapper-GAL4> is used in (I, J). cortex glial overexpression of htlACT but not InRwt or EgfrACT causes an increase in glial cell (Repo+) numbers quantified in (K) (n=10, 10, 10; 7, 12 brain lobes) L) Glial (repo-GAL4>) overexpression of InRwt but not htlACT or EgfrACT significantly reduces the number of CB NBs (n= 10, 10, 8, 11 brain lobes). M) Glial (repo-GAL4>) overexpression of htlACT but not InRwt or EgfrACT significantly reduces the pH3 index of CB NBs