EMBO/sd-character-level-ner huggingface dataset:
Rab8a is required for autophagy‐activated IL‐1β secretion.Figure 3(A) Colocalization of Rab8a with the basal autophagic machinery factor LC3 and IL‐1β. Fluorescence; LC3 (green, Alexa488), IL‐1β (red, Alexa568), Rab8a (blue, Alexa633). BMMs from GFP-LC3 knock‐in mice were pretreated with LPS and analysed by immunofluorescence microscopy. Arrows indicate triple colocalization. (B) Line tracing analysis of fluorescence signal intensity. (C) Pearson's colocalization coefficient for IL‐1β and Rab8a. Pearson's coefficients were derived from three completely independent experiments with >5 fields per experiment, for a total of ⩾15 fields contributing to the cumulative result. (D) BMMs were transfected with siRNAs against Rab8a or scramble (Scr) control. At 24 h after the first transfection, cells were transfected again with siRNA, treated with LPS and the day after subjected to nigericin in full medium for 1 h, and IL‐1β secretion measured. (E) Immunoblot analysis of Rab8a knockdown in BMMs. (F) RAW 264.7 macrophages were transfected with GFP‐tagged Rab8a constructs (WT, wild type; S22N, dominant‐negative mutant), treated overnight with LPS and stimulated for 1 h with 20 μM nigericin along with induction of autophagy by starvation. IL‐1β secretion was measured by ELISA. Data represent mean values±s.d. (n≥3); *P0.05. Figure source data can be found in Supplementary data.
Figure 3Immunofluorescence labeling of GLUT1 (white), early endosomes (EEA1, green) and nuclei (blue) in HUVECs treated with VEGF-B186 for 10 min, 30 min or 2 h. Scale bar, 10 µm. GLUT1 protein expression in HUVECs treated with VEGF-B186 for 15 min or 2 h. Data presented as mean ± StDev of 3-4 independent experiments relative to β-Actin expression. Statistical evaluation using t-test revealed no significant changes. Western blots shown in right panel. Sucrose gradient (15-50% sucrose) fractions of HUVEC cellular lysates after 2 h VEGF-B186 stimulation were analyzed with Western blots shown in Appendix Fig S2. Different cellular sub-compartments were identified by accumulation of specific protein markers: EEA1 for early endosomes, LIMPII for late endosomes/lysosomes, flotillin for plasma membrane/lipid rafts and calnexin for endoplasmic reticulum (ER) (left panels). Amount and sub-cellular distribution of VEGFR1, NRP1 and GLUT1 protein in control or VEGF-B186 stimulated HUVECs shown in panels to the right. Data represent % protein relative to total protein of interest measured from a representative experiment. Quantification of GLUT1 protein in % relative to total measured protein in HUVECs after 2 h VEGF-B186 stimulation in different sucrose fractions (upper panel). Data from two independent experiments are shown. Green arrows mark fractions with increased GLUT1 content and red arrows mark fractions with decreased GLUT1 content after VEGF-B186 stimulation. Changes in GLUT1 content in response to VEGF-B treatment are shown in the table (bottom panel).
Luciferase sequestration as an assay for in vitro uptake.Figure 2Luciferase sequestration as an assay for in vitro uptake. (A) Luciferase cofractionating with vacuoles is proteinase K protected. In vitro uptake reactions of three times the standard volume were performed (60 min, 27°C) in the presence or absence of the ATP-regenerating system. The vacuoles were reisolated, washed, resuspended in 150 mM KCl in PS buffer to a concentration of 0.23 mg/ml, and split into aliquots. After addition of 1% Triton X-100 or buffer, the mixture was digested with the indicated concentrations of proteinase K (15 min, 0°C). Proteinase treatment was stopped by adding one volume of 0.5 mM PMSF in 150 mM KCl in PS buffer. Then, luciferase activity was assayed. (B) Luciferase can be released from the vacuolar lumen by freeze-thaw treatment. An uptake reaction was performed (60 min, 27°C). The vacuoles were reisolated and washed as described in the Materials and Methods section. The final pellet was resuspended in 90 μl PS buffer with 150 mM KCl and split into two aliquots. One was frozen at −80°C and thawed again slowly, whereas the other remained on ice. The samples were centrifuged (15 min, 145,000 g, 4°C). The supernatants (Sup) were recovered and the pellets were resuspended in an equal volume of PS with 150 mM KCl. Luciferase activity in both fractions was determined. The reaction tubes for this experiment had been coated with BSA (0.5 mg/ml, 15 min at room temperature) to reduce unspecific binding of luciferase to the surface.
ACE2 expression is upregulated in human bronchial epithelial cells (HBECs) after IFNβ and H1N1 influenza stimulation.Figure 7. ACE2 expression is upregulated in human bronchial epithelial cells (HBECs) after IFNβ and H1N1 influenza stimulation.HBECs were treated with IFNβ (1000U/ml) or wild type H1N1 influenza (A/PR/8/34). Control samples were incubated with fresh medium under the same conditions. HBECs were harvested at indicated time points (0.25, 0.5, 1, 1.5, 2, 4, 6, 8, 12, and 18 hours post-treatment). Total RNA was extracted and analyzed using the Affymetrix High Throughput Array. ACE2 expression data was retrieved from GEO microarray database (NCBI Accession: PRJNA121751; GEO: GSE19392).
Identification of γ-secretase cleavage sites in TREM2 wild-type and selected K186 variants.Figure 5(a) Schematic overview of the in vitro γ-secretase assay to determine the ε-cleavage site(s) of TREM2. (b) Immunoblot detection of ICD accumulation of TREM2 wildtype, K186A, K186P, and K186L variants mediated by γ-secretase cleavage upon overnight incubation of membrane preparations at 37 °C. ICD formation is blocked by 1 µM of the transition state analogue L-685,458. Note that inhibition of ICD formation from wt TREM2 was less efficient. Due to the charge within the TMH wt TREM2 is less efficiently transported to the cell surface (Lanier et al, 1998) and therefore retained within the endoplasmic reticulum, where other proteases not related to γ-secretase and therefore not inhabitable by L-685,458 may cleave. In vitro generation of the ICD of APP (AICD) was used as a positive control. Immunoblot detection of mature nicastrin and the presenilin 1 NTF, which results from presenilin endoproteolysis necessary to activate γ-secretase (Thinakaran et al, 1996), proves comparable amounts of γ-secretase in the individual assays. (c) MALDI MS analysis of the ε-cleavage sites of wt and mutant TREM2 C-terminal fragments. Mass spectrometry reveals ε-cleavage C-terminal of A192 for the wt CTF. MS determination of the ε-cleavage site of TREM2 CTF K186A reveals major and minor ε-cleavage sites C-terminal of A195 and L193, respectively. The exact same cleavage pattern is observed for the K186L mutant. MS analysis of the cleavage pattern of the K186P mutant reveals major and minor ε-cleavage sites C-terminal of L193 and S191. Minor peaks that could not be assigned are indicated by an asterisk. (d) Schematic representation of the ε-cleavage sites of CTF wildtype and selected K186 mutants. Residue 186 is indicated in bold. The location of helices is indicated by boxes. (e) Table listing the calculated and observed masses of both wildtype and mutant ICDs as determined by MALDI MS. Note that molecular weights are increased by 58 Da due to alkylation of C213. As a positive control, we ran immunoblots confirming reduction of APP ICD levels upon γ-secretase inhibition
Figure 6A, B, Representative images of scaf1-/- and scaf1+/+ females (A) and males (B) fed with the indicated diets. C, D, Length of females (C) and males (D) after the indicated diets (Δ1 +/+ n=5, Δ1 -/- n=5, Δ2 +/+ n=5, Δ2 -/- n=5). E, F, Weight of females (E) and males (F) after the indicated diets (Δ1 +/+ n=5, Δ1 -/- n=5, Δ2 +/+ n=5, Δ2 -/- n=5). G, number of eggs per clutch (standard diet: Δ1 +/+ n=4, Δ1 -/- n=3, Δ2 +/+ n=3, Δ2 -/- n=2, double diet: Δ1 +/+ n=5, Δ1 -/-, n=5 Δ2 +/+ n=1, Δ2 -/- n=2). H, Quantification of mature ovary follicles per ovary section in haematoxylin-eosin (H&E) histological sections (average of three sections/biological sample) (Δ1 n=3, Δ2 n=3). adipose tissue quantification in H&E sections (Δ1 n=3, Δ2 n=3) in females and males : adipose tissue area (I, J) adipose tissue quantification in H&E sections (Δ1 n=3, Δ2 n=3) in females and males adipocyte size (K, L).
Figure 2Cavities of TTC0263_TPR, PILF_TPR, CUT9_TPR, and PTPIP51_TPR. The cavities were calculated as solvent-accessible regions using the CASTp program. The surface properties of the PTPIP51_TPR structure. Hydrophilic and hydrophobic residues are shown in pink and yellow, respectively. Sliced views of PTPIP51_TPR showing the cavity and its hydrophobic property. The α8 helix is highlighted with a dotted line in the sliced view. Detailed view of the electron density map indicative of a tube-shaped molecule. Hydrophilic and hydrophobic amino acid residues adjacent to the electron density map are shown in pink and yellow, respectively. The mFo-DFc omit maps were generated using the program 'phenix.composite_omit_map' in the Phenix program suite. The contour level of the omit map is 0.8 σ.
Fig.Figure 2(A) A schematic drawing of GPR56 protein structure, with a N-terminal fragment (NTF), a seven transmembrane domain (7-TM) and a C-terminal fragment (CTF). (B) A diagram shows the hFc tag was added to the c-terminal of GPR56-NTF (NTF-hFC) or GAIN domain (GAIN-hFc). (C) A flow chart showing the experimental design of flow cytometry analysis. Briefly, Ba/F3 cells are treated with A23187 to externalize PS. For the binding assay, Alexa Fluor 647-conjugated hFc, GAIN-hFc, or NTF-hFc were incubated with PS-externalized Ba/F3 cells. For the competition assay, Alexa Fluor 647-conjugated hFc, GAIN-hFc, or NTF-hFc were used to compete with FITC-conjugated Annexin V binding. (D) The binding experiment using flow cytometry show that only the GAIN domain binds to PS, similar as Annexin V binding. (E) In the competition experiment, AF647 channel shows that GAIN domain binds 60.1% of the PS+ Ba/F3 cells (left). Correspondingly, FITC channel reveals 39.7% Annexin V binding to PS+ Ba/F3 cells (right). (F) A diagram of membrane lipid strips spotted with fifteen different lipids. DAG, diacylglycerol; PA, phosphatidic acid; PE, phosphatidylethanolamine; PC, phosphatidylcholine; PG, phosphatidylglycerol; PI, phosphatidylinositol. (G) Direct binding of hFc-tagged GPR56 GAIN proteins to PS and other phospholipids. HFc was used as a negative control, and GAS6 as a positive control.
Translational stress results in unfolded proteins.Figure 5A Representative fluorescence confocal images of live HeLa cells expressing Fluc-EGFP and FlucR188Q-EGFP maintained in SF compared to SF+q. Scale bars, 10 μm. Data information: SF (serum free medium), q (queuine) B Fluorescence intensity of aggregates was quantified as a fraction of fluorescence of the entire cell. The number of cells showing at least one aggregate is indicated. 10 cells were imaged for Fluc-EGFP and 20 cells for FlucR188Q-EGFP from 3 independent transfections; *p < 0.05 (t-test). Data information: S (standard medium), SF (serum free medium), q (queuine), AU (arbitrary units). C Specific luminescence activity of EGFP-tagged sensor proteins maintained in SF compared to SF+q. Luci­ferase activity was normalized to the luciferase protein content in the sample. Error bars: ±SD; *p < 0.05 (t-test); n = 8. Data information: SF (serum free medium), q (queuine)
Transplantable KRAS/TP53-mutant murine mesothelioma (KPM) cell lines.Figure 7A KPM cell culture showing anoikis (white arrows) and spindle-shaped morphology (black arrows). B Representative colonies of KPM1 cells (7.5 x 103 cells/vessel) seeded on a soft agar-containing 60 mm Petri dish and stained with crystal violet after a month (n = 3/group). C Data summaries from in vitro MTT reduction (top; 2 x 104 cells/well; n = 6 independent experiments) and in vivo subcutaneous tumor growth after injection of 106 cells per C57BL/6 mouse (bottom; n = 5/group). KRAS/Kras mRNA Sanger sequencing shows wild-type Kras (KrasWT) of PMC and mutant murine Kras/human KRAS alleles (KRASG12D and KrasG12C) of KPM and AE17 cells (arrows). E, F RT-PCR (E) and qPCR (F) of KPM cells and parental tumors show Trp53f/f allele deletion (Δ) and Bap1 and Cdkn2a overexpression compared with PMC.
Figure 2Sequence alignment of the MICU1 protein from nine vertebrate species. The Akt consensus phosphorylation motif, R-X-R-X-X-S/T, is marked in yellow HeLa cells treated with vehicle or 1 μM rapamycin for 4 h were stained for phosphorylated (S473) Akt (p-Akt) or HSP60 (mitochondrial marker). Merged images are indicated (merge). Scale bar 10 μm Analysis of the number of cells, expressed as a percentage, showing obvious mitochondrial staining of activated (S473 phosphorylated) Akt (p-Akt) (n=3 independent experiments; 350-355 cells) Hek293T cells treated with vehicle or 1 μM rapamycin (Rapa.) for 4 h were fractionated into Cytosol (Cyt.) or mitochondrial (Mito.) extracts and analyzed by western blotting. E-cadherin: plasma membrane marker; β-tubulin: cytosolic marker; VDAC: mitochondrial marker; Homo: cell homogenate Mouse livers treated with vehicle or 1 μM rapamycin (Rapa.) for 16 h by an intraperitoneal injection were fractionated into cytosol (Cyt.) or mitochondrial (Mito.) extracts and analyzed by western blotting. Phosphorylated (S2468) mTOR (p-mTOR) was used to assess the rapamycin activity. E-cadherin: plasma membrane marker; β-tubulin: cytosolic marker; VDAC: mitochondrial marker; Homo: cell homogenate Mitochondria isolated from Hek293T cells were subjected to the indicated treatments and analyzed by western blotting against Akt, phosphorylated (S473) Akt (p-Akt), and mitochondrial proteins with known localizations. Osmotic swelling through the removal of sucrose from the buffer was used to induce OMM rupture. OMM: outer mitochondrial membrane; IMS: intermembrane space Western blot analysis of the supernatant (S) and insoluble pellet (P) fractions of vehicle- or rapamycin (Rapa., 1 μM for 4 h)-treated Hek293T cell mitochondria following carbonate extraction at pH 11.5. Akt, phosphorylated (S473) Akt (p-Akt), MICU1, the established integral membrane proteins TIM23 and MCU, and the soluble protein cytochrome c (Cyt. c) was analyzed Hek293T cells were transfected with the GFP-tagged wild-type (WT) MICU1 and then treated with 1 μM rapamycin (Rapa.) alone for 4 h or in combination with the Akt inhibitor triciribine (10 μM). GFP-MICU1 immunocomplexes were precipitated with a GFP antibody and analyzed with PAS (phospho-Akt substrate) and phosphorylated (S473) Akt (p-Akt) antibodies by western blotting Hek293T cells were transfected with either the GFP-tagged wild-type (WT) MICU1 or MICU1 S124A-GFP mutant. MICU1-GFP immunocomplexes were precipitated with a GFP antibody and analyzed with PAS (phospho-Akt substrate) antibody by western blotting.
a–e) Primary (1st) and secondary (2nd) neurospheres from Ctrl and FIP200GFAP cKO mice at P0 and P28.figf3(a-e) Primary (1st) and secondary (2nd) neurospheres from Ctrl and FIP200GFAP cKO mice at P0 and P28. Representative P0 phase contrast images are shown in a. Mean ± s.e.m. of number (b,d) and size (c,e) of primary and secondary neurospheres from three independent experiments are shown (n = 3 mice for each). (f) Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) and DAPI staining of the SVZ and dentate gyrus (DG) from Ctrl and FIP200GFAP cKO mice at P28. Lines indicate the boundaries of the SVZ. Numbered boxed areas are shown in more detail as insets for staining of TUNEL and DAPI in the SVZ and DG. (g) Short-term BrdU incorporation in the SVZ and DG of Ctrl and FIP200GFAPcKO mice at P28. Dotted lines indicate the boundaries of the SVZ and granular zone (GZ). Boxed areas are shown in more detail in the insets. Lines indicate the boundaries of the SVZ and GZ. Arrows mark BrdU+ cells. CC, corpus callosum; LV, lateral ventricle; ST, striatum. *P 0.05; **P 0.01; ***P 0.001. (h) Long-term retention of BrdU in the SVZ and dentate gyrus of Ctrl and FIP200GFAP cKO mice at P31. Lines indicate the boundaries of the SVZ and GZ. Arrows mark BrdU+ cells. CC, corpus callosum; LV, lateral ventricle; ST, striatum. *P 0.05; **P 0.01; ***P 0.001.
Figure 6(A) Animals were implanted with recording electrodes in lateral geniculate nucleus (LGN), superior colliculus (SC), and visual cortex (V1) and stimulated with a flashing stroboscope. (B) Nissl-stained sections illustrating the location of the recording electrodes (arrows). dhc, dorsal hippocampal commissure; PAC, periaqueductal grey; Th, thalamus. (C) Examples of field potentials (averaged ≥ 20 times) evoked by flash stimulation in the recording sites for WT and HET mice. Dotted lines indicate the beginning of evoked field potentials. (D) Latency of evoked field potentials for WT (N=13) and HET (N=18) mice. (E) In a first series of experiments, animals [WT (N=13) and HET (N=18)] were trained to press a lever to obtain a pellet of food in an illuminated Skinner box with a fixed-ratio (1:1) schedule. After ten days of training, they were transferred to a light on/light off protocol where lever presses were only rewarded when a small light bulb located over the lever was switched on. Lever presses while the bulb was off were punished with a time penalty of ≤ 10 s during which the bulb would not turn on. (F,G) Mean days to criterion (F) and mean number of lever presses during the last two training sessions (G) for the fixed-ratio (1:1) paradigm. No significant differences were observed. (H) Performance of WT (N=13) and HET (N=18) mice in the light on/light off test. Criterion (dotted line) was that the animal had to press the lever more times during the small bulb light on period than during the light off one. WT mice performed better than HET animals and reached the criterion by the 6th session. The HET did not reached criterion for the duration of the test. While a linear fit to WT data increased significantly across training, the same linear fit to HET performance did not present any significant change. (I) The percentage of lever presses during the light on and light off periods for WT and HET mice was also different, because the WT group pressed the lever preferentially during the light period. Data were averaged from the 9th and 10th sessions. For statistical significance (*P<0.05; **P<0.01; ***P<0.001).
Figure 9Data in this study support the existence of a YAP-LATS2 negative feedback loop consisting of oncogene YAP and its upstream suppressor LATS2. Under normal physiological condition, the feedback loop is functional, activation of YAP leads to increased expression of LATS2, which may inactivate YAP via phosphorylating YAP and retain YAP in the cytoplasm, leading to the blockage of its proliferative action on hOSE cells (A). Under certain circumstance, YAP protein may be over-activated by the intrinsic and extrinsic stresses (e.g. YAP gene amplification). If the Hippo pathway is still functional and the YAP-LATS2 negative feedback is still exist in these cells, hyperactivated YAP may induce cellular senescence via high expression of LATS2 (B). However, if the Hippo pathway is dysfunctional and the YAP-LATS2 feedback loop is deficient, YAP may induce transformation and carcinogenesis of OSE cells (C).
FYCO1 colocalizes with Rab7 and decorates the limiting membranes of autophagosomes, LEs, and lysosomes.Figure 4.(A) HeLa cells transfected with the indicated constructs were imaged by confocal microscopy 24 h after transfection. Insets show an enlarged field of interest. (B-D) Immunoelectron micrographs of HeLa cells transfected with GFP-FYCO1 and stained with anti-GFP polyclonal antibody (10-nm gold particle).
Figure 1A Time course images of the hippocampal CA1 region of Siglechdtr/dtr mice after DT administration. Sections were stained with an anti-Iba1 antibody (green) and DAPI (cyan). Arrow: live microglia with normal nucleus. Double arrow: apoptotic microglia with pyknotic nucleus. Arrowhead: microglial debris with no nucleus. Scale bar, 50 µm. B Number of apoptotic microglia with pyknotic or fragmented nucleus (n = 5 animals per group, Kruskal-Wallis test with post hoc Dunn's test). C Number of live microglia with normal nucleus (n = 5 animals per group, one-way ANOVA with post hoc Tukey's test). D Number of pieces of microglial debris (Iba1+ spheres with a diameter > 2 µm and no nucleus) (n = 5 animals per group, Kruskal-Wallis test with post hoc Dunn's test). E A representative image of a survived microglial cell in the hippocampal CA1 region of Siglechdtr/dtr mice 2 days after DT administration. Sections were stained with an anti-Iba1 antibody (green) and DAPI (cyan). A 3D image (rightmost panel) was reconstructed using Imaris software. Arrow: live microglia. Arrowhead: microglial debris. Scale bar, 10 µm.
Figure 9BRAF-mutated melanoma cells adapt to BRAF/MEK inhibition by turning on a drug tolerant pathway that is initiated by collagen-rich environments interacting with cancer cell DDR. Clustered DDR activates the non-canonical NFκB2 (p52/RelB) pathway that is therapeutically targetable with clinically approved DDR inhibitor such as Imatinib or with pre-clinically tested NIK inhibitors.
Figure 9A) Heatmaps and cumulative distributions showing ZEB1 genomic distribution in the high-grade PDAC cell line PANC1. For comparison, the acetylation and methylation levels at the same regions are also shown in the low-grade PDAC cell line CFPAC1. B) PWMs identified by de novo motif discovery at the top 2,500 ZEB1-bound genomic regions. The MEME E-values of individual PWMs are indicated. C) Two representative snapshots showing (left) a ZEB1 peak upstream of the MIR200A/B cluster and immediately downstream of the ADAM8 gene (right).
Figure 2A BL-E5(+) cells were individually transduced with shRNA-directed against TET1 (TET1-sh1 and TET1-sh3), TET2 (TET2-sh4) or control shRNA. Knockdown efficiency was assessed through qRT-PCR relative to expression of the housekeeping gene RPS16. B 5hmC MedIP and qPCR for CD45 exon 5 DNA relative to input in control-transduced and TET1 or TET2 depleted BL-E5(+) cells. C qRT-PCR in shTET1, shTET2 and control-transduced BL-E5(+) cells, showing reduced detection of exon 5/6 junctions relative to a constitutive CD45 junction (exon 7/8) in TET-depleted cells. D Cell-surface staining for CD45RA (exon 4-containing isoforms), CD45RB (exon 5-containing isoforms) and total CD45 (pan) in TET1 or TET2 depleted BL-E5(+) cells. RNAi against either TET protein specifically reduced detection of exon-5 containing CD45 isoforms relative to control. E TET1 and TET2 RNAi in unmethylated BJAB cells. qRT-PCR for knockdown efficiency (top) and cell-surface staining for exon-5 containing CD45 isoforms (CD45RB, bottom) relative to control-transduced cells. F Large scale depletion of TET1 in BL-E5(+) cells. MedIP (left and center) and CTCF-ChIP (right) followed by qPCR with the indicated CD45 primers in shTET1 (red, throughout) and sh-control (gray, throughout) cells. MedIP showed decreased 5hmC and increased 5mC, whereas CTCF-ChIP indicated reduced binding to CD45 exon 5 in response to TET1 depletion, as compared to control-transduced cells. Rabbit Ig ChIP served as control.