EMBO/sd-character-level-ner huggingface dataset:
Figure 6A) Representative blood smears of patients with TUBB1 mutations (P1 and P3 homozygous for p.P160L, P4 heterozygous for p.Y106X, and P6 and P7 heterozygous for c.35delG) after May-Grünwald Giemsa staining. Variable platelet size in patients was observed with all three mutations with presence of macroplatelets (arrows) B) Transmission electron microscopy (TEM) analysis of platelet ultrastructure in the patients. The results confirm the abnormal platelet morphology C) Representative images of proplatelet formation in P1 and P4. Cultured control and patient megakaryocytes (MKs) after thrombopoietin-induced differentiation were spread over a BSA-coated coverslip on day 10. On day 13 or 14, MKs were fixed (4% paraformaldehyde) and proplatelet structure was observed after β-tubulin staining. Scale bars: 20 μm. Only P1 and P4 could be studied. The graphs show the mean ± SEM of the shaft thickness of proplatelet extensions (left) and the diameter of coiled elements (right) of the future proplatelets, both of which were significantly increased in the patients, indicating that the TUBB1 mutations (homozygous p.P160L and heterozygous p.Y106X) affected proplatelet morphology. Statistical significance was determined by one-way ANOVA, followed by Dunnett"s multiple comparisons test (*p<0.05, ***p<0.001; 20-60 MKs were analysed/pat
in human carcinomasA.Figure 8A. Representative micrographs of tissue sections derived from oral squamous cell carcinomas (OSCC) and esophageal squamous cell carcinomas (ESCC) stained with the indicated antibodies by immunohistochemistry (IHC). Top panels show routine H&E staining. Black squares point to regions magnified in the adjacent panels to the right. B. IHC staining of HSPA1 and HSPH1 was performed for the indicated numbers of normal, OSCC and ESCC tissues. Staining intensity was scored as described in Materials and Methods. C. Kaplan-Meier plots showing the correlation between HSPH1 mRNA expression level and the survival of patients with ESCC. The expression data was obtained from and visualized with KM Plotter (www.kmplot.com, (Nagy et al, 2018) using the Pan Cancer algorithm at default settings. D. KYSE150 and KYSE150HSPH1-/- cells were injected into nude mice, and xenograft tumor growth was monitored over time (left panel; mean tumor volume +/- SEM, n = 4 with 5 animals per n). Mean tumor weights at the end of the experiments were determined (means +/- SD, n = 4 with 5 animals per n, number above the bar graph represent p values, * p<0.05, **p<0.01 (calculated with the two-stage linear step-up procedure of Benjamini, Krieger and Yekutieli)). E. Macroscopic images of representative tumors derived from KYSE150 and KYSE150HSPH1-/- cells (shown are tumors from two of the four independent experiments).
Figure 1(A) RT-qPCR analysis of Myh7b/miR-499 and Myh7/miR-208b levels during differentiation of myoblasts into mature myotubes (n = 3 independent experiments). *p = 0.0005 (Myh7b), *p < 0.0001 (miR-499), *p = 0.0002 (Myh7), *p < 0.0001 (miR-208b). (B) Oxygen consumption rates (OCR) in primary myoblasts and differentiated myotubes. Basal OCR was first measured, followed by administration of 10 mM sodium pyruvate, and 2 μM oligomycin (to inhibit ATP synthase), uncoupler FCCP (2 μM), or rotenone/antimycin (Rot/A) (1 μM) as indicted. n = 3 separate experiments done with 10 biological replicates. *p < 0.0001 (FCCP). (C) Mean expression levels (RT-qPCR) in white vastus (WV) and soleus (Sol) muscle from 8-week-old male wild type mice (n = 5 mice per group). *p < 0.0001 (Myh7b), *p < 0.0001 (miR-499), *p < 0.0001 (Myh7), *p < 0.0001 (miR-208b). (D) Mitochondrial respiration rates were determined from mitochondria isolated from indicated muscle using pyruvate as substrate. ADP-dependent respiration, oligomycin-induced (oligo), uncoupler FCCP, and antimycin A (AA) are shown. n = 3 separate experiments done with 7-8 biological replicates. *p < 0.05 (ADP). (E) (Left) Schematic depicts the increments of speed over time. (Right) Respiratory exchange ratio (RER) during a graded exercise regimen as described in Materials and Methods (n = 5 mice per group). Notably, MCK-miR-499 mice were able to exercise at a higher speed before exhaustion. *p < 0.05. (F) Bars represent mean blood lactate levels for 3-month and 15-month old male MCK-miR-499 and NTG mice following a 25 min run on a motorized treadmill. For 3-month blood lactate, NTG, n = 9; MCK-miR-499, n = 10. For 15-month blood lactate, NTG, n = 7; MCK-miR-499, n = 5. *p = 0.039 (3-month), *p = 0.018 (15-month). (G) Mitochondrial respiration rates were determined from the plantaris muscle of the indicated genotypes using pyruvate/malate as substrate. Pyruvate/malate (Py/M)-stimulated, ADP-dependent respiration, oligomycin-induced (oligo) and the respiratory control ratio (RC) are shown. NTG, n = 6; MCK-miR-499, n = 4. *p = 0.015 (Py/M), *p = 0.017 (ADP), *p = 0.001 (Oligo).
Figure 1A-C. SPF C57BL/6Jmice were co-colonized with E1 and N1 wild type, (WT, A, n=5 mice) N1 T6SS mutant, (ΔtssC, B, n=4) or N1 complemented (ΔtssC pTssC, C, n=5). FecalCFU was quantified for E1 (closed squares) and N1 (open squares) weekly. D. Four weeks post-colonization, E1 fecal recovery was compared between the N1 WT, ΔtssC, and ΔtssC pTssC groups.
p62 bodies are degraded by autophagy.Figure 5.(A) Comparison of p62 degradation using pulse-chase labeling with 35S-methionine and immunoblotting after cycloheximide (CHX) treatment. HeLa cells were pulsed with 35S-methionine and incubated for the indicated times in nonradioactive medium. After immunopurification, the amount of radioactive p62 was determined by autoradiography, and the total amount of p62 was determined by an immunoblot of the same membrane. The p62 level in total cellular lysates after different times of 10 μg/ml cycloheximide treatment determined by immunoblotting (bottom). (B) The levels of p62 and LC3-II change with autophagic activity. HeLa or S-GFP-p62 cells were either left untreated or rapamycin (10 μg/ml) or bafilomycin A1 (Baf. A1; 10 μg/ml) was added for 18 h. Immunoblots were sequentially probed using LC3, p62, and actin antibodies. (C and D) The amount of p62 located to cytoplasmic bodies increases upon inhibition of autophagy. HeLa cells or S-GFP-p62 HeLa cells were left untreated or bafilomycin A1 was added for 18 h, the cells were fixed, and p62 was either stained red using a p62 mAb (C) or imaged directly (D). Nuclei were visualized using the Draq5 DNA stain. The settings for imaging were identical for the treated cells and the untreated control. (E) The majority of S-GFP-p62 cytoplasmic bodies are stained with antibodies recognizing transiently expressed LC3. S-GFP-p62 cells were transiently transfected with myc-LC3. Myc-LC3 was stained red using an anti-myc tag mAb. The boxed area indicates the part of the cell that is shown to the left at a higher magnification. Bars, 5 μm.
Figure 8Figure 8. Model of Akt-Inversin Interaction in Nephronophthisis type IIWe found that Akt interacts with and phosphorylates the ciliary proteinINVS, which is mutated in human NPHP2. Lack of phosphorylation of INVS leads to misalignment of the spindle axis and distorted luminal formation, underscoring the physiological significance of the Akt-INVS interaction in the development of abnormal cysts in NPHP2.
a) Thr172-phosphorylated (p-) and total AMPK-α subunit determined by immunoblotting.figf4(a) Thr172-phosphorylated (p-) and total AMPK-α subunit determined by immunoblotting. Resting or LPS-primed BMMs were untreated or stimulated with PA-BSA (0.5 mM) for 24 h. (b-e) Analysis of LPS-primed BMMs stimulated with PA-BSA (0.5 mM) in the absence or presence of AMPK agonist AICAR. (b) ROS production determined using the fluoroprobe DHR. (b-e) Analysis of LPS-primed BMMs stimulated with PA-BSA (0.5 mM) in the absence or presence of AMPK agonist AICAR. (c,d) IL-1β (c) and IL-6 (d) in supernatants, measured by ELISA. (b-e) Analysis of LPS-primed BMMs stimulated with PA-BSA (0.5 mM) in the absence or presence of AMPK agonist AICAR. (e) Immunoblotting for caspase-1, IL-1β and AMPK signaling molecules. (f) Transfection of BMMs with empty vector (EV) or constitutively active (CA) AMPK-α1 subunit. N, non-transfected. Efficiency was determined 16 h later by flow cytometric analysis of GFP expression in macrophages cotransfected with CA AMPK-α1 and pmaxGFP. Values are percentages of GFP-positive cells in the ranges indicated by the bars. (g) ELISA for IL-1β in supernatants of cells transfected as in f and stimulated with PA-BSA (0.5 mM) for 24 h. In each of a, b and f, one of two independent experiments is shown. In each of c-e and g, the results are representative of three independent experiments. Values are expressed as mean ± s.d. *P 0.05 versus controls.
Figure 4(A, B) Mature S. aureus biofilms were allowed to develop for 24 h in polystyrene plates and then treated for the indicated time intervals with cell suspensions, or culture supernatants of the CV2 or the CV2-DispB strains (A) or the WT or the WT-DispB strain (B). Biofilm presence was assessed by crystal violet staining and included a negative staining control (i.e., crystal violet without biofilm). The results are expressed as mean ± s.d. of OD 595 nm absorbance values obtained from three different biological replicates (n=3) and statistically compared by one-sided ANOVA followed by the post-hoc Fisher's PLSD test: *P ≤ 0.05; **P ≤ 0.005; ***P ≤ 0.0005; ****P ≤ 0.00005
Complex-centric detection of COP9 Signalosome variant CSN1/3/8.Figure 5For nearly half the CORUM complex hypotheses queried, two or more distinct subunit co-elution signals were detected (See methods and Appendix information on CCprofiler). Among the four distinct co-elution signals detected from the eight canonical CSN subunits' chromatograms (here with CSN7A, not CSN7B) are two distinct signals indicating distinct co-elution of two different complex variants. Distinct co-elution of holo-CSN (observed at the expected fraction 35) and Mini-CSN CSN1/3/8 (observed eluting offset only one fraction late, F45, of the expected fraction, F44). Expected fractions are estimated from the cumulative sum of one copy per component and external size calibration. Coloring adapted to highlight subversion components and their partitioning across holo- and sub-complex. CSN1/3/8 interact and form a submodule within the CSN holo-complex structure (PDB accession 4D10). The observations are consistent between the two whole workflow replicates (see Appendix Figure S6A). Lower right panel, validation of distinct elution behavior of holo-CSN exclusive (CSN7A) and shared subunit (CSN8) by immunoblotting. For full immunoblotting data (CSN1, CSN3, CSN4, CSN5, CSN7A and CSN8) see Appendix Figure S7C). CSN1/3/8 display distinct fractionation patterns in co-fractionation experiments performed in other laboratories, specifically in orthogonal ion exchange fractionation of HEK293 lysates (Wan et al. 2015, upper panels) and size exclusion chromatographic fractionation of U2OS lysates (Kirkwood et al. 2013, lower panel), in line with the CSN1/3/8 as distinct entity. Baculoviral co-expression of human CSN1, CSN3 and CSN8 in Sf21VM insect cells, with CSN8 N-terminally Strep(II)- and CSN1 & CSN3 N-terminally His6-tagged, followed by affinity purification and SDS-PAGE displays banding pattern in line with the formation of a stable trimer CSN1/3/8.
Figure 6A. Schematic of DNA repair involving generation of the fragments S1 (BspCNI-SmaI), S2 (BglII-BglII) and L (SalI-EcoRI) as products of SSA (see Figure 4A and 5A for more explanation). B. Comparative analysis of fragment S1, S2, and L production in SRS2 and srs2Δ cells during a time-course experiment (Combined data from Figs 4D and 5B). Blue two-ended arrow indicates a ~ 0.5 h time difference between the formation of fragments S2 and L in srs2Δ mutants. Since the distance between the BlgII site on the right and the EcoRI site is ~6 kb the rate of restoration of dsDNA in srs2Δ mutants can be roughly estimated at 12 kb/h. The rate of re-synthesis is much faster in wild-type cells as the difference between S2 and L is not even detectable at the 1 h time point. . Strains used: NK4691-4693; NK4805-4808.
Bak is shifted into the cytosol in parallel to Bax by retrotranslocation.Figure 3A Western blot analysis of GFP-Bak localization in cytosol (C) and heavy membrane fraction (HM) of HCT116 Bax/Bak DKO cells with or without overexpressed wild-type Bcl-xL or Bcl-xL G138A. GAPDH and Tom20 serve as fractionation controls. n = 10. Bak shuttling by Bcl-xL is independent of the GFP fusion (Fig. E2a).B Quantification of predominant mitochondrial localization (light grey bars) and mixed cytosolic and mitochondrial localization (dark grey bars) of Bak in HCT116 Bax/Bak DKO cells expressing GFP-Bak with or without Bcl-xL overexpression. Data represent averages ± SD from 3 independent experiments with n ≥ 100. C Retrotranslocation rates measured for wild-type Bak or Bak D83R in the presence or the absence of Bcl-2, Mcl-1, wild-type Bcl-xL or Bcl-xL G138A. Data represent averages ± SD. D Analysis of endogenous Bak localization in cytosol (C) and heavy membrane fraction (HM) of HeLa cells expressing degradation-prone DD-FLAG-Bcl-xL. In the absence of Shield-1 DD-FLAG-Bcl-xL is readily degraded, resulting in unaltered Bcl-xL levels. Addition of 0.5 µM Shield-1 instantly stabilizes DD-FLAG-Bcl-xL resulting in elevated Bcl-xL levels that increase cytosolic levels of endogenous Bak (Fig. E3C, D). Akt1 and Tom20 serve as fractionation controls. n = 3.
Figure 1(A) Summary and representative analysis of NET formation of pPMNs stimulated by 50 nM PMA for 3 h, detected by fluorescent microplate reader and confocal microscopy, respectively. Scale bar 20 µm. (B) Representative confocal microscopy images of pPMNs that were treated without (control) or with 50 nM PMA for 3 h, and stained for lamin B and DNA as described in panel B. The light blue arrows indicate the release of decondensed DNA associated with lamin B molecules from ruptured nuclear envelope. Scale bar 10 µm. (C) Schematic cross-section of a cell and portion of the nucleus and the nuclear envelope, as well as the ones with corresponding overexpression of lamin B. The two lipid bilayers of the nuclear envelope are the inner and outer nuclear membranes (INM and ONM, respectively). The meshwork, nuclear lamin B is anchored to INM through lamin B receptor (LBR). (D) Representative and summary of immunoblots of lamin B of bone marrow neutrophils from WT and Lmnb1TG mice. Vinculin served as loading control. (E) Summary analysis of PAF-induced NET formation in mPMNs from WT vs Lmnb1TG mice that were stimulated without or with 10 µM PAF for 3 h, then fixed by 2% PFA and stained by Sytox Green, following by detection with fluorescent microplate reader. (F) The endpoint analysis of NET-DNA release index was detected by coincubation of primary mouse peritoneal mPMNs from WT and Lmnb1TG mice without (control) or with 10 µM PAF in medium containing 1 μM Sytox Green dye with recording by a microplate reader at the 3 h time point. The NET-DNA release index was reported in comparison to an assigned value of 100% for the total DNA released by neutrophils lysed by 0.5% (v/v) Triton-X-100. (G,H) Representative images (G) and summary analysis (H) of PAF-induced NET formation in mPMNs from WT vs Lmnb1TG mice that were stimulated without or with 10 µM PAF for 3 h, and stained with both cell permeable SYTO Red and cell impermeable Sytox Green, without fixation. Images were taken by Olympus confocal microscopy, followed by automated quantification of NETs on 5-6 non-overlapping area per well using ImageJ for calculation of % cells with NET formation. (G) Scale bars, 40 μm.
Figure 7Left panel, in normal cells, both VPS4A and VPS4B act redundantly in several essential intracellular processes. So, a single depletion of any VPS4 paralog (e.g. VPS4A) is tolerated, as unperturbed expression of the other paralog alone (e.g. VPS4B) suffices to substitute for its downregulated counterpart. Right panel, cells that have lost VPS4B expression, e.g. due to oncogenic genome rearrangements, rely exclusively on VPS4A activity. So, inactivation of VPS4A in these cells leads to synthetic lethality that is accompanied by strong induction of an inflammatory response and release of immunogenic DAMPs. Immunomodulatory molecules released by dying VPS4A+B-deficient cancer cells can elicit paracrine effects on primary immune cells, e.g. reprogramming of macrophages toward the M1 anti-tumor phenotype.
Figure 1(A) Scheme showing the major cyclin-substrate interactions of Cdk1 complex. (B) Scheme showing the CDK phosphorylation sites and the potential Clb5 docking region in the disordered N terminus of Far1 (residues 1-150). (C) Co-immunoprecipitation of Cdc4 using either unphosphorylated or Clb5-Cdk1-phosphorylated Far1(85-150) as bait. The experiment was repeated twice, a representative example is shown. (D) Time-lapse fluorescence images showing the expression and degradation of wild-type and S87A S91A mutant Far1(1-150)-GFP and nuclear-cytoplasmic shuttling of Whi5-mCherry. Nuclear export of Whi5-mCherry takes place at 0 min (Start point) and was used to synchronize the cells for quantitative analysis. (E, F) Mean±SEM fluorescence intensities of the indicated Far1(1-150)-GFP mutants over the cell cycle. (G) Time from Start to degradation of 50% of the indicated Far1-GFP sensor in single cells. The bars show median ±95% confidence intervals. The numbers above the plot show median values. **** indicates p-value <0.0001, *** <0.001 and ns >0.05 for pair-wise comparisons with wild-type using Mann Whitney U test. (H) In vitro kinase assay showing the effect of N130A, L131A, L135A and L136A mutations on Far1(1-150)-GFP phosphorylation by Clb5-Cdk1. WT/mut shows the decrease in phosphorylation rate of the mutant compared to wild-type. The experiment was repeated twice, a representative example is shown.
Figure 2(A) Schematic depicting ER localization of EI24. TM, transmembrane domain; N-term, N-terminus; C-term, C-terminus. (B) EI24 persistently localizes on the ER regardless of the presence or absence of ER stress. HeLa cells stably expressing Flag-EI24 were treated with or without 4 μM tunicamycin (TM) for 2 hr. Colocalization of EI24 with ER was analyzed. KDEL-mCherry, ER marker. Scale bar, 10 μm. (C) Schematic depicting EI24 truncation mutants. FL, full length; △, deletion of the indicated regions. (D) The C-terminus of EI24 is required for IRE1 binding. HeLa cells transfected with the indicated EI24 mutants were treated as in (B), after which protein-protein interactions were analyzed by immunoprecipitation (IP). The star represents IgG light chains. (E) The C-terminus of EI24 binds to IRE1 in a persistent fashion. HeLa cells transfected with the indicated EI24 domains were treated as in (B). Protein-protein interactions were analyzed by IP. The star represents IgG light chains. ( (F) Expression of the EI24 C-terminus in EI24 knockout cells prevents TM-induced IRE1 activation. HeLa cells transfected with full-length EI24 or the C-terminus alone were treated with 4 μM TM for the indicated times. Protein expressions were analyzed by immunoblotting. (G) EI24 binds to the kinase domain of IRE1. HeLa cells transfected with the indicated IRE1 domains were analyzed for protein-protein interactions by IP. FL, full-length. (H) EI24 cannot bind to the phospho-mimicking (3SE) IRE1 mutant. HeLa cells transfected with wild-type HA-IRE1 or the 3SE mutant were treated as in (B). Protein-protein interactions were analyzed by IP. (I) EI24 overexpression cannot prevent the activation of the phospho-mimicking (3SE) IRE1 mutant. Wild-type and IRE1 3SE knock-in HeLa cells were transfected with empty vector or Flag-EI24. Transfected cells were treated as in (B). Protein expressions were analyzed by immunoblotting. (J The TM34 domain of EI24 is required for ER stress sensing. HeLa cells transfected with the indicated EI24 mutants were treated with TM as indicated, after which protein-protein interactions were analyzed by IP. The star represents IgG light chains. K) The TM34 domain of EI24 is required for ER stress sensing. HeLa cells transfected with the indicated EI24 mutants were treated with TM as indicated, after which protein-protein interactions were analyzed by IP. The star represents IgG light chains. (L) Expression of full-length EI24 or the △TM34 or △C mutant in EI24 knockout HeLa cells for comparison of their functions. HeLa cells transfected with the indicated EI24 mutants were treated as in (B). Protein expressions were analyzed by immunoblotting. (M) Schematic depicting ER stress sensing and IRE1 activation.
Delayed Parkin recruitment to mitochondria in MiroDKO cells upon mitochondrial damage.Figure 1(A) Representative images (and details from boxed regions) of WT MEF cells transfected with YFPParkin (green) and treated with FCCP (10 μM) to induce mitophagy. Cells were immunostained with anti-Tom20 (magenta) to reveal the mitochondrial network. Each row represents one stage in the process of mitophagy that has been used to score the progression of mitophagy at different time-points for the different conditions tested (the specific time-point of each example is indicated in the image). From top to bottom: i) "Diffuse Parkin" defines cells in where Parkin expression is homogenously distributed throughout the cytoplasm (quantified in B). ii) Soon after mitochondrial damage Parkin appears enriched in small "puncta" throughout the cytoplasm localizing within isolated mitochondria (quantified in C). iii) Later in the process these puncta structures aggregate in bigger structures accumulating in perinuclear regions (quantified in D). iv) The process of Parkin translocation eventually affects all the mitochondrial population and usually collapses in few large structures possibly forming part of big autolysosomes in the perinuclear region (quantified in D). Scoring was performed from 3 independent experiments (n=3; one way ANOVA with Dunnett post test). Error bars represent s.e.m. Significance: *p<0.05 and **p<0.01 (F) Representative images (and details from boxed regions) of WT MEFs cells transfected with GFPPINK1 (green) and treated with FCCP (10 μM) to induce mitophagy. Cells were immunostained with anti-Tom20 (magenta) to reveal the mitochondrial network. Each row represents one of the three categories of GFP signal used to describe PINK1 translocation to mitochondria after mitochondrial damage (cytoplasmic, intermediate and mitochondrial). The scoring of these categories was used to describe PINK1 translocation to mitochondria in WT and MiroDKO cells at different time-points shortly after mitochondrial insult (G). Data collected from 3 independent experiments (n=3). Error bars represent s.e.m. Significance: *p<0.05 and **p<0.01
M45 inhibits TLR- and IL-1R-mediated NF-κB but not p38 activation.Figure 1(A) NIH-3T3 cells were transduced with retroviral vectors expressing M45 or GFP. Two days after transduction cells were stimulated with the TLR4 agonist LPS (10 µg/ml), the TLR2 agonist LTA-SA (10 µg/ml), or IL-1β (20 ng/ml). IκBα levels were determined by immunoblotting. (B) NIH-3T3 cells were transduced with an M45-expressing or an empty retroviral vector. 30 min after stimulation with IL-1β (10 ng/ml) cells were fixed, and subcellular localization of the NF-κB p65 subunit was analyzed by immunofluorescence. (C) NIH-3T3 cells expressing an NF-κB-dependent secreted alkaline phosphatase (SEAP) reporter were transduced with retroviral vectors expressing M45 or GFP and stimulated 78 h later with the TLR2 agonist Pam3CSK4 (Pam.,1 µg/ml), the TLR4 agonist LPS (0.1 µg/ml), or IL-1β (5 ng/ml). SEAP activity in the supernatant was quantified 15 h after stimulation and is shown as fold induction of SEAP activity of stimulated cells compared to non-stimulated cells (mean ± SD) (D) NIH-3T3 cells transduced with retroviral vectors expressing M45 or GFP were stimulated with IL-1β (20 ng/ml, 15 min) or TNFα (10 ng/ml, 5 min). Phosphorylated and total p38 in cell lysates was detected by immunoblotting.