EMBO/sd-character-level-ner huggingface dataset:
Ykt6 phosphorylation inhibits autophagy flux.Figure 2A) ykt6ts pho8∆60 cells containing an empty plasmid, GFP-Ykt6 or 3HA-Ykt6 on a centromeric plasmid and pho8∆60 cells containing an empty plasmid were grown at permissive temperature (23 ºC) to late exponential phase in selective medium. Cells were shifted to the restrictive temperature (37 °C) for 1 hour (light grey bars) and subsequently starved for 4 hours in SD-N medium at restrictive temperature (black bars). Pho8Δ60 alkaline phosphatase activity was measured in three independent biological experiments and the mean was plotted normalized to starved Pho8Δ60 alkaline phosphatase activity. Error bars represent the standard deviation. B) Trichloroacetic acid extracts were prepared from ykt6ts cells containing an empty plasmid, 3HA-Ykt6 wild type, 3HA-tagged Ykt6 mutant variants or atg1∆ and wild type cells as controls. Cells were grown at the permissive temperature (23 ºC) to late exponential phase in selective medium. Cells were shifted to the restrictive temperature (37 °C) for 1 hour preincubation to inactivate the endogenous Ykt6 temperature sensitive protein, and harvested (0) or further incubated at 37 ºC for an additional 4 hours (4). Samples were analyzed by anti-Ape1 and anti-HA Western blotting. One representative experiment out of three biological replicates is shown. Wild type or atg1∆ cells served as controls. Note that the temperature shift results in enhanced Ape1 processing. C) ykt6ts pho8∆60 cells containing an empty plasmid, 3HA-Ykt6 or 3HA-tagged Ykt6 mutant variants as indicated were analyzed as in A). D) ykt6ts pho8∆60 cells containing an empty plasmid, 3HA-Ykt6, or ADH1 promoter driven 3HA-Ykt6 wild type or 3HA-tagged Ykt6 mutant variants as indicated were grown and Pho8∆60 activity was monitored as described in A). E) Ykt6 levels were analyzed from the strains shown in D) and corresponding cell extracts were analyzed by anti-HA and anti-Pgk1 Western blotting.
Moderate CoQ deficiency in Coq9Q95X mice leads to impaired mitochondrial bioenergetics function.Figure 6A-C Mitochondrial CoQ9 levels from cerebrum (A), kidney (B) and skeletal muscle (C) of Coq9+/+ and Coq9Q95X males and females. n = 8 for each group. D-F CI+CIII activity in cerebrum (D), kidney (E) and skeletal muscle (F) of male and female Coq9+/+ and Coq9Q95X mice. n = 6 for each group.G-I CII+CIII activity in cerebrum (G), kidney (H) and skeletal muscle (I) of male and female Coq9+/+ and Coq9Q95X mice. n = 6 for each group. J-L Blue-native gel electrophoresis (BNGE) followed by immunoblotting analysis of mitochondrial supercomplexes from Coq9+/+ (n = 3) and Coq9Q95X mice (n = 4) at 3 months of age.
Figure 5A. At P14, afferent terminals below IHCs are slightly swollen in the mutants (neurofilament labelling in green, arrowheads to compare; CtBP2 labels ribbons and IHCnuclei in red). Scale bars: 10 μm. At P28,Neurofilament/CtBP2 labelling in the organ of Corti of 4 week old Wbp2-deficient mice and littermate controls shows severe swelling of IHCafferent terminals in the mutants, especially in the 24kHz region (yellow arrowheads). The pre-synaptic ribbons do not look as well aligned to the terminals in the mutants (white arrows). At this stage, we also observe swelling of OHCafferent terminals in the 24kHz region (empty arrowheads). Scale bars: 5 μm. ihc: IHCnucleus; p: pillar side; m: modiolar side.B. Counts of pre-synaptic ribbons per IHC in the 8, 18 and 24kHz regions, showing no difference between mutants and controls at P28. C. TEM of the organ of Corti performed at P28 showing swollen afferent terminals below inner and outer hair cells (arrowheads for comparisons between mutants and controls). Scale bars: 5 μm. D. Neurofilament/CtBP2 labelling shows swollen and retracting terminals (white arrows), especially in the 24kHz regions in the mutants at 8 weeks (arrows). Scale bars: 10μm.E. Counts of pre-synaptic ribbons in the 8, 18 and 24kHz regions, showing no difference in their number per IHC in the mutants compared to littermate controls at 8 weeks.
OPCML interacts with AXL.Figure 1 (A) Western blotting of AXL and OPCML protein levels in AXL-expressing, OPCML-null SKOV3 and PEA1 cell lines transduced with OPCML "O" or control Empty vector "E" (B) Mammalian 2-Hybrid assay between OPCML and AXL Data in (A) to (M) are representative of at least three experiments with graphs depicting means ± SEM s; *P < 0.05, ***P < 0.01, and ****P < 0.0001 by Student"s t tests (C) Western blotting of the OPCML-GST pull down. Input: 1/20 of SKOV3-Empty whole cell lysate (D) Western blotting of the anti-AXL immunoprecipitation. Input: 1/100 of SKOV3-OPCML whole cell lysate. IP: Immunoprecipitated protein, IB: Immunoblotted protein (E) Immunostaining of OPCML (red) and AXL (green) in SKOV3-OPCML and PEA1-OPCML cells. Scale bar = 10μm Data i M, (E) was performed once due to the limited numbers of primary cells;  Data in (A) to (M) are representative of at least three experiments with graphs depicting means ± SEM s; *P < 0.05, ***P < 0.01, and ****P < 0.0001 by Student"s t tests (G) FRET efficiency for AXL-OPCML interaction in SKOV3-Empty "E" and SKOV3-OPCML "O", using AXL-AXL FRET as a positive control. Cells were labeled with Anti-Axl rabbit (conjugated to donor probe) and Anti-OPCML mouse (conjugated to acceptor probe), or Anti-Axl mouse (conjugated to acceptor probe) and Anti-Axl rabbit (conjugated to donor probe) Data in (A) to (M) are representative of at least three experiments with graphs depicting means ± SE s; *P < 0.05, ***P < 0.01, and ****P < 0.0001 by Student"s t tests (H) PLA of AXL-OPCML (red) in SKOV3 and PEA1 cells transduced with empty vector "E" or OPCML "O". Scale bar = 50μm Primary ovarian tumour cells were characterised for AXL and OPCML levels by (I) Western blottin PAX8 was used to identify ovarian cancer cells Primary ovarian tumour cells were characterised for AXL and OPCML levels b (J) Immunostaining. PAX8 was used to identify ovarian cancer cells (K) Quantitation of AXL-OPCML PLA in primary cells Data in (A) to (M) are representative of at least three experiments with graphs depicting means ± SEM s; *P < 0.05, ***P < 0.01, and ****P < 0.0001 by Student"s t tests (L) Western blotting of membrane fractionation of SKOV3-Empty cells: total cell lysate "T", liquid-disordered soluble fraction "S", detergent resistant membrane fraction "R". Caveolin-1 is used as a marker of the R fraction and Calnexin is used as a marker for the S fraction, E: SKOV3-Empty, O: SKOV3-OPCML (M) Densitometry of AXL band intensities in (J) in S and R, normalized to AXL intensity in input. Data in (A) to (M) are representative of at least three experiments with graphs depicting means ± SE s; *P < 0.05, ***P < 0.01, and ****P < 0.0001 by Student"s t tests
K8 filaments extend in the ExE regionA-D.Figure 2A-D. Bright-field pictures of K8-eYFP (Left) and Z-projections of time series (Right) of live LB K8-eYFP embryos imaged using two-photon laser with time intervals between 20 and 30 min, focusing on regions corresponding to dashed white areas. A. Lateral view showing extension of K8 containing cables (white arrows) attached to the amnion and ExE wall (n=10). B. Anterior view showing chorion retraction at the Anterior Separation Point (white arrows) (n=6). C. Lateral and D. Posterior views showing allantois growth over time (n=12 and 3). Allantois cortex and mesenchyme are indicated with yellow and magenta arrows, respectively. E. Spectral images at high magnification of ExE region from fixed K8-eYFP LB/NP embryos (n=20). E: lateral view where white arrows point to allantois edge and red arrows to amnion. E': lateral view where blue arrow shows Anterior Separation Point and red arrow the amnion. E'': posterior view focused on allantois where yellow arrow indicates allantois cortex and magenta arrow the mesenchyme. K8-eYFP appears in yellow and DAPI in cyan. F. Immunostaining on sagittal section (anterior to the left) of a LB K8-eYFP embryo (n=20) with anti-eGFP antibody (yellow) focusing on ExE region (F); higher magnification images of allantois edge (F', yellow arrow) and base (F'', magenta arrow). G-G'. Immunostaining on sagittal section of a LB wild-type embryo (n=32) with anti-Krt8 antibody (white) (G); higher magnification acquisition centred on allantois (G') where yellow arrows indicate allantois cortex and magenta arrows point to allantois mesenchyme. Da
Proposed mechanism of action for ETO/CTH and SAMHD1 regulation ETO/CTH induced DNA damage appears to promote a p53/p21-dependent pathway.Figure 6 Proposed mechanism of action for ETO/CTH and SAMHD1 regulation ETO/CTH induced DNA damage appears to promote a p53/p21-dependent pathway. This is associated with cell cycle arrest, evidenced by decrease in MCM2 expression. Furthermore, CDK1 - responsible for SAMHD1 phosphorylation - is downregulated. As a result SAMHD1 is not phosphorylated and becomes active against HIV-1 infection. Dephosphorylated, active SAMHD1 induced by DNA damage appears to block 2LTR and provirus formation.
Figure 4A-C ROC analysis was performed to test the accuracy to discriminate between Aβ-positive (A+) from Aβ-negative (A-) individuals. Aβ positivity was defined as CSF Aβ42/40 < 0.071 (A), Aβ PET positive visual read (B) or Aβ PET Centiloid (CL) > 12 (C). Abbreviations: CSF, cerebrospinal fluid; Mid, mid-region; NfL, neurofilament light; N, N-terminal; p-tau, phosphorylated tau; t-tau, total tau.
Figure 4A. The interaction between overexpressed Merlin and LRP6 was reduced under serum starvation condition. HEK293T cells were transfected with Flag-Merlin and LRP6-EGFP. After transfection, cells were incubated in serum-free medium for overnight with Bafilomycin A1 (20 nM). The cell lysates were immunoprecipitated with anti-EGFP antibody and analyzed by immunoblotting with the indicated antibodies. WCL, whole-cell lysates. The interaction between endogenous Merlin and LRP6 was reduced under serum starvation. HEK293T cells were incubated in serum-free medium for 4 h with Bafilomycin A1 (100 nM). The cell lysates were immunoprecipitated with anti-LRP6 antibody and analyzed by immunoblotting with the indicated antibodies. WCL, whole-cell lysates. The interaction between endogenous Merlin and LRP6 was reduced under serum starvation. HEK293T cells were incubated in serum-free medium for 4 h with Bafilomycin A1 (100 nM). The cell lysates were immunoprecipitated with anti-Merlin antibody and analyzed by immunoblotting with the indicated antibodies. WCL, whole-cell lysates. D. The interaction between overexpressed Merlin and LATS1 was increased under serum starvation. HEK293T cells were transfected with Myc-LATS1 and EGFP-Merlin. After transfection, cells were incubated in serum-free medium for overnight and then were lysed. The cell lysates were immunoprecipitated with anti-EGFP antibody and analyzed by immunoblotting. WCL, whole-cell lysates. E. Knockdown of LRP6 enhanced the interaction between overexpressed Merlin and LATS1. HEK293T cells were transfected with Myc-LATS1, EGFP-Merlin and LRP6 siRNA. Cells were lysed and the cell lysates were immunoprecipitated with anti-EGFP antibody and analyzed by immunoblotting. WCL, whole-cell lysates. F. Knockdown of LRP6 enhances the interaction between endogenous Merlin and LATS1. HEK293T cells were transfected with siRNA for GFP or LRP6. After transfection, cells were lysed and the cell lysates were immunoprecipitated with anti-Merlin antibody and analyzed by immunoblotting. WCL, whole-cell lysates. G. Schematic summary of Figure 4 experimental results.
a) HeLa cells stably expressing GFP-VPS35 WT and D620N were treated with bafilomycin A1 or DMSO vehicle control.f4a) HeLa cells stably expressing GFP-VPS35 WT and D620N were treated with bafilomycin A1 or DMSO vehicle control. Endogenous LC3-II and tubulin levels were examined by western blot. A representative experiment of six experiments is shown. (b) Quantification of the representative experiment in triplicate shown in a, in which endogenous LC3-II levels are normalized to tubulin and expressed as a ratio of levels in WT. ***P=7.86 × 10−6 (DMSO) and 3.81 × 10−5 (Baf) by 2-tailed Student's t-test. c) HeLa cells stably expressing GFP-VPS35 WT and D620N were transfected with mRFP-LC3. The number of LC3 vesicles was quantified by Cellomics automated fluorescence microscopy. A representative experiment of three independent experiments is shown, with 344 (WT) and 401 (D620N) cells analysed. ***P0.0001 by 2-tailed Student's t-test. (d) GFP-VPS35 WT and D620N-expressing cells were transfected with HA-Q74 and immunostained for HA. The percentage of transfected cells with aggregates was counted by a blinded experimenter. The quantification shows the mean of three experiments in triplicate with minimum 200 cells per replicate. ***P=0.00086 by 1-tailed Student's t-test. (e) Confocal images representative of the experiment described in d. Scale bar, 20 μm. (f) Cells stably expressing WT and D620N GFP-VPS35 were transfected with GFP-α-synuclein A53T and GFP for 48 h and analyzed by western blotting. g) Quantification of the representative experiment in triplicate in f, in which the level of α-synuclein was expressed as a ratio to GFP. A representative experiment of two independent experiments is shown. **P=0.0047 by 2-tailed Student's t-test. (h) VPS35 was knocked down with two individual siRNA nucleotides in HeLa cells, and cells were treated with bafilomycin A1 and lysed as in a. A representative experiment is shown in triplicate (i) Quantification of three independent experiments in triplicate. *P=0.026; other results non-significant by 2-tailed Student's t-test. (j) Protein levels of CSC, including VPS35, VPS26 and VPS29), were assessed upon VPS35 knockdown, confirming previous results that knockdown of one component destabilizes the CSC. All error bars indicate s.e.m.
An inflamed intestinal environment induces cellular stress and caspase activation, thereby enhancing caspase-3-mediated cleavage of the T300A variant of ATG16L1.f6An inflamed intestinal environment induces cellular stress and caspase activation, thereby enhancing caspase-3-mediated cleavage of the T300A variant of ATG16L1. This results in defective stress-induced autophagy and bacterial clearance by xenophagy, establishing a chronic inflammatory state.
Figure 3Steady state levels of endogenous AK2 in presence and absence of MIA40. 72 h before experiments, HeLa cells were transfected with control siRNA or siRNAs directed against MIA40 or ALR. Cell lysates were analysed by immunoblot against the indicated proteins. MIA40 depletion led to a strong decrease in AK2 and ALR levels but not of the control proteins. ALR depletion did not affect AK2 levels. Reported values are the mean of 3 independent experiments; error bars represent ±SD. Student's t-test was performed. * represents p<0.05,. Stability of endogenous AK2 in the presence and absence of MIA40. 72 h before experiments HeLa, cells were transfected with control siRNA or siRNAs directed against MIA40. Cells were pulse labeled for 10 min with [35S]-methionine and chased with cold methionine for the indicated times. The chase was stopped by cell lysis, and AK2 was isolated by denaturing immunoprecipitation. Eluates were analyzed by SDS-PAGE and autoradiography. Stability of endogenous AK2 is significantly decreased upon depletion of MIA40. Reported values are the mean of 3 independent experiments; error bars represent ±SD. Student's t-test was performed; * represents p-value <0.05 compared to wild-type. Stability of cytosolic AK2-HA cysteine variant. Experiment was performed as in (B) except that cells expressing AK2C40S,C42S,C92S-HA were analysed by IP against HA. AK2C40S,C42S,C92S-HA is unstable but becomes stabilized by application of the proteasomal inhibitor MG132. Reported values are the mean of 3 independent experiments; error bars represent ±SD. Student's t-test was performed. * represents p<0.05,. Steady state levels of AK2-HA cysteine variants. AK2C40S,C42S,C92S-HA and AK2WT-HA were expressed in the presence or absence of proteasomal inhibitor MG132. Protein levels at steady state were analysed by immunoblot. All variants are stabilized by proteasomal inhibition. Reported values are the mean of 3 (AK2WT-HA) or 5 (AK2C40S,C42S,C92S-HA) independent experiments; error bars represent ±SD. ANOVA/Tukey's post-hoc test was performed. *** represents p<0.001. Steady state levels of AK2-HA cysteine variant. 72 h before experiments HEK293 cells were transfected with control siRNA or siRNAs directed against the proteasomal subunits PSMB3 and PSMC5. AK2C40S,C42S,C92S-HA was stabilized by decreased proteasome levels. Reported values are the mean of 4 independent experiments; error bars represent ±SD. Student's t-test was performed. * represents p<0.05.
Figure 4Box plot showing the quantitative protein expression results of nanoLC-MS/MS analysis reveals the significantly higher presence of Nid1 and Fbln2 in the Ndrg4-/- compared to the Ndrg4+/+ ENS cell secretome (n=4; NSAF, normalized spectral abundance factor). Data are analyzed with R version 3.5.2 and the ibb R package. Each dot within the box plot represents the NSAF of an individual sample; the inside band reflects the median, and the bottom and top of the box the first and third quartile, respectively. The whiskers reflect the minimum and maximal values within 1.5× the interquartile range. NSAF values were compared using the Mann-Whitney U test. Data derived from the Linnarsson mouse database (Linnarsson) displays the expression of Nid1 and Fbln2 within cells of the central, peripheral and enteric nervous system. Unit color scale: Expression (log(UMI)); UMI, unique molecular identifier. Abbreviations: ENS, enteric nervous system (i.e. enteric neurons and glial cells); OL, oligodendrocytes; Exc N, excitatory neurons in CNS; Hippo, hippocampus; MB, midbrain; HB, hindbrain; Str, striatum; Hyp, hypothalamus; BG, basal ganglia; OB, olfactory bulb; CB, cerebellum; SC, spinal cord; PNS, peripheral nervous system; Glia, glial cells; Vasc, vasculature. Representative immunohistochemistry (C) and more detailed immunofluorescence (D, E) labeling indicate that Nid1 (C, D) and Fbln2 (C, E) are expressed within the myenteric plexus and by primary ENS-cells ((C), black arrowheads, scale bars, 50 µm; (D, E), n=3, scale bars, 10 µm).
Model for the connection of HY5 to the GLK1 genetic pathways through the action of PAP8 during the dark-to-light transition.Figure 8The yellow spark represents the initial exposure of the seedling to light. Photo-initiation is mediated by PAP8 and PAP5 rapid accumulation in early photo-morphogenesis (φMG): the Pr state of phytochrome B (PHYBPr) is converted in the Pfr state (PHYBPfr), which enters the nucleus, accumulates in early photobodies (small green discs) that regroup in late photobodies (large green discs) while promoting the destabilization of PIFs (PIF1 and PIF3 in particular). The red arrow in the timeline represents the albino block observed in pap8-1 that may represent a key feature of the PAP syndrome. The dark induces ubiquitination and degradation of HY5Ub impeding recognition of a cis-regulatory element in the promoter of PAP8 (yellow box). Light induces transcriptional activation of PAP8 in palisade cells likely through the HY5 pathway (dashed yellow arrow on PAP8 promoter); this part of the model is supported by transient assays, in vitro experiments, and in vivo genome-wide ChIP sequencing data. The cTP pre-sequence of PAP8 allows plastid import and then PAP8 assembly within the PEP-PAP complex. Using an unknown trafficking route such as travelling across the plastid envelope (?), part of the processed PAP8 pool is found in the nucleus where its action could be necessary for the PHYB-mediated transcriptional activation of GLK1 directly through HY530 (dashed yellow arrow) or other light responsive factors (LR), which in turn can activate the Photosynthetic Associated Nuclear Genes (PhANGs) concomitantly to the (PEP-PAP)-driven expression of the Photosynthetic Associated Plastid Genes (PhAPGs) essential for the building of the photosynthetic apparatus (PS) in the functional chloroplast.
Figure 6A, PTPN3 and PTPN3 (D811A), but not PTPN3 (L232R), enhance Smad2 and Smad3 phosphorylation. HaCaT cells were stimulated with TGF-β (2 ng/mL) for 1 h. Levels of P-Smad2, P-Smad3, Smad2, Smad3, PTPN3 and GAPDH were detected by Western blotting. B, PTPN3 (L232R) inhibits TGF-β-induced reporter gene activity. HepG2 cells were transfected with expression plasmids for PTPN3, PTPN3 mutants and CAGA-luc reporter, and Renilla-luc reporter. Cells were harvested for luciferase assay after 8 h of TGF-β (2 ng/mL) treatment. Data are shown as mean ± SEM; n = 3. Statistical analysis was performed using two-tailed Student's t-test. *P<0.05, **P<0.01. C, PTPN3 and PTPN3 (D811A), but not PTPN3 (L232R), enhance TGF-β-induced growth inhibitory effect. HepG2 cells stably expressing PTPN3, PTPN3 (D811A) or PTPN3 (L232R) were treated with or without TGF-β (1 ng/mL) for indicated days. Cells were analyzed for cell proliferation by MTS assay. Data are shown as mean ± SEM; n = 3. D, Depletion of PTPN3 attenuates TGF-β-induced growth inhibitory effect. HepG2 cells stably expressing shPTPN3 were treated with or without TGF-β (1 ng/mL) for indicated days. Cells were analyzed for cell proliferation by using MTS assay. Data are shown as mean ± SEM; n = 3. E, PTPN3 (L232R) attenuates TGF-β-induced growth inhibition on colony formation in HepG2 cells. Cells were treated with or without TGF-β (1 ng/mL) treatment for 10 days. Clonogenic assay Quantification of the results in clonogenic assays was analyzed by Image J. Data are shown as mean ± SEM; n = 3. F, Depletion of PTPN3 attenuates TGF-β-induced growth inhibitory effect in HepG2 cells. Cells with stable knockdown of PTPN3 were treated with or without TGF-β (1 ng/mL) for 10 days. Clonogenic assay Quantification of the results in clonogenic assays were analyzed by Image J. Knockdown efficiency of PTPN3 in HepG2 cells were analyzed by Western blotting with the anti-PTPN3 antibody. Data are shown as mean ± SEM; n = 3. G, Depletion of PTPN3 attenuates TGF-β-induced growth inhibitory effect in Huh7 cells. Experiments and analysis were similarly done as in (F). Data are shown as mean ± SEM; n = 3.
PINK1 accumulates upon knockdown of Lon.Figure 1(A) Western blot analysis of whole head homogenates from transgenic flies expressing a Myc-tagged PINK1 genomic construct and a UAS-RNAi construct driven by elav-GAL4. The RNAi constructs used were as follows: the non-specific control mCherry-RNAi (Control-R), Lon-RNAi1 (Lon-R1), and Lon-RNAi2 (Lon-R2). An anti-Myc antibody was used to detect PINK1 and an anti-Actin antibody was used as a protein loading control. (B) Densitometry of the PINK1-Myc bands from the indicated genotypes was performed using Fiji software. The PINK1-Myc band intensities were then normalized to their respective Actin loading controls, and these ratios were in turn normalized to the Control-R PINK1/Actin ratio. (C) Western blot analysis of Lon protease (Lon), Mitochondrial transcription factor A (TFAM), Heat shock protein 60 (Hsp60) and Actin in whole head homogenate from control and Lon deficient animals. (D) Quantification of the Lon band intensities from the indicated genotypes performed as described in B. (E) Quantification of the TFAM bands from the indicated genotypes performed as described in B. (F) Quantification of the Hsp60 bands from the indicated genotypes performed as described in B. (G) Flow cytometry analysis of dissociated cells from the brains of flies expressing the indicated RNAi constructs. The graph shows the normalized fluorescence intensity of TMRE, an indicator of mitochondrial membrane potential, relative to age-matched control animals. Fluorescence intensity was normalized to control samples prepared and analyzed on the same day as experimental samples. The mitochondrial membrane potential uncoupling agent CCCP was added to samples of the indicated genotypes to illustrate the effect of mitochondrial depolarization on TMRE signal intensity. All experiments described in this figure were repeated at least three times per genotype. Error bars represent the standard error of the mean (s.e.m.). *p<0.05, **p<0.005 by Student t test.