EMBO/sd-character-level-ner huggingface dataset:
Figure 6A-E, Capacitance increments recorded from Otof+/+ (black) and OtofI515T/I515T (red) IHCs. Individual cells recorded at indicated temperatures (light circles), mean ±SEM for perforated patch clamp experiments (t-test). F,G, Summary of the capacitance changes and Ca2+-current integrals for Otof+/+ (F) and OtofI515T/I515T IHCs (G) at the different temperatures illustrates the drastic increase in exocytosis for physiological temperature. *Significant differences compared to RT measurements are indicated with colors of the respective temperature, and between PT and high temperature in violet (t-test). H-J, Otoferlin immunofluorescence in Otof+/+ IHCs (upper panel) and OtofI515T/I515T IHCs (middle and lower panels) of explanted organs of Corti at P7-P8 after incubation at indicated temperatures for 24h; maximum projections of z-stacks, inverted images, scale bar 10µm. The same imaging settings have been applied in all experiments, and the same lookup table was applied for the upper and middle panels. Images of lower panels are enhanced compared to middle panels (lookup table covering full data range of only this genotype) to visualize otoferlin distribution. H-J, Otoferlin immunofluorescence in Otof+/+ IHCs (upper panel) and OtofI515T/I515T IHCs (middle and lower panels) of explanted organs of Corti at P7-P8 after incubation at indicated temperatures for 24h; maximum projections of z-stacks, inverted images, scale bar 10µm. The same imaging settings have been applied in all experiments, and the same lookup table was applied for the upper and middle panels. Images of lower panels are enhanced compared to middle panels (lookup table covering full data range of only this genotype) to visualize otoferlin distribution.K, Quantification of otoferlin immunofluorescence in Otof+/+ IHCs (black bars, n=98-137 cells, 4-5 experiments) and OtofI515T/I515T IHCs (red bars, n=90-97 cells; Kruskal-Wallis test as in L-N), indicate reduced protein levels with increasing temperature.L, Apical/basal otoferlin protein distribution, revealing a significant apical shift of otoferlin and Vglut3 for OtofI515T/I515T IHCs (red symbols) at 38.5°C compared to Otof+/+ (grey/black symbols).M, Relative levels of membrane bound otoferlin were lowered with increasing temperature.N, Absolute otoferlin membrane immunostaining strongly decreased with temperature.
Figure 8(A) Model for ERS-mediated loss of hepatic identity. Initial loss in LIVER-ID TF activities involves reduced protein levels linked to PERK and SRC signaling ❶, which is secondarily amplified by decommissioning of BRD4 SE ❷ and impaired feedback loops within the LIVER-ID TF hepatic network ❸. This consequently leads to loss of hepatic molecular identity and partial dedifferentiation of hepatocytes ❸. Induction of NFIL3 operates as an additional mechanism further contributing to active repression of the LIVER-ID TF network target genes, especially those involved in xenobiotic metabolism. (B) Implications of the competitive equilibrium between the hepatic and ERS transcriptional programs in liver pathophysiology. Please also refer to the discussion.
Figure 2a) Histogram of CSPs between free 15N-UbcH7 and 15N-UbcH7-O-Ub identifies UbcH7 residues affected by conjugation to Ub. Active site (Ser86) is indicated with a star and cross-over helix residues (101-113) are marked with a gray cylinder. INSET. CSPs greater than 1 stdv (> 0.115 ppm) are highlighted in yellow on a surface representation of UbcH7 (PDB 1fbv). The view on the left shows the surface that contains the UbcH7 cross-over helix. b) Region of 1H-15N-HSQC TROSY spectra containing resonances of the cross-over helix residues S107 and Q106 are overlaid: unconjugated 15N-UbcH7 (blue), 15N-UbcH7-O-Ub (black), and 15N-UbcH7-O-UbI44A (red). Peaks representing S107 and Q106 in the context of UbcH7~Ub-I44A (red) are closer to peaks observed in free UbcH7 (blue) than UbcH7~Ub-WT (black) indicating that I44A of Ub disrupts interactions with the cross-over helix of UbcH7.
a) ITT performed 2 h after IL-1β injection.figf7(a) ITT performed 2 h after IL-1β injection. Eight-week old male C57BL/6 mice were intraperitoneally injected with saline or mouse recombinant IL-1β (1 μg/kg). (b) ITT in WT mice on regular diet (RD) or HFD for 12 weeks, or Il1b-/- mice on HFD for 12 weeks. (c,d) ITT (c) and serum IL-1β (d, left) and TNF (d, right) determined by ELISA, 2 h after IL-1β injection. Recombinant mouse IL-1β was administered to WT and Tnfa−/− mice (n = 5 for each group). One of two independent experiments is shown (mean ± s.d.). *P 0.05 versus controls.
Replication barrier bypass in Chk1-deficient cells does not alleviate DNA damage.Figure 6Scheme of the tools used to revert DNA replication phenotypes caused by Chk1 deficiency. CDK inhibition rescues impaired fork elongation and excess origin firing; NS supplementation or GFP-Polη overexpression specifically rescue reduced fork elongation; CDT1 depletion or CDC7 depletion/inhibition specifically rescue excess origin firing. Percentage of γH2AX-positive U2OS cells (mean+SD) after 5 h treatment with the Chk1 inhibitor Gö6976 (Chk1i), in the presence of 5 μM NS, 1,25 μM CDC7i or 10 μM roscovitine. >900 (DMSO) or >2000 (Chk1i) cells/sample were analyzed in 2 independent experiments. Total γH2AX was used to perform the statistics. Representative images of data shown in (B). Scale bar: 50 μm. DSB accumulation measured by neutral comet assay after 8 h treatment with Chk1i, in the presence of 5 μM NS, siCDC7 or 10 μM roscovitine. 85-170 cells/sample were analyzed in 2 independent experiments. Representative images of data shown in (D). Scale bar: 25 μm. Percentage of parental ssDNA-positive U2OS cells (mean+SD) after 5 h treatment with Chk1i, in the presence of 5 μM NS, 1,25 μM CDC7i or 10 μM roscovitine. >450 cells/sample were analyzed in 2 independent experiments. The induction of ssDNA on control samples was null. Representative images of data shown in (F). Scale bar: 25 μm.
Figure 9A model illustrating the mechanism by which proteasome inhibitors (PIs) selectively kill GSCs. A key factor in low-dosage proteasome inhibition and its selective efficacy against GSCs lies in the sustained accumulation of ubiquitinated conjugates due to the failure of the proteasome (red cross). The resulting proteotoxicity then can activate the apoptotic cascade, among which genes such as ATF3, CHOP and JNK are upregulated and translated into their respective protein counterparts. ATF3 and CHOP also form a heterodimer which serves to maintain the apoptotic cascade. The ATF3-CHOP axis-induced cell death is independent of classical ER stress-associated apoptosis, and may instead directly play a role via NOXA to induce mitochondrial dysfunction (yellow lightning) and eventually cell death.
Figure 5A Amylose pulldown of MBP (control) or MBP-tagged MMS22L-TONSL and RAD51. Eluate proteins were detected by silver staining. MT: MBP-MMS22L-GST-TONSL.B Quantitative analysis of RAD51 binding to MMS22L-TONSL such as shown in Figure EV6A. Averages shown, n = 2; error bars, SEM. C, D Yeast two-hybrid analysis of interactions between LexA DNA binding domain-tagged MMS22L and indicated proteins fused to GAL4 activating domain (GAD) assessed by survival on media containing 3-AT in the absence of histidine and by X-Gal assays. C, D Yeast two-hybrid analysis of interactions between LexA DNA binding domain-tagged MMS22L and indicated proteins fused to GAL4 activating domain (GAD) assessed by survival on media containing 3-AT in the absence of histidine and by X-Gal assays. E, F Streptavidinpulldowns of indicated recombinant proteins and biotinylated ssDNA. The proteins in the input and eluates were detected by western blotting. E, F Streptavidinpulldowns of indicated recombinant proteins and biotinylated ssDNA. The proteins in the input and eluates were detected by western blotting. G Streptavidinpulldown of HSS-tagged MMS22L variants stably integrated and overexpressed in U2OS cells. Input and co-precipitated proteins were detected by immunoblotting. H Representative immunofluorescence images of CPT-induced foci in stable U2OS cell lines depleted of endogenous MMS22L and overexpressing indicated siRNA-resistant variants of HSS-MMS22L treated with 50 nM CPT for 1 hour. Scale bar: 5 μm. I-K Quantification of the effect of indicated MMS22L mutations on RAD51 foci in U2OS cells treated with 50 nM CPT for 1 h (I; n = 3), 50 nM CPT for 18 h (J; n = 3) or 5 µM ETP for 1 h followed by 3 hours incubation without the drug (K; n = 3). Graphs represent averaged median values; error bars indicate SEM. Statistical analysis according to one-way ANOVA with Bonferroni post-test; *** P ≤ 0.001; ** P ≤ 0. 01; ns, not significant. Representative immunofluorescence images are shown in (H), Figures EV6G and EV6H, respectively.
Transcriptional landscape at HIF-1 and HIF-2 binding sites in HepG2 cells.Figure 8Heatmaps showing average (n = 2, independent ChIP-seq experiments) ChIP-seq signal (read counts per million mapped reads, CPM; expressed as colour intensity) at canonical HIF-1 and HIF-2 binding sites and across the flanking ±5kb regions (x-axis) in HepG2 cells incubated in 0.5% oxygen for 16 hours. Sites are ordered on the y-axis according to HIF signal derived from HepG2 cells incubated in hypoxia. ChIP-seq signal for histone modifications was obtained from ENCODE and was derived from HepG2 cells incubated in normoxia Bar chart showing enrichment of transcription factor binding sites (TFBS) at canonical HIF-1 and HIF-2 sites in HepG2 cells (identified as in Figure 4). TFBSs for 61 additional DNA-binding proteins were determined from ENCODE ChIP-seq data in normoxic HepG2 cells. The significance of the overlap between each set of binding sites and canonical HIF-1 and HIF-2 sites, was determined using a hypergeometric test. Accessible sites determined from ENCODE DNAse-seq data in HepG2 cells was used as a negative control
Figure 5Immunoblot analysis of the indicated phosphorylated (p-) and total proteins in whole-cell lysates of wildtype (WT) or Peli1-KO (KO) OT-I CD8 T cells that were activated for 16 h with plate-coated anti-CD3 (1 μg/ml) plus anti-CD28, starved for 6 h in serum-free RPMI medium, and then treated for indicated time periods with FBS (10%) (A) Immunoblot analysis of the indicated phosphorylated (p-) and total proteins in whole-cell lysates of wildtype (WT) or Peli1-KO (KO) OT-I CD8 T cells that were activated for 16 h with plate-coated anti-CD3 (1 μg/ml) plus anti-CD28, starved for 6 h in serum-free RPMI medium, and then treated for indicated time periods with insulin (B). Immunoblot analysis of the indicated phosphorylated (p-) and total proteins in whole-cell lysates of wildtype (WT) or Peli1-KO (KO) MEFs that were starved for 16 h in serum-free DMEM medium and then treated for the indicated time periods with FBS (C) Immunoblot analysis of the indicated phosphorylated (p-) and total proteins in whole-cell lysates of wildtype (WT) or Peli1-KO (KO) MEFs that were starved for 16 h in serum-free DMEM medium and then treated for the indicated time periods with insulin (D) Immunoblot analysis of the indicated phosphorylated (p-) and total proteins in whole-cell lysates of wildtype (WT) or Peli1-KO (KO) MEFs that were starved for 16 h in serum-free DMEM medium and then treated for the indicated time periods with EGF (E).
Rab7 forms a complex with FYCO1 and induces the recruitment of FYCO1 to intracellular membranes.Figure 7.(A) Rab7Q67L recruits FYCO11,038-1,233 and FYCO11,091-1,233 but not FYCO11,156-1,233 to the cytosolic vesicles. HeLa cells transfected with GFP-FYCO11,038-1,233 (top), GFP-FYCO11,091-1,233 (bottom), or GFP-FYCO11,156-1,233 (middle right) with or without mCherry-Rab7Q67L were imaged 24 h after transfection. The graph shows the relative number of cells ± SD with GFP-FYCO11,038-1,233 recruited to intracellular membranes without and with cotransfected mCherry-Rab7Q67L. (B) Rab7Q67L recruits the 2xFYVEFYCO1 construct containing a short CC region in front of the first FYVE domain (FYCO11,091-1,233+1,156-1,233) but not a 2xFYVE alone to the perinuclear vesicles. HeLa cells transfected with GFP-FYCO11,091-1,233+1,156-1,233 or GFP-FYCO11,156-1,233+1,156-1,233 with or without mCherry-Rab7Q67L were imaged 24 h after transfection. (C) Rab7 coimmunoprecipitates with FYCO1 from HEK293 cell lysate. HEK293 cells stably transfected with 3xFlag-FYCO1 were cotransfected with GTP-locked mutants of Rab5, -7, -11, and -24 and lysed 24 h after transfection, and protein complexes were immunoprecipitated with anti-Flag M2 agarose affinity gel. Bound proteins were eluted with Flag peptide, resolved by SDS-PAGE, and visualized by immunoblotting with anti-GFP and anti-Flag antibody. IP, immunoprecipitation; WB, Western blot. (D) Leucine-1151 and tryptophan-1152 are important for the recruitment of FYCO1 to Rab7-containing vesicles. HeLa cells transfected with GFP-FYCO1990-1,233L1151A/W1152A with or without mCherry-Rab7Q67L were imaged 24 h after transfection. (B and D) Insets show an enlarged field of interest. (E) RILP but not Rab7 or ORP1L competes with FYCO1 for recruitment to intracellular vesicles. (right) HeLa cells cotransfected with GFP-RILP together with mCherry-FYCO1 and stained with Draq5 were imaged 24 h after transfection. (left) Relative number of HeLa cells cotransfected with mCherry-FYCO1 and the indicated constructs, in which FYCO1 is recruited to the intracellular vesicles. Error bars represent SD. Bars, 10 µm.
Identification of sialic acid‐independent Siglec‐5 ligandsHuman tissue sections were probed with Siglec‐5 Fc and Siglec‐5 R119A Fc to determine the presence of potential sialic acid‐independent ligands.Figure 1Human tissue sections were probed with Siglec‐5 Fc and Siglec‐5 R119A Fc to determine the presence of potential sialic acid‐independent ligands. Positive signals (in blue) for such ligands were identified in the lumen of the lung bronchioles and prostate ducts, as well as in pancreatic tissues. Pancreatic tissue lysate or lung BAL were incubated with Siglec‐5 R119A Fc (Sig5‐RA Fc) or Siglec‐7 Fc (Sig7Fc) immobilized onto Protein G beads. The proteins captured by these complexes were identified by mass spectrometric analysis. Hsp70 was the only protein that was captured by the Sig5‐RA Fc bead complex from pancreas lysate and bronchoalveolar lavage, but not captured by Sig7Fc bead complex.
Figure 8(A) Expression of KUP9-GFP in BY-2 tobacco cells. HDEL-mCherry was used as an ER marker. Scale bar, 10 μm. (B) 3H-IAA accumulation in the BY-2 cells expressing the indicated proteins. PIN4-GFP and KUP10-GFP were used as positive control and negative control, respectively. Data are means ± SE (n = 3, biological replicates; each replicate contains 3-4 mL suspension cells). Student's t-test (*P < 0.05, **P < 0.01) was used to analyze statistical significance, and "#" represent control. (C) Expression of KUP9-GFP and OST4-GFP-KUP9 in Xenopus oocytes. Scale bar, 100 μm. (D) 3H-IAA accumulation in the oocytes expressing KUP9-GFP and PIN4 at the indicated times. PIN4 was used as a positive control. The oocytes were incubated in the bath solution containing 3H-IAA for the indicated times. Data are means ± SE (n = 6, biological replicates; each replicate contains 6 oocytes). (E) IAA content in the oocytes expressing the indicated proteins. The oocytes were injected with the same amount of IAA and incubated in IAA-free bath solution for 6 h. IAA content was measured using UPLC-MS. Data are means ± SE (n = 3, biological replicates; each replicate contains 6 oocytes). (F) K+ efflux activity of the oocytes expressing KUP9 and NRT1.5. The oocytes were not injected with IAA. The oocytes were incubated in K+-free bath solution for 6 h. Data are means ± SE (n = 3, biological replicates; each replicate contains 6 oocytes). (G) K+ efflux activity of the oocytes expressing the indicated proteins. The oocytes were injected with H2O, IAA, or BA. Data are means ± SE (n = 3, biological replicates; each replicate contains 6 oocytes). (H) K+ efflux activity of the oocytes expressing the indicated proteins. IAA was injected into the oocytes. Then the oocytes were incubated in K+-free bath solutions at different pH for 6 h. Data are means ± SE (n = 3-6, biological replicates; each replicate contains 6 oocytes). (I) IAA efflux activity of oocytes expressing the indicated proteins under different external K+ conditions (-K, 0 mM; +K, 50 mM). The oocytes were injected with the same amount of IAA, and then incubated in IAA-free bath solution for 6 h. IAA content was measured using UPLC-MS. Data are means ± SE (n = 3, biological replicates; each replicate contains 6 oocytes). Student's t-test (*P < 0.05, **P < 0.01) was used to analyze statistical significance, and "#" represent control.
Figure 6(A) Acid denatures UgpB. The secondary structure of 2 µM UgpB was monitored by circular dichroism (CD) spectroscopy in 10 mM sodium phosphate buffer at pH 2 and pH 7. (B) G3P binds to acid-stripped UgpB as observed by monitoring the tryptophan fluorescence spectrum, either at pH7 or after transition from pH 2 to 7 (see Methods). Transitions from pH 2 to 7 in the presence of G3P are accompanied by a blue shift, indicating G3P binding. (C) The anti-aggregation activity of UgpB is monitored by CHO-induced MDH aggregation assay. Light scattering values were monitored. The anti-aggregation activity of UgpB is activated after transition from pH 2 to 7 and the addition of G3P (120 µM) can again inactivate its chaperone activity. (D) Addition of G3P to UgpB inhibits its anti-aggregation chaperone activity
The presence of caldendrin does not impair the ability of myosin V to bind actin filaments in vitro and in live COS7 cells.Figure 4A Commasie blue stained SDS-PAGE showing results of F-actin co-sedimentation assay. In vitro polymerized actin filaments sediment irrespectively of the presence and absence of calcium ions. BSA was used as a control to estimate the amount of "passively" co-sedimented non-actin-binding protein and/or residual non-actin-binding protein in the pellet. There was no difference between the amount of BSA in the pellet with or without F-actin. B Co-sedimentation assay of GFP-tagged myosin fragment lacking the globular tail domain (GFP-myoVa-motor+CC) with F-actin shows that the presence of Ca2+-caldendrin does not prevent myoV from binding to actin filaments. GFP-myoVa-motor+CC, as visualized by an anti-GFP antibody via western blot, co-sediments with F-actin (comassie blue stained SDS-PAGE), both in presence and absence of Ca2+, regardless of the presence of caldendrin. C Schematic representation of the inducible transport assay in COS7 cells. Three plasmids are co-expressed in COS7 cells: a constitutively active kinesin-2 motor (KIF17) fused to a GFP-tagged peroxisome targeting sequence (KIF17-GFP-PEX13), an RFP-tagged PEX13 fused to an FKBP protein (PEX-RFP-FKBP), and a constitutively active myosin Vb motor fused to an FRB protein (myoV-GFP-FRB). Addition of the small molecule rapalog leads to dimerization of the FKBP and FRB, thus recruiting myoV to the peroxisome. Additionally, the cells were transfected with untagged caldendrin (or an empty control vector) to assess the effect of caldendrin binding to myoV on its activity in living cells. D Left: Representative image of COS7 cell expressing the inducible dimerization system described in C. Red: Peroxisomes (PEX-RFP-FKBP), Green: KIF17-GFP-PEX13 and myoV-GFP-FRB. Scale bar = 10 µm. Middle and right: Individual tracks of kinesin-driven peroxisome movements reconstructed over 20 seconds (yellow) before (middle) and after (right) addition of rapalog indicate that the recruitment of myoV slows down the motility of peroxisomes. Blue outlines represent manually drawn cellular border and nucleus (excluded from analysis). E Analysis of the mean squared displacement (MSD) of peroxisomes over time (left) suggests a linear increase for all conditions. Slope analysis for MSD curves for individual cells (right) indicates that addition of rapalog induces efficient stalling (2-way ANOVA, *** p<0.0001 as compared before and after rapalog). However, no difference between control and caldendrin (cald) transfected cells could be observed (2-way ANOVA, Mean ± SEM; n control=14 cells, n cald=23 cells; 3 experiments).
Figure 5(A) During fasting, inhibition of Dab2-Akt association reduces the interaction of Akt with USP1-PHLPP1-TSC1 complex. Left: TSC1 was immunoprecipitated from the soluble fraction of muscles expressing shLacz or Dab2-DN from fed or fasted mice. Mouse IgG was used as a control for non-specific binding. Precipitates were analyzed by immunoblotting. Right: densitometric measurement of PHLPP1 and TSC1 blots, and data is presented as PHLPP1/TSC1 ratio. (B) Dab2 promotes Akt deubiquitination in vivo. Left: Akt was immunoprecipitated from the soluble fraction of muscles expressing shLacz or Dab2-DN from fed and fasted mice using a specific antibody. Mouse IgG was used as a control for non-specific binding. Protein precipitates were analyzed by Western blot analysis using Akt antibody. Right: Densitometric measurements of presented blots. Data is presented as the ratio between ubiquitinated Akt to total Akt in each lane (n=2). (C( Dab2 promotes cleavage of K63-linked polyubiquitin chains on Akt in vivo. Akt was immunoprecipitated from the soluble fraction of muscles expressing shLacz or Dab2-DN from fed and fasted mice using a specific antibody. Mouse IgG was used as a control for non-specific binding. Protein precipitates were subjected to immunoblotting with an antibody against K63-linked polyubiquitin conjugates. (D) Inhibition of Dab2-Akt association enhances Akt phosphorylation in fasting. Soluble fraction of TA muscles transfected with shLacz or Dab2-DN from fed and fasted mice were analyzed by SDS-PAGE and immunoblotting using the indicated antibodies. Right: densitometric measurement of presented blots. Data are mean ±SEM, n=3. * p<0.05. vs. shLacz in fed conditions, by one-tailed t-test.
Figure 8(A) BW25113 Δ8EDs harboring a plasmid carrying the mepM gene under the control of the PrhaBAD L-rhamnose-inducible promoter with the "weak" TIS2 translation initiation signal (plasmid pHV53) was grown on BHI agar in the absence or presence of 0.2% L-rhamnose. Growth was dependent upon induction of the mepM copy carried by pHV53. (B) The plating efficiency assay was performed with derivatives of BW25113 Δ8EDs pHV53(mepM) harboring the vector pHV7 or recombinant plasmids carrying each of the eight endopeptidase genes under the control of the ParaBAD promoter. In this assay, functional replacement of MepM is detected based on growth in media containing 0.2% or 1% L-arabinose for expression of the endopeptidase gene carried by vector pHV7, while by-passing the requirement for induction of the mepM copy of pHV53 by L-rhamnose. Complementation was observed with both concentrations of inducer for mepH, mepS, and pbpG. Overproduction of mepM encoded by the pHV7 derivative in the presence of the high dose of L-arabinose (1%) was lethal. The right panel presents the growth control performed in the presence of 0.2% L-rhamnose for induction of the mepM copy carried by pHV53.