EMBO/sd-character-level-ner huggingface dataset:
RHIM requires the intact core tetrad.Figure 4Representative fluorescence intensity traces collected over 50 s for mixtures of (A) mCHERRY-M451-90 and YPet‑only; (B) mCHERRY-M451-90 and YPet‑RIPK1497‑583; (C) mCHERRY-M451-90 and YPet-RIPK3387‑518; (D) mCHERRY-M451-90 and YPet-ZBP1170-355; (E) mCHERRY-M451-90AAAA and YPet‑only; (F) mCHERRY-M451-90AAAA and YPet-RIPK1497-583; (G) mCHERRY-M451-90AAAA and YPet-RIPK3387‑518 and (H) mCHERRY-M451-90 and YPet-ZBP1170-355. Independent replicates performed at least twice
Figure 4A Cells transfected with FLAG-tagged p73 isoforms were treated with 20 μM AGK2 or DMSO for 6 h and subjected to immunoprecipitation with anti-FLAG antibody followed by immunoblotting with antibody against acetylated lysine or FLAG. B Three conserved lysine residues are located in the most C-terminal region of p73. Amino acid sequences of p73 of the indicated species and human p53 and p63 are aligned. *, conserved and acetylated lysine residues, black frame, highly conserved residues, gray frame, conserved residues. Schematic representation of TAp73α and K3R. K3R is a mutant TAp73α in which three lysine (K) residues in the C-terminal region are replaced with arginine (R). Cells transfected with the indicated constructs were treated with 20 μM AGK2 or DMSO, respectively, for 6 h and subjected to immunoprecipitation with anti-FLAG antibody followed by immunoblotting with antibody against acetylated lysine or FLAG. E HEK293T cells transfected with FLAG-tagged TAp73α were treated with 20 μM AGK2 (pre-AGK2 +) or DMSO (pre-AGK2 -) for 6 h. p73 was purified by immunoprecipitation and incubated with recombinant SIRT2 (10 U), NAD (1 mM) and/or AGK2 (3.5 μM) as indicated. F qRT-PCR analysis of PUMA and GADD45 mRNA in GB2 cells infected with the indicated lentiviruses. Bars indicate mean ± s.e.m. (n = 4-5). G Expression of p73 in (F) was determined by immunoblotting with anti-p73 antibody. RFP was used as a control. H GB glioblastima neurospheres [5 × 104 cells (indicated by the dashed line)] were infected with the indicated lentiviruses. After 96 h, the number of viable cells was counted. Bars indicate mean ± s.d. (n = 4). GB2 cells infected with the indicated lentiviruses were intracranially transplanted into immunocompromised mice. After 8 weeks, mice (3 to 5 animals, see number of dots) were sacrificed and the expression levels of human β-actin mRNA were quantified by qRT-PCR. SIRT2-mediated inactivation of p73 is critical for the proliferation and tumorigenicity of glioblastoma cells. SIRT2 regulates the transcriptional activity of the tumor suppressor p73 by deacetylating its C-terminal lysine residues.
Alternative MPC complexes are found dependent on the carbon sourceMpc1 and Mpc2 are present under fermentative conditions, while Mpc1 and Mpc3 are present under respiratory conditions.Figure 1A Mpc1 and Mpc2 are present under fermentative conditions, while Mpc1 and Mpc3 are present under respiratory conditions. MPC subunits were detected by Western blot in mitochondria isolated from yeast cells grown in glucose (YPD) or glycerol (YPG). Mitochondrial matrix protein aconitase (Aco1), 23‐kDa translocase of the inner membrane subunit (Tim23), and 70‐kDa translocase of the outer membrane subunit (Tom70) were used as loading controls. B Kinetics of the switch from Mpc2 to Mpc3. Yeast cells were grown in YPD until mid‐log phase and then shifted to YPG medium. Whole cell extracts were taken at the indicated time points after the shift, and HA‐tagged Mpc2 and Mpc3 detected by Western blotting. Mpc3 is induced very rapidly by switch from YPD to YPG, while Mpc2 levels decrease only gradually. C Alternative pyruvate carrier complexes MPCFERM and MPCOX are detected by BN‐PAGE. Isolated mitochondria expressing HA‐tagged MPC proteins were run on blue native gels, and MPC complexes detected with an HA‐specific antibody. MPCFERM consisting of Mpc1 and Mpc2 is present under fermentative conditions, and MPCOX consisting of Mpc1 and Mpc3 is present under respiratory conditions. In addition, a complex of ˜300 kDa is observed under respiratory conditions that contains Mpc1 and Mpc3. Non‐specific reactivity of the anti‐HA antibody toward respiratory chain complexes that are highly abundant in oxidative growth conditions is denoted by an asterisk (*).
Involvement of p62 in the selective clearance of α-synuclein inclusions.Figure 5(A) HEK293 cells stably expressing GFP-LC3 were transfected with p62-siRNA (right panels) or control-siRNA (left panels). Phosphorylated α-synuclein-positive inclusions (P-αSyn) are indicated by arrowheads. In p62-knockdown cells, the expression and accumulation of p62 were not detected, and inclusions were never sequestrated by GFP-LC3 autophagosomes. Scale bars, 20 µm. (B) The number of GFP-LC3-positive α-synuclein inclusions (%) in Fig. 5A were assessed in randomly chosen fields with 32-44 inclusions (n = 3). Data represent mean ± s.e.m. Statistical analysis was performed with t-test (*p<0.05). (C) α-Synuclein fibrils were introduced into control (Cont) or p62-knockdown (p62) cells. Four hours after introduction, cell lysates were subjected to immunoblotting analysis using anti-LC3 (upper panel) or anti-actin (lower panel) antibodies. Quantification of the relative levels of LC3-II/Actin is shown with the ratio. Relative level of LC3-II/Actin was represented by mean ± s.d. as a graph. Statistical analysis was performed with one-way ANOVA (post-hoc Tukey's test). *p<0.01. This experiment was repeated three times. (D) Clearance of α-synuclein inclusions in control (solid bar) or p62 (open bar) knockdown cells was measured at 4 h and 24 h after α-synuclein fibrils. The number of phosphorylated α-synuclein-positive cells was assessed in randomly chosen fields (n = 5). Data from each experiment were normalized to Cont-siRNA-4 h (100%) and represented as relative value of α-synuclein inclusions ± s.e.m. Statistical analysis was performed with one-way ANOVA (post-hoc Tukey's test). *p<0.01.
Figure 6Proliferation assay results of MGC-803 (A), MKN-45 (B), NCI-N87 (C) cells treated with oxaliplatin along with indicated treatments (n = 3 biological replicates, mean ± standard error of mean (SEM); *P = 0.0204 (MGC-803), *P = 0.0498 (MKN-45), *P = 0.0473 (NCI-N87), and *P = 0.0019 (LSD1 KO MGC-803); two-tailed unpaired Student's t-test). LSD1 knockout (KO) MGC-803 (D) cells treated with oxaliplatin along with indicated treatments (n = 3 biological replicates, mean ± standard error of mean (SEM); *P = 0.0204 (MGC-803), *P = 0.0498 (MKN-45), *P = 0.0473 (NCI-N87), and *P = 0.0019 (LSD1 KO MGC-803); two-tailed unpaired Student's t-test). Representative images of tumors (E) and the tumor weight (F) of mice treated with oxaliplatin in the presence or absence of sEVs (n = 3 biological replicates; mean ± SEM; *P = 0.0488; two-tailed unpaired Student's t-test). (G) Tumor growth rate in mice treated with oxaliplatin in the presence or absence of sEVs. Tumor growth rate was measured according to tumor volume (n = 3 biological replicates; mean ± SEM; *P = 0.0439; two-tailed unpaired Student's t-test). (H) Proliferation assay results of MGC-803 cells treated with oxaliplatin in the presence or absence of sEVs from the plasma of patients with gastric cancer or healthy individuals (n = 3 biological replicates; mean ± SEM; *P = 0.0487; two-tailed unpaired Student's t-test). (I) Schematic model for the shuttling of LSD1 from parent cells to recipient cells using sEVs as vehicles. The sEV-delivered LSD1 promotes recipient gastric cancer stemness and chemoresistance.
Figure 6A Lysates from WT and SNX10 KO Caco-2 cells treated with or without 100 μg/mL OMVs for 24 h were analyzed by immunoblots with the indicated antibodies. B Co-staining of SNX10-Flag and PIKfyve in Caco-2 cells transfected with SNX10-Flag. Scale bar: 20 μm. C WT and SNX10 KO Caco-2 cells were treated with or without OMVs (100 μg/mL) for 24h and then subjected to IP with anti-PIKfyve antibody. D, E Co-staining of PIKfyve and caspase-5 in WT and SNX10 KO Caco-2 cells treated with or without OMVs (100 μg/mL) for 24 h (D). Anti-PIKfyve and anti-caspase-5 antibodies were used to detect endogenous PIKfyve and caspase-5 respectively. Scale bar: 20 μm. Colocalized PIKfyve and caspase-5 were quantified by ImageJ software (E) (n = 3 independent experiments, n = 18 fields analysed). Data information: Data are means ± SD. One-way ANOVA followed by Bonferroni post hoc test was used for statistical analyzes. ***p < 0.001. F Caco-2 cells were stimulated by OMVs (100 μg/mL) with or without apilimod (30 nM) for 24h. Proteins of lysates extracted from Caco-2 cells were subjected to immunoblots with the indicated antibodies. G, H Co-staining of PIKfyve and Rab5 in WT and SNX10 KO Caco-2 cells treated with or without OMVs (100 μg/mL) for 24 h (G). Anti-PIKfyve and anti-Rab5 antibodies were used to detect endogenous PIKfyve and Rab5 respectively. Scale bar: 20 μm. Colocalized PIKfyve and caspase-5 were quantified by ImageJ software (H) (n = 3 independent experiments, n = 18 fields analysed). Data information: Data are means ± SD. One-way ANOVA followed by Bonferroni post hoc test was used for statistical analyzes. ***p < 0.001. I LPS levels in the cytosolic and residual fractions of Caco-2 cells stimulated by OMVs (100 μg/mL) with or without apilimod (30 nM) for the indicated time were detected by LAL assay (n = 6 independent experiments). J Caco-2 cells transfected with control siRNA or CASP5 siRNA were treated with OMVs (100 μg/mL) for the indicated time. LPS levels in the cytosolic and residual fractions of Caco-2 cells were detected by LAL assay (n = 6 independent experiments). K LPS levels in the cytosolic and residual fractions of Caco-2 cells stimulated by OMVs (100μg/mL) with or without bafetinib (1μM) for the indicated time were detected by LAL assay (n = 6 independent experiments). Data information: Data are means ± SD. One-way ANOVA followed by Bonferroni post hoc test was used for statistical analyzes. ***p < 0.001.
siRNA screen for identifying regulators of AGR2 dimer formation.Figure 2A) The first screen was carried out using HEK293T cells transfected with the ERMIT bait/prey, system, the XBP1 splicing reporter and the siRNA library (n = 5). The counter screen was carried out using the same experimental setup without the ERMIT bait/prey system (n = 4). B) Volcano plot representing the statistical analysis of the data from the screen, where the y axis represents statistical criteria and the x axis the fold change in luminescence units. C) Number of genes associated with the indicated processes, as revealed by functional analysis based on Gene Ontology and Reactome terms, (*): Apoptotic signaling pathway (p=0.0083), ER-associated degradation (p=0.0076), and lipid biosynthetic process (p=0.0059), respectively; (**): ER stress (p=0.0044), Protein folding (p=0.003), Calcium homeostasis (p=0.0022), N-glycosylation (p=0.0018), respectively. D and E) Enrichment of AGR2 homodimer enhancers in functions associated with ER homeostasis (green) and of AGR2 homodimer inhibitors in functions associated with ER homeostasis imbalance (red).
p62 accumulation in ECs from patients with CAD and Tchp knock-out mice A, Expression of TCHP, p62, IL-1β, IL-8, and IL-6 in ECs from vessels wall from patients with CAD (n=8 patients per group, unpaired t-test; TCHP **p <0.Figure 5Expression of TCHP, p62, IL-1β, IL-8, and IL-6 in ECs from vessels wall from patients with CAD (n=8 patients per group, unpaired t-test; TCHP **p <0.0001; p62 **p = 0.0007; IL-1β **p <0.0001; IL-8 **p <0.0001; and IL-6 **p <0.0001 vs. Healthy subject). Staining, and quantification of p62 in ECs from healthy subject and patients either treated with vehicle (DMSO) or BAY. Scale bars, 50 µm; (n=3 patients per group; n=80 cells; one-way ANOVA; **p<0.0001 vs healthy DMSO; ##p=0.003 vs patients DMSO). Expression of IL-6, IL-8 and IL-1β (n=3 patients per group; one-way ANOVA; IL-6: *p = 0.0135 vs healthy DMSO; #p = 0.0180 vs patients DMSO; IL-8: *p = 0.0139 vs healthy DMSO; ##p = 0.0024 vs patients DMSO; IL-1β: *p = 0.0104 vs healthy DMSO; ##p = 0.0069 vs patients DMSO). migration speed, in ECs from healthy subject and patients either treated with vehicle (DMSO) or BAY. (n=3 patients per group; one-way ANOVA; *p=0.0325 vs healthy DMSO; #p=0.0336 vs patients DMSO). Left panels: representative images of the heart of wild-type and Tchp knock-out mice stained for CD31 (green) and p62 (magenta). Scale bars, 100µm, and 50µm for the inset. Right panels: Quantification of the percentage of cardiac area (n=4 mice per group; unpaired t-test; **p=0.083 vs wild-type) or vessels positive for p62 (n=4 mice per group; unpaired t-test; **p=0.058 vs wild-type) and vessel density (n=4 mice per group; unpaired t-test; **p=0.0097 vs wild-type).
Figure 5A. Enzymatic activity of respiratory complex CII in Skm isolated mitochondria from wt and LowOXPHOS mice fed with chow or HFD (wt, n= 4; LowOXPHOS, n= 4; wt + HFD, n= 4; LowOXPHOS + HFD, n= 4). B. Polarographic profiles of isolated mitochondria from wt (lower trace) and LowOXPHOS (upper trace) animals using succinate (left graph) or palmitoyl-carnitine (right graph) as a substrate. Quantification of maximal respiration in the right histogram. Bars are the mean ± s.e.m. of n= 3 mice/genotype, 3 traces/mouse; OL, oligomycin; Ant A, antimycin A. Data information: *, # p <0.05 when compared to wt or LowOXPHOS, respectively, by ANOVA and Student's t-test. (C-E) 2D-PAGE of Skm extracts (C, D) and acetylated proteins (E) from wt (n= 4) and LowOXPHOS (n= 3) mice. The isoelectric point (pI) of the CII subunits SDHB (C) and SDHA (D) was calculated by protein migration in pH 3-10 NL strips. F. Immunocapture (IP) of SDHA blotted with anti-acetyl-K antibody (upper panel) and of acetylated proteins blotted with anti-SDHA antibody (middle panel) in Skm isolated mitochondria from wt (n= 3) and LowOXPHOS (n= 3) mice. (G PyMOL cartoon representations of acetylated lysines in SDHA related to CII activity. 6 acetylated-K are shown. H). PyMOL cartoon representations of acetylated lysines in SDHA related to CII activity. 6 acetylated-K are shown. I. MitoSox staining in myocytes expressing or not ATPIF1H49K. The left scheme illustrates where each ETC inhibitor works. The right histogram shows the quantification of mitochondrial ROS. Bars are the mean ± s.e.m. of n= 3 experiments, 12 replicas/condition. Data information: *, # p <0.05 when compared to wt or LowOXPHOS, respectively, by ANOVA and Student's t-test. J. MitoSox staining in myocytes expressing or not ATPIF1H49K. The left scheme illustrates where each CII inhibitor works. The right histogram shows the quantification of mitochondrial ROS. Bars are the mean ± s.e.m. of n= 3 experiments, 12 replicas/condition. Data information: *, # p <0.05 when compared to wt or LowOXPHOS, respectively, by ANOVA and Student's t-test. K. Representative blue native immunoblots (BN) and clear native in-gel activity (CN) of Skm mitochondrial membrane proteins from wt (n= 3) and LowOXPHOS (n= 3) 2-month-old mice. The migration of the respiratory complexes/supercomplexes CI-V is indicated. Subassembly of CI (with no activity, suggesting degradation) was observed in LowOXPHOS mice. VDAC is shown as a loading control.
Figure 4Figure 4. Topological analysis of cell cycle hub-genes in a protein-protein interaction (PPI) network. Mapping of the top 30 hub-genes relevant to TBV measures in smaller and bigger ASD and control brains. A) Hub-genes that are normally active in control toddlers. B) Hub-genes that are active in both control and ASD toddlers. C) Hub-genes that are abnormally active in ASD toddlers. Node size (circle) is proportional to the number of actual biologically driven PPIs. Blue and red nodes are hub-genes that displayed PPIs and were in the top 30 lists in control and ASD, respectively. Grey nodes are genes in the cell cycle PPIs network that were not ranked within the top 30 genes. Black nodes are genes that fall out from the top 30-gene list in ASD compared to control.
Figure 2A Schematic overview of scRNA-seq strategies in Lepr-Cre; tdTomato mice. Young adult: 8-week-old mice (4 males and 2 females); Aging: 12-month-old mice (4 females); Rosiglitazone diet: 10-week-old mice (feeding started at 5-week-old, 2 females); Irradiation: 8-week-old mice (sub-lethally irradiated at 7-week-old, 2 males and 2 females); Fracture: 8-week-old mice (femur fractured at 7-week-old, 2 males and 2 females). t-SNE plots showing 17224 CD45- Ter119- Tie2- tdTomato+ single cells from all 5 conditions. Cells are colored by conditions (B) Top 20 PCs were chosen for the clustering. t-SNE plots showing 17224 CD45- Ter119- Tie2- tdTomato+ single cells from all 5 conditions. Cells are colored by clustering (C). The adipogenic, osteogenic, periosteal, and cycling cells were highlighted by dotted lines in (C). Top 20 PCs were chosen for the clustering. D Heatmap showing the relative expression levels (row-wide Z score) of the 20 most significant markers for each cluster (rows) across cells in the 8 clusters (columns). Bars on the top were colored as in (C). E Violin plots showing the expression of feature genes for each cluster. F Immunofluorescent images of the distal femur in 8-week-old Lepr-Cre; tdTomato mice. Arrowheads indicated tdTomato+ periosteal cells. Two representative periosteal regions were magnified (a and b). GP: Growth plate. B: Bone. BM: Bone marrow. Peri: Periosteum. Scale bars were 100 µm. G Immunofluorescent images of the distal femur in 8-week-old Lepr-Cre; tdTomato mice 7 days after fracture. Aggrecan (green), tdTomato (red), DAPI (blue). Arrowheads indicated tdTomato+ periosteal cells. Two representative regions were magnified (a and b). Scale bars were 100 µm.
Over-expression of UVRAG enhanced the maturation of autophagosomes and reduced HCV RNA replication.Fig 4Huh7.5 cells were transfected with the control vector or the Flag-tagged UVRAG expression plasmid for 24 hours followed by infection with HCV. (A) Western-blot analysis of cell lysates at different time points after infection. Mock-infected cells were lysed at 48 hours post-transfection. Huh7.5 cells were transfected with the control vector or the Flag-tagged UVRAG expression plasmid for 24 hours followed by infection with HCV. (B) Real-time RT-PCR analysis of HCV RNA at 24 and 48 hours post-infection. *, p < 0.05. Huh7.5 cells were transfected with the control vector or the Flag-tagged UVRAG expression plasmid for 24 hours followed by infection with HCV. (C) RFP and GFP puncta in cells with the over-expression of UVRAG at different time points after HCV infection. Merged images are shown to the right. (D) Percentages of RFP puncta that were also positive for GFP in Huh7.5 cells transfected with either the control vector or the UVRAG expression plasmid. The results represent the average of >50 cells.
SH-BC-893 normalizes adipokine levels in mice consuming a HFD.Figure 5Plasma adiponectin (A) or leptin (B) in mice maintained on a SD or HFD for 8-9 weeks then gavaged with vehicle or 120 mg/kg SH-BC-893 4 h prior to blood collection. Pair-fed mice were provided with the amount of food consumed by SH-BC-893-treated mice in 4 h but left untreated; n=8-10. The adiponectin:leptin ratio was calculated for the mice in A,B. As in (A,B) but in mice maintained on the HFD for 10 weeks and then gavaged Mondays, Wednesdays, and Fridays for 3 weeks with vehicle or 120 mg/kg SH-BC-893; n=9. Rx, treatment number of a total of nine doses. The adiponectin:leptin ratio was calculated for the mice in (D,E); n=9. Mice were treated as in D-F, sacrificed 4 h after the ninth dose, and the levels of Socs3 (G) or Lepr (H) mRNA measured in the hypothalamus; n=8. Body weights of mice fed the HFD for the indicated interval. Two cohorts were utilized, one measured at 4 and 8 wk and the other at 10 and 16 wk; n=16-20. Plasma adiponectin and leptin were measured in the mice in (I) 4 h after a single dose of 120 mg/kg SH-BC-893; n=7-10. The adiponectin:leptin ratio was calculated for the mice in J,K; n=7-10.
Figure 1A. LRP6 level is reduced during serum starvation. HEK293 cells were incubated in serum-free medium for the indicated hours. Cells were lysed and the cell lysates were analyzed by immunoblotting. B. Serum starvation reduced the amount of LRP6. HEK293 cells were incubated in serum-free medium for 3 h. Then cells were additionally incubated for 1 h after addition of 10% serum. Cells were lysed and the cell lysates were analyzed by immunoblotting (left panel). The ratio of LRP6/β-actin and p-YAP/YAP of three independent immunoblots was quantified (biological replicates, middle and right panel, respectively). N.S.: non-significant, **P < 0.01 and ***P < 0.001. Error bars indicate standard deviation of biological triplicate measurements. Student's t-test was used for statistical analysis. C. Loss of LRP6 in serum starvation was seen in different cell lines. Cell lines were incubated in serum-free medium for 2 h. Cells were lysed, and the cell lysates were analyzed by immunoblotting. S.E., short exposure; L.E., long exposure. D. Glucose starvation decreased LRP6. HEK293 cells were incubated in glucose-free medium for 4 h. Cells were lysed and the cell lysates were analyzed by immunoblotting. E. The decrease of LRP6 by serum starvation was mediated by the lysosomal degradation pathway. HEK293 cells were incubated in serum-free medium for 4 h with DMSO or Bafilomycin A1 (100 nM). Cells were lysed and the cell lysates were analyzed by immunoblotting. Loss of LRP6 by serum starvation was mediated in a clathrin HEK293 cells were transfected with clathrin siRNA and incubated in serum-free medium for 4 h. Cells were lysed and the cell lysates were analyzed by immunoblotting. G. Loss of LRP6 by serum starvation was mediated in a caveolin-1-dependent manner. HEK293 cells were transfected with caveolin siRNA and incubated in serum-free medium for 4 h. Cells were lysed and the cell lysates were analyzed by immunoblotting. H. Lipid components of serum were essential for maintaining LRP6. HEK293 cells were incubated in serum-free medium with A2058 BSA (Sigma) or Fraction V BSA for 4 h. Cells were lysed and the cell lysates were analyzed by immunoblotting (left panel). The ratio of LRP6/β-actin of three independent immunoblots was quantified (biological replicates, right panel). Error bars indicate standard deviation of biological triplicate measurements. N.S.: non-significant and *P < 0.05. Student's t-test was used for statistical analysis.
Figure 4A. Western blot and quantification of phosphorylated PKD (S916/S876) in small intestine of Pkd2flox/flox and Pkd2gutΔ/Δ male mice. n=3. B. Western blot and quantification of phosphorylated PKD (S744-748) in small intestine of Pkd2flox/flox and Pkd2gutΔ/Δ male mice. n=3. C C. Lipids tolerance test after oral gavage of olive oil to Pkd2flox/flox and Pkd2gutΔ/Δ male mice after 6 weeks in HFD. n=10. D. Body weight gain of male mice of indicated genotypes in HFD. n=10. E. Quantification of fat, free fluid and lean mass by nuclear magnetic resonance of mice in panel B. n=10. F. Glucose tolerance test of male mice with indicated genotypes after 16 weeks in HFD. n=10. G. Free-fatty acids and H. Triglycerides content in serum of male mice after 17 weeks in HFD. n=10. I. Differences in bacterial alpha diversity as indicated by Shannon Index. n=7. J. Changes in specific Operational taxonomic unit (OTU) in small intestinal samples. n=7.