EMBO/sd-character-level-ner huggingface dataset:
Figure 2A Representative MS/MS fragmentation spectrum of tryptic phosphorylated PDI peptide (Ile351-Lys370) depicting PDI Ser357 phosphorylation enriched from HepG2 cells treated with 5 μM Tg for 10 min. B (Upper) Domain organization of the human PDI molecule. The residue numbering is for human PDI with signal sequence. The "CGHC" motifs in the a and a' domains are active sites; carboxyl-terminal KDEL motif is the ER retention sequence. Ser357 located in the x-linker region is marked in red. (Lower) Amino acid sequence alignments of PDI x-linker in several species. Residue positions are indicated by numbering with signal sequences. Human PDI Ser357 and its counterparts are shown in red. C Time-dependent incorporation of phosphate group into PDI WT or S357A catalyzed by recombinant Fam20C. Phosphorylated PDI was determined by anti-pS357-PDI immunoblotting on both regular and phostag gels. D Phosphorylated PDI catalyzed by Fam20C were analyzed by dual-color immunoblotting with anti-PDI (red) and anti-pS357-PDI (green) antibodies. The yellow signal depicts the phosphorylated PDI species. Detection of pS357-PDI in HepG2 cells treated with 5 μM Tg (E) for indicated times. Detection of pS357-PDI in HepG2 cells treated with 2.5 μg/ml Tm (F) for indicated times. Detection of pS357-PDI in HepG2 cells treated with 1 mM DTT (G) for indicated times. (Left) Detection of p-PDI in WT and PDI KO HepG2 cells (H) treated with 5 μM Tg for indicated times by protein immunoblotting on both regular and phostag gels. (Right) Quantification of p-PDI percentage based on band intensities on phostag gels. Data information: Data were shown as mean ± SEM of three (H) independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001 (one-way ANOVA, the post hoc Tukey's HSD test). (Left) Detection of p-PDI in WT and Fam20C KO HeLa cells (I) treated with 5 μM Tg for indicated times by protein immunoblotting on both regular and phostag gels. (Right) Quantification of p-PDI percentage based on band intensities on phostag gels. Data information: Data were shown as mean ± SEM of four (I) independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001 (one-way ANOVA, the post hoc Tukey's HSD test).
Figure 3A Immunoblot analysis of extracts of 293T cells transfected with expression vector for Flag-Beclin-1 and increasing doses of expression vector for HA-USP19 (wedge). B HeLa cell line harboring inducible Flag-USP19 was induced with indicated concentration of Doxy for overnight, and the protein was harvested for immunoblot analysis. C BECN1 mRNA levels of the same sample (B) were detected by real-time PCR. Bars represented mean ± SEM of triplicate samples. D Human PBMCs were transfected with USP19-specific or scramble siRNA and treated with rapamycin (250 nM) for 18 h. The extracts were analyzed by immunoblot with indicated antibodies. E Wild-type (WT) and USP19 KO A549 cells were cultured in EBSS for indicated time points, and the protein expression levels of Beclin-1 and ULK1 were detected by immunoblot. F USP13 KO HEK293T cells were transfected with plasmids encoding Myc-USP5, Myc-USP19 and Myc-USP38 and the cellular extracts were analyzed by immunoblot. G 293T cells transfected with plasmid expressing Flag-Beclin-1 and empty vector or Myc-tagged USP19 (WT, CS, CS/HA or ΔTMD), the lysates were analyzed by immunoblot. H 293T cells were transfected with plasmid encoding Flag-Beclin-1 and treated with CHX (100 μg/ml) alone or with MG132 (10 μM), 3MA (10 mM), CQ (50 μM) as combination for 6 h or transfected with the HA-USP19 plasmid. The cell lysates were analyzed by immunoblot.
Fig.Figure 6(A) Representative images of root explants from WT, atg2-2 and atg5-1 in CIM (21 days) or CIM + SIM (21+ 21 days or 21+ 35 days). Scale bar: 2 cm (B and C) Fresh weights of calli after incubation on CIM (B) or CIM + SIM (21+ 21) (C). Box plots: centerlines show the medians; box limits indicate the 25th and 75th percentiles; whiskers extend to the minimum and maximum. Results were obtained from 3 independent experiments with at least 20 calli per condition, asteriscs mark statistical significance to WT according to the T -test (** P<0.001; *** P<0.0001).
Figure 4(A) Serum concentrations of BAM15 over a 24-hour period (N=3 animals per time point) (B) peak tissue concentrations of BAM15 (N=6 per tissue). C, D (C) Daily body weight (day 8: P=0.0454, day 9: P=0.0353, day 10: P=0.0251, day 11: P=0.0131, day 12: 0.0038, day 13: P=0.0008, day 14: P=0.0005, day 15: P=0.0003, day 16-21: P<0.0001) and (D) cumulative food intake over the 3-week treatment in CTRL and BAM15 treated animals (N=16 per group). (E) Whole body temperature before (pre), one hour (1 hr Post), and 24 hours (24 hrs Post) after intraperitoneal injection of corn oil, vehicle (corn oil and saline), 0.1, 0.5, and, 1 mg BAM15 per kg of body weight (N=11 per group), Whole body temperature (F) daily during chronic BAM15 treatment (N=8 per group). G, H (G) Change in fat mass (%) (P<0.0001) and (H) lean mass (%) (P<0.0001) from baseline to 3-weeks in CTRL and BAM15 treated animals (N=8 per group). (I) Fasting plasma glucose (N=8 per group; P=0.007) and (J) insulin (N=7 per group; P=0.0024) after 3 weeks of treatment in CTRL and BAM15 treated animals. (K) Plasma glucose concentrations at 0, 15, 30, 60, 90, and 120 minutes following intraperitoneal injection of glucose (2 g glucose/kg of body weight; N=8 per group) (L) total area under the curve glucose after 3-weeks in CTRL and BAM15 treated animals (N=8 per group; P=0.0106). M-O (M) Oxygen consumption rates (N) energy expenditure (daily: P=0.0357, dark phase: P=0.030), and (O) respiratory exchange ratio (daily: P=0.0424, dark phase: P=0.0016) measured by continuous high-resolution indirect calorimetry after 3-weeks of CTRL or BAM15 treatment (N=8 per group). (P) Fecal energy density (% recovered in feces) after 3-weeks of CTRL or BAM15 treatment (N=8 per group).
OCRL deficiency does not affect centrosomal PI(4,5)P2 poolA RPE-1 cells transfected with nontargeting control siRNA or OCRL siRNA, were left for 48 h, then subjected to IF microscopy to visualize PI(4,5)P2 (green), γ-tubulin (red) and nuclei (blue, DAPI staining).Figure 5A RPE-1 cells transfected with nontargeting control siRNA or OCRL siRNA, were left for 48 h, then subjected to IF microscopy to visualize PI(4,5) P2 (green), γ-tubulin (red) and nuclei (blue, DAPI staining). Scale bar, 10 μm. B Quantitative analyses of centrosomal PI(4,5) P2 fluorescence intensity in the experiments shown in A. n = 3 per group in which 50 cells were counted per condition per experiment. C Data information: data are presented as mean ± SEM. n.s., not significant, calculated by calculated by two-tailed Student's t-test. C NHF, Lowe 1676 and Lowe 3265 cells were subjected to IF microscopy to visualize PI(4,5) P2 (green), gamma-tubulin (red) and nuclei (blue, DAPI staining). Scale bar, 10 μm. D The fluorescence intensity of PI (4,5) P2 on centrosomes in C was measured. n = 3 per group in which 50 cells were counted per condition per experiment. D Data information: data are presented as mean ± SEM. n.s., not significant, calculated by calculated by two-tailed Student's t-test.
Figure 2(A) Schematic representation of the thiol trapping assay. Mitochondria were solubilized in sample buffer with either dithiothreitol (DTT), iodoacetamide (IAA), or 4-acetamido-4-maleimidylstilbene-2,2-disulfonic acid (AMS). The samples were analyzed by SDS-PAGE and Western blot. (B) Indirect thiol trapping assay. Mitochondria were pretreated with IAA as indicated to block free cysteine residues, and disulfide bonds were subsequently reduced by DTT. Mitochondria were solubilized in sample buffer with AMS. (C) Localization of mitochondrial proteins analyzed by limited degradation by proteinase K in intact mitochondria (250 mM sucrose), mitoplasts (5 mM sucrose), and mitochondrial lysates (1 % Triton X-100). The samples were analyzed by SDS-PAGE and Western blot. Mitos, mitochondria; Sup, post-mitochondria supernatant; OM, outer membrane; IM, inner membrane; IMS, intermembrane space. (D) N-SIM super-resolution micrographs of one Z-stack (0.15 μm) orthogonal section (XYZ) of HeLa cells or HeLa cells that stably expressed COA7-HA transfected with different markers TOMM20-DsRed (OM), COX8A-DsRed (IM), and matrix targeted photoactivatable GFP (Matrix target) labeled with anti-HA, anti-COA7 and Smac/Diablo (IMS) antibodies. The picture represents the majority population of cells from three independent experiments. Scale bar = 2 μm, scale bar in the magnified insert = 0.5 μm. The panel shows Pearson's coefficient in a co-localized volume of different subcompartment combinations with anti-HA or anti-COA7. The data are expressed as a mean ± SD (n=5). ***p<0.001 [IMSxEnd vs. IMxEnd p=0.0001; IMSxEnd vs. MxEnd p=0.0002], ****p<0.0001 [OMxHA vs. IMSxHA p<0.0001; IMSxHA vs. IMxHA p<0.0001] (one-way ANOVA). M, matrix; End, endogenous COA7; HA, COA7-HA.
Figure 2 A. Schematic depicting the experimental setup for in vivo xenograft treatment of MCC tumors with GSK-LSD1 in NSG mice. GSK-LSD1 or vehicle treatment was started 22 days after PeTa cell injection, when tumor volume was ≥ 50mm3  B. Individual tumor growth in GSK-LSD1 (n = 9) or vehicle-treated (n = 8) mice. Red arrow: start of therapy, day 22  C. Representative picture of mice treated with vehicle or GSK-LSD1 at day 41 after PeTa cell injection. Tumor location is indicated with a circle  D. Kaplan-Meier curve of GSK-LSD1 (n = 9) or vehicle-treated (n = 8) mice. Mice were sacrificed when tumors reached a volume ≥ 1.5cm3 or the greatest dimension was ≥ 1.5cm. Red arrow: start of therapy, day 22. ****p < 0.0001 (log-rank Mantel-Cox test)  E. Schematic depicting the experimental setup for in vivo xenograft treatment of MCC "micrometastases" with GSK-LSD1 in NSG mice. GSK-LSD1 or vehicle treatment was started one day after tumor injection (D1)  F. Individual tumor growth in GSK-LSD1 (n = 8) or vehicle-treated (n = 8) mice. Red arrow: start of therapy, day 1  G. Relative mouse weight (%) during treatment of GSK-LSD1 (n = 8) or vehicle-treated (n = 8) mice  H. Kaplan-Meier curve of GSK-LSD1 (n = 8) or vehicle-treated (n = 8) mice. Mice were sacrificed when tumors reached a volume ≥ 1.5cm3 or greatest dimension ≥ 1.5cm. Red arrow: start of therapy, day 1. ****p < 0.0001 (log-rank Mantel-Cox test)
Figure 7A. H&amp;amp;E stained sections of cell invasion into collagen matrix after 8 days, in the absence (top) or presence of 10 µM GM6001 (bottom). Scale bar = 100 µm.B. Invasion index of KPflC cells. Means ± SEM (n=4), p value by unpaired t test.C. Quantification of cell number at the collagen matrix surface per 0.046 mm2 field. Means ± SEM (n=20), p value by unpaired t test. D. Cell proliferation determined by Ki67 immunofluorescence. Scale bar: 20 µm.E. Ki67 positive cell percentages at the collagen matrix surface. Means ± SEM (n=20; n=19 for GFP:ER/vehicle, n=18 for GFP/GM6001), one-way ANOVA with multiplicity adjusted exact p value by post hoc Tukey multiple comparison test.F. Ki67 positive cell percentages in collagen matrix. Means ± SEM (n=20; n=8 for GFP:ER/ vehicle, n=10 for GFP:ER/GM6001, n=17 for ROCK1:ER/GM6001, n=15 for ROCK2:ER/GM6001), one-way ANOVA with multiplicity adjusted exact p value by post hoc Tukey multiple comparison test. G. The increase in viable cell numbers of cells plated on uncoated plastic surfaces and treated with 1 µM 4HT for 24 h was not affected by 10 µm GM6001. Means ± SEM (n=3), one-way ANOVA with multiplicity adjusted exact p value by post hoc Tukey multiple comparison test.H. The increase in viable cell numbers of cells plated on collagen1 (Col1) coated surfaces and treated with 1 µM 4HT for 24 h was not affected by 10 µM GM6001. Means ± SEM (n=3), one-way ANOVA with multiplicity adjusted exact p value by post hoc Tukey multiple comparison test.
Figure 4A. Western blot for control sgRNA (Ctrl) and cGAS-KO MC38 cells, assayed with cGAS and Gapdh antibodies. Mice were injected with Ctrl or cGAS-KO MC38 cells and treated with PBS or 5-FU. (B) Pictures of tumors and spleens from a representative experiment. Image panels were cropped from the same picture. Mice were injected with Ctrl or cGAS-KO MC38 cells and treated with PBS or 5-FU. (C) Tumor volumes were quantified at the indicated days post cancer cell injection. N=4 to N=5, as shown in (B). Mice were injected with Ctrl or cGAS-KO MC38 cells and treated with PBS or 5-FU. (D) Tumor and spleen weights at the endpoint for (C), with each dot representing a mouse.
MEK1T55delinsRT is a preexisting mutation.Figure 6gDNA was isolated from another fragment of the tumor resection, and the PCR with the insertion-specific primers was repeated. Primers amplifying exon 2 of MEK1WT were used as an input control.Source data are available online for this figure.
Figure 4(A) Principle of dual-luciferase co-IP. In all experiments IP is performed against the Flag-tag fused to the Firefly Luciferase (FFL). FFL (unfused to Yki) is used as reference to normalize each interaction. The prey protein is fused to Renilla Luciferase (RL) in C-term for most proteins or in N-term (for Wbp2 and Mor). (B) Drawing of the monitored protein-protein interactions. Interactions previously shown to involve WW domains are indicated by orange arrows, whereas other interactions are schematized by blue arrows. (C) Comparison of Yki isoforms binding capabilities by dual-luciferase co-IP. Graphs show the normalized co-IP (NcoIP) results for 13 proteins tested against the two Yki isoforms and the negative control (Flag-Firefly). (D) Interaction between Sd and Tgi in absence or presence of Yki isoforms. (E) Interaction between Sd and Wbp2 in absence or presence of Yki isoforms. (F) Interaction between Tgi and Wbp2 in absence or presence of Yki isoforms. In C, D, E and F, bars represent mean and error bars represent SD, n=4. p-values: *P < 0.05, **P < 0.01, ***P<0.001, ****P<0.0001 (unpaired two-tailed t-tests). (G) Luciferase assay using 3xSd-Luciferase reporter in S2 cells, transfected with single or combination of Yki isoforms. Error bars represent SD, n=6. * p-value<0.05; **** p-value<0.0001 (unpaired two-tailed t-tests). (H) Co-overexpression of Yki isoforms using GMR-Gal4 driver provides evidence of competition between Yki isoforms.
Figure 5A-C H&E staining of miniature pig canine frontal sections from embryonic day 60 (E60 group) (A) and after the mandible slices were infected with RUNX2 overexpression lentiviral vector with a Myc tag (overexpression group) (B) or overexpression control lentiviral vector with a Myc tag (O-control group) (C); (A'-C') are magnifications of boxed regions in the corresponding figure panels. Data information: Data represent the means ± SEM. n = 3 for all experiments. Scale bars = 50 µm D-F Immunohistochemical (IHC) staining of Myc expression in mandible slices from the E60, overexpression, and O-control groups. Data information: Data represent the means ± SEM. n = 3 for all experiments. Scale bars = 100 µm (D-F) G-I IF staining of RUNX2 expression (yellow arrows) in mandible slices of the E60, overexpression, and O-control groups. Data information: Data represent the means ± SEM. n = 3 for all experiments. Scale bars = 50 µm J, K Relative expression levels of Myc and RUNX2 in the E60, O-control, and overexpression groups. Data information: Data represent the means ± SEM. n = 3 for all experiments. One-way ANOVA (Newman-Keuls test for post-hoc comparisons between two groups) for J, K , *P < .05, **P < .01, ***P < .001. L, M IF staining of RUNX2 expression after infecting mandible slices with scrambled shRNA lentiviral vector (knockdown control, K-control) (L) or RUNX2 shRNA lentiviral vector (knockdown) (M); (L'-M') are magnifications of boxed regions in the corresponding figure panels. Data information: Data represent the means ± SEM. n = 3 for all experiments. Scale bars = 50 µm (other panels). N Relative RUNX2 expression levels between K-control and knockdown groups. Data information: Data represent the means ± SEM. n = 3 for all experiments. Unpaired t-tests for N , *P < .05, **P < .01, ***P < .001. O-Q H&E staining of mandible slice sections from the K-control, knockdown + force (3 kPa), and K-control + force (3 kPa) groups. (O'-Q') are magnifications of boxed regions in the corresponding figure panels. Data information: Data represent the means ± SEM. n = 3 for all experiments. Scale bars = 50 µm R-T IF staining of RUNX2 expression (yellow arrows) of mandible slice sections from the K-control, knockdown + force (3 kPa), and K-control + force (3 kPa) groups. (R'-T') are magnifications of boxed regions in the corresponding figure panels. Data information: Data represent the means ± SEM. n = 3 for all experiments. Scale bars = 25 µm (R'-T') U Relative RUNX2 expression levels among the three groups Data information: Data represent the means ± SEM. n = 3 for all experiments. One-way ANOVA (Newman-Keuls test for post-hoc comparisons between two groups) for U, *P < .05, **P < .01, ***P < .001. V PC epithelium areas in the overexpression, O-control, knockdown, knockdown + force, K-control, and K-control + force groups. Data information: Data represent the means ± SEM. n = 3 for all experiments. Unpaired t-tests for V; , *P < .05, **P < .01, ***P < .001.
Vote counting analysis in cancer tissue.Figure 3Vote counting of cancer tissue metabolites (reported in at least six cohorts) showed consistently deregulated metabolites. Blue bars: decreased metabolites; red bars: increased metabolites. * (asterisk) in front of the name of the metabolite indicates a statistical trend (p < 0.1); red arrows denote key metabolites mentioned in the main text (see Table EV5 for full annotation).
Nutrient deprivation induces autophagy at region 2 within the germarium and in dying midstage egg chambers.Figure 3.(A) GFP-LC3 proteins were expressed in nurse cells (NC) but not in follicle cells (FC) by using the UASp/nanos Gal4 system. DAPI staining of nuclei is shown in blue. (B) UASp-GFP-LC3; nanos GAL4 flies were conditioned on yeast paste and had a diffuse GFP-LC3 pattern. Numerous GFP-LC3 puncta (green) at region 2 within germarium were observed in nutrient-deprived flies. Ovaries were stained with LTR in w1118 flies. Germarium of nutrient-deprived w1118 flies had an increase in punctate LTR staining (red) compared with well-fed germarium. (C) Degenerating stage 8 egg chambers (arrows) had numerous GFP-LC3 puncta (green) and an increase in LTR-positive dots (red) compared with healthy egg chambers (arrowheads). DAPI (blue) staining of nuclei is shown in the two panels on the right. (D) Degenerating stage 8 egg chambers (arrows) of nutrient-deprived Atg7 mutants (Atg7d77/Atg7d14) showed a dramatic decrease in LTR staining. DAPI staining of nuclei is shown in blue.
Figure 1Expression level of Shox2, Tbx15, HoxC8, and HoxC5, mRNA was compared using quantitative real-time PCR (qPCR) of RNA isolated from different fat depots. Two subcutaneous depots (inguinal and scapular white), three intra-abdominal fat depots (perigonadal, perirenal, and mesenteric), and interscapular brown adipose tissue of 8 week-old male C57BL/6 mice. Data are shown as mean ± SEM of six samples. Expression level of Shox2, Tbx15, HoxC8, and HoxC5, mRNA was compared using qPCR of RNA isolated from cultured stromovascular cells derived from adipose tissue of the Immortomouse™. The depot-specific cell lines were made from inguinal, scapular white, perigonadal, perirenal, mesenteric, and interscapular brown adipose tissue of 8 week-old male mice. Data are shown as mean ± SEM of cell lines from three individual mice. Bright-field image of depot-derived cell lines after in vitro adipogenic differentiation and Oil Red O staining (Lee et al., 2017a). Original photographs were taken at 10x magnification (left panel). Scale bar = 100 μm. qPCR analysis for adiponectin (Adipoq), peroxisome proliferator-activated receptor gamma (Pparγ2), and fatty acid synthase (FAS) in RNA isolated from depot-derived cell lines after in vitro differentiation. Data are shown as mean ± SEM of 3 cell lines/group.
Kinetics of p53 degradation (a) and induction of GFP–LC3 puncta (b) in WT HCT116 cells cultured in complete medium in the presence of rapamycin (Rapa), tunicamycin (Tn), lithium (Li) or in nutrient-free medium (NF).figf7Kinetics of p53 degradation (a) and induction of GFP-LC3 puncta (b) in WT HCT116 cells cultured in complete medium in the presence of rapamycin (Rapa), tunicamycin (Tn), lithium (Li) or in nutrient-free medium (NF). (c) WT HCT116 cells transfected with control or HDM2-targeted siRNAs were cultured in the presence or absence of MG132 for 3 h, followed by the treatment with autophagy inducers for 6 h (Tp, thapsigargin; other abbreviations as in b). The abundance of p53 and HDM2 was determined by immunoblotting analysis and the percentage of cells showing accumulation of GFP-LC3 in vacuoles (GFP-LC3vac) was determined. (d) Effects of HDM2 inhibitors on p53 protein levels and autophagy. WT HCT116 were left untreated or treated with Nutlin-3, RITA and/or the indicated autophagy inducers, followed by the p53 immunoblot or immunofluorescence microscopy to visualize GFP-LC3 redistribution. (e) Effect of MG132 and HDM2 siRNA on autophagy of WT and p53−/− HCT116 cells. (f) Failure of HDM2 inhibitors to prevent autophagy induced by the absence of p53. Nutlin-3 and RITA were added to p53−/− HCT116 cells, followed by quantification of GFP-LC3 puncta. (g) Effect of WT and ubiquitination-resistant p53 on autophagy. p53−/− HCT116 cells were transfected with GFP-LC3 alone or in combination with different concentrations of pcDNA3.1, WT p53 or p53 lacking the ubiquitination site at amino acids 13-19 (p53ΔI), then treated with rapamycin, tunicamycin or thapsigargin. Thereafter, p53 expression and GFP-LC3 puncta were quantified. (h) Depletion of Atg5 or Beclin 1 does not prevent p53 degradation triggered by rapamycin or ER stressors. In WT HCT116 cells transfected with siRNA targeting Atg5 or Beclin 1, p53 levels were determined by immunoblot analysis after 6 h of treatment with rapamycin, tunicamycin or thapsigargin.
Figure 6A. mRNA levels of indicated genes in colon and cultured ISEMFs from wild-type mice (n=4) based on qRT-PCR analysis. Inta5, Integrin α5; Intb1, Integrin β1. B. Representative IHC of integrin α5 and β1 in colon from wild-type mouse. Scale bar = 50 μm. C, D. mRNA levels of Bmp2 and Bmp7 in ISEMFs from wild-type mice 24 h after treatment with integrinα5β1 antibody or various signaling pathway inhibitors. n=3. U0126, ERK inhibitor; LY294002, PI3K inhibitor; BAY11-7085, NF-κB inhibitor.