EMBO/sd-character-level-ner huggingface dataset:
a) Flow cytometry of LPS-stimulated B cells stained with LysoTracker Red.figf1(a) Flow cytometry of LPS-stimulated B cells stained with LysoTracker Red. (b) Autophagic flux of GFP-LC3 splenic B cells stimulated with LPS and then left untreated (UT) or treated for 1 h with bafimolycin A1 (BafA1), assessed by live-cell fluorescence microscopy (left) and automated quantification of spots (right). Scale bar (left), 5 μm. (c) Immunoblot analysis of the processing of endogenous unconjugated LC3-I to lipid-conjugated LC3-II in LPS-stimulated B cells with (+NH4Cl) or without treatment with NH4Cl (left). Actin serves as a loading control throughout. Right, quantification of LC3-II band intensity in the absence of NH4Cl, presented relative to that of actin. (d) Quantitative RT-PCR analysis of transcripts encoding autophagy-related molecules in ASCs 3 d after stimulation with LPS, presented relative to that in unstimulated B cells. (e) Immunoblot analysis of p62 in LPS-stimulated B cells (bottom). Above, quantification of band intensity (as in c). (f) Live-cell fluorescence microscopy of CD138+ PCs purified from the spleen of GFP-LC3 mice 5 d after the injection of NP-Ficoll (PCs) and of splenic B cells (B cells) and bone marrow CD138+ PCs (BM PCs) purified from unstimulated GFP-LC3 mice (right) and automated quantification of spots (left). Scale bar (right), 5 μm. *P 0.05, **P 0.01 and ***P 0.001 (Student's t-test). Data are representative of three or more independent experiments (a,c-e) or one experiment or more with >40 cells (b,f; average and s.e.m.).
UBE2QL1 and its homologue UBC-25 in C.Figure 7A Control or UBE2QL1-depleted (for 60 h) HeLa cells were stained with DAPI and an antibody against TFEB and automatically analyzed by confocal microscopy. Note nuclear translocation of TFEB upon loss of UBE2QL1. Scale bar: 20µm. B Automated quantification The intensity ratio of the cytoplasmic and nuclear TFEB signals was determined in both conditions. Graph represents data from four independent experiments with ≥ 100 cells per condition (mean ± SD). ***P<0.001; ****P<0.0001 (One-way-ANOVA with Bonferroni's multiple comparison test). C Cells were stained for LAMP1 and mTOR. Note the increase in lysosomes and lysosomal dissociation of mTOR in UBE2QL1 depleted cells. Scale bar: 20 µm. D Automated quantification The colocalization of mTOR and LAMP1 is shown as Pearson correlation coefficients (P.C.C.) of the signals. Graph represents data from three independent experiments with ≥ 70 cells per condition (mean ± SD). *P<0.05 (One-way-ANOVA with Bonferroni's multiple comparison test). E Automated quantification Number of LAMP1-positive vesicles per cell is shown. Graph represents data from three independent experiments with ≥ 70 cells per condition (mean ± SD). F Western blot analysis of lysates of cells treated As a positive control, cells were treated with the mTOR-inhibitor Torin1 (1 µM) for 2 h. GAPDH was probed as loading control. G C.elegans wild-type (N2) and mutant strains for ubc-25(ok1732), for scav-3(qx193) or the ubc-25 and scav-3 double mutant (ok1732;qx193) carrying a EGFP-Gal3 transgene were imaged in adult worms (L4 + 24 h). 3D reconstructions of the hypodermis. Small and big units represent 1 and 5 µm, respectively. H Average volume of permeabilized lysosomes within the region of hypodermis corresponding to 100 µm of body length was quantified. At least 20 animals were scored in each strain. *P<0.05; **P<0.01; ****P<0.0001 (One-way-ANOVA with Dunnett's multiple comparison test or Student's two-tailed unpaired t test). I Populations of worms with different extent of lysosome permeabilization were analyzed. Graph represents percentage of worms with less than 5, 5 to 20 or more than 20 permeabilized lysosomes in the region of hypodermis corresponding to 100 µm of body length.
Figure 2Expression of YAP1 in SCP‑1 cells transduced with FUS‑DDIT3 or EV and liposarcoma cell lines. One of at least two independent experiments with similar results is shown. FUS‑DDIT3‑expressing cell types are indicated in red. Expression of YAP1 in cytoplasmic (yellow) and nuclear (blue) fractions from SCP-1 cells transduced with FUS-DDIT3 or EV and liposarcoma cell lines. One of at least two independent experiments with similar results is shown. FUS‑DDIT3‑expressing cell types are indicated in red. Expression of FOXM1 and PLK1 in MLS cell lines. One of at least two independent experiments with similar results is shown. Strong nuclear expression of YAP1, FOXM1, and PLK1 in MLS patient samples (original magnification, x10 [inset, x20]). Intensity of nuclear YAP1 expression in liposarcoma patient samples. Immunoreactivity was assessed using a semi‑quantitative score (0, negative; 1, weak; 2, moderate; and 3, strong) defining the staining intensity in the positive control (hepatocellular carcinoma) as strong. Only tumors with at least moderate staining (semi‑quantitative score ≥2) and ≥30% YAP positive cells were considered positive for the purposes of the study. Proportion of cells with nuclear YAP1 expression in liposarcoma patient samples. Boxes represent mean values and lower and upper quartiles. Whiskers represent minimum and maximum values.
Fig.Figure 6(A) Western blots showed NDUFS3 expression in KO-NDUFS3 and GFP expression in the KO-GFP quadriceps samples. PGC-1α expression was normalized in the KO-NDUFS3 mice which was abnormally elevated in the KO-GFP. The same was observed for CIV (COX1), CIII (Core 2) and CII (SDHA). (B) Quantification of western blots showed in A. Statistical analysis was performed by One-way ANOVA followed by Tukey post-test. Data represent means ±SEM. (C) BN-PAGE western blots showed recovery of Complex I assembly into complex and supercomplexes in KO-NDUFS3 injected mice. CIV and CII were similar to wild-type in the KO-NDUFS3 samples, at 6 months. (D) Quantification of BN-PAGE western blots Statistical analysis was performed by One-way ANOVA followed by Tukey post-test. Data represent means ±SEM. (E) BN-PAGE In-gel CI and IV activities of KO-NDUFS3 mice were also reverted to levels similar to the WTFlx samples (F) Quantification of in-gel activities. Statistical analysis was performed by One-way ANOVA followed by Tukey post-test. Data represent means ±SEM. (G) Complex I/Citrate synthase activity of KO-NDUFS3 samples measured spectrophotometrically were similar to WTFlx samples and increased in comparison to KO-GFP mice. Statistical analysis was performed by One-way ANOVA followed by Tukey post-test. Data represent means ±SEM. (H) Total mtDNA levels were determined by qPCR. MtDNA levels in KO-NDUFS3 mice were decreased when compared to KO-GFP mice and not different from the levels in WTFlx mice. Statistical analysis was performed by One-way ANOVA followed by Bonferroni post-test. Statistical analysis was performed by One-way ANOVA followed by Tukey post-test. Data represent means ±SEM. (I) The serum lactate levels were similar to WTFlx in KO-NDUFS3 samples and significantly decreased when compared to KO-GFP levels. The individual KO-NDUFS3 animals are color coded. Green triangles represent the animals injected with rAAV9-eGFP whereas black triangles represent animals which did not receive an rAAV9-eGFP injection. Statistical analysis was performed by One-way ANOVA followed by Tukey post-test. Data represent means ±SEM.
Figure 4A. Schematic diagram of constructing chromosomal mutations in rpoC gene in E. coli based on a CRIPSR/Cas9 system. A 20-nt guide RNA targeting a region between K74 and K87 on β′ subunit is shown in purple. Double-strand DNA fragments, which contains synonymous mutations (yellow background) in sgRNA targeting region to facilitate clone selection and aimed mutations at K74 or K87 or both residues (only example for constructing the Ec-K74A-K87A strain is shown), were used as donor templates. B, C. Promoter activities of tarp (B) and fliCp (C) in E. coli wild-type (Ec-WT) and its mutants expressing ZBD-mutated RNAP (Ec-K74A, Ec-K87A and Ec-K74A-K87A) shown as mean ± SD from three replicates. D. Volcano plot showing log2-fold change of mRNA abundance as a ratio of Ec-K74A-K87A and Ec-WT strains. Blue dots represent genes with significant difference between these two strains. Red dots represent flagellar genes. Data are shown as mean values of three independent colonies. E. Relative mRNA levels of flagellar genes in Ec-K74A-K87A strain comparing with that in Ec-WT (normalized to 1 for each gene). Diagram for the role of σ28 (fliA gene) in flagella regulon hierarchy Clarke & Sperandio, 2005Fitzgerald, 2014(; ) is shown in the left panel. Data are mean ± SEM from three replicates. F. Motilities of Ec-K74A-K87A strains with over-expression of wild-type rpoC gene (Ec-K74A-K87A-C) or carrying the control plasmid (Ec-K74A-K87A-V). G. Activities of fliCp in Ec-K74A-K87A-C and Ec-K74A-K87A-V strains. L-arabinose was used at final concentration of 0.002%. Data are mean ± SD from three colonies.
Metabolic stressors inactivate HSF1 and disrupt proteostasis in tumor cells.Figure 6A, B Following treatment with 10 μM metformin for 3 days, the levels of HSF1 binding to endogenous HSP promoters were quantitated by chromatin IP (mean ± SD, n = 3, one-way ANOVA, **P < 0.01, ***P < 0.001). Normal rabbit IgG served as the negative control (A). Following the same treatment, HSP mRNA levels were quantitated by qRT-PCR (mean ± SD, n = 3, Student's t-test, ***P < 0.001) (B). C, D Following transfection with AMPKα1/2-targeting siRNAs for 2 days, WM115 cells were treated with 10 μM metformin for 3 days. HSP mRNA levels were quantitated by qRT-PCR (mean ± SD, n = 6, Student's t-test, n.s., not significant, *P < 0.05, **P < 0.01). C, D Following transfection with AMPKα1/2-targeting siRNAs for 2 days, WM115 cells were treated with 10 μM metformin for 3 days. HSP mRNA levels were quantitated by qRT-PCR (mean ± SD, n = 6, Student's t-test, n.s., not significant, *P < 0.05, **P < 0.01). E Following treatment with 10 μM metformin for 7 days, protein levels in WM115 and S462 cells were measured by immunoblotting. F, G Following treatment with 10 μM metformin for 7 days, levels of polyubiquitinated proteins were detected in both detergent-soluble and detergent-insoluble fractions in WM115 and A2058 cells using a Lys48-specific polyubiquitin antibody. H A2058 cells were treated with 10 μM metformin alone or co-treated with 2 μM compound C for 7 days. Individual proteins were detected by immunoblotting. I HEK293T cells stably expressing either scramble or HSF1-targeting shRNAs were treated with 10 μM metformin for 7 days. Individual proteins were detected by immunoblotting. J In HEK293T cells, a plasmid encoding HA-polyQ79-GFP was co-transfected with the indicated plasmids. Following treatment with and without 10 μM metformin for 5 days, the sizes of aggregates in detergent-insoluble fractions were quantitated by a Multisizer™ 3 Coulter Counter. Experimental details are described in Materials and Methods. K, L Immortalized and RAS-transformed MEFs were treated with either 10 μM metformin (K) or different concentrations of glucose (L) for 7 days. Individual proteins were detected by immunoblotting. M Following transfection with LacZ or polyQ79, HEK293T cells were grown under different concentrations of glucose for 5 days before measuring aggregate size.
Figure 3A. Confocal images of mitochondrial morphology in Drp1-/- 293T cells transfected with scrambled siRNA (control) or with hFis1-siRNA and then stained with MitoTracker (red). B. Confocal images of mitochondrial morphology in Drp1-/- 293T cells transfected with either Mfn1-Myc, Mfn2-Myc or OPA1-Myc, stained with MitoTracker (red) followed by immunostaining with anti-Myc antibody (green). C. Percentages (mean ± s.e.m.) of cells with indicated mitochondrial morphologies in Drp1-/- 293T cells transfected with either scrambled siRNA (Ctr), hFis1 siRNA, Mfn1-Myc, Mfn2-Myc or OPA1-Myc in three independent experiments for each condition (n represents the number of cells analyzed). D. hFis1 interacts with Mfn1, Mfn2 and OPA1 as well as Drp1, but not with Dyn2 at endogenous levels following chemical crosslinking. Wild-type (WT) and Drp1-/- 293T cells were in vivo crosslinked with 1% formaldehyde (FA) and cell lysates were used for co-immunoprecipitation (IP) with Protein G beads bound to rabbit normal IgG (negative control) or rabbit anti-hFis1 antibody as indicated, followed by immunoblotting with indicated antibodies. hFis1 binds to Mfn1, Mfn2 and OPA1 at endogenous levels also in the absence of chemical crosslinking. Cell lysates prepared from WT 293T (E) cells without chemical crosslinking were used for co-immunoprecipitation (IP) with Protein G beads bound to rabbit normal IgG (negative control) or rabbit anti-hFis1 antibody as indicated, followed by Western blotting with indicated antibodies. F. hFis1 binds to Mfn1, Mfn2 and OPA1 at endogenous levels also in the absence of chemical crosslinking. Cell lysates prepared from HeLa (F) cells without chemical crosslinking were used for co-immunoprecipitation (IP) with Protein G beads bound to rabbit normal IgG (negative control) or rabbit anti-hFis1 antibody as indicated, followed by Western blotting with indicated antibodies. G, H. Interaction of hFis1 with Mfn1/2 and with OPA1 are independent events. WT 293T cells were treated with control, OPA1 (G) or Mfn1 plus Mfn2 (H) siRNA, followed by in vivo crosslinking with 1% FA. Cell lysates were used for co-IP with Protein G beads bound to rabbit normal IgG (negative control) or rabbit anti-hFis1 antibody as indicated, followed by immunoblotting with indicated antibodies. I. Interactions between Mfn1/2 and OPA1 occur independent of hFis1. WT 293T cells were treated with control or hFis1 siRNA, followed by in vivo crosslinking with 1% FA. Cell lysates were used for co-IP with Protein G beads bound to mouse normal IgG (negative control) or mouse anti-OPA1 antibody as indicated, followed by immunoblotting with indicated antibodies. J. Interaction between Mfn1 and Mfn2 is not affected by hFis1 overexpression. 293T cells were transfected with empty vector or Myc-hFis1, followed by in vivo crosslinking with 1% FA. Cell lysates were used for co-IP with Protein G beads bound to mouse normal IgG or mouse anti-Mfn1 antibody, followed by immunoblotting with indicated antibodies.
a, AMBRA1–BECN1 interaction by yeast two-hybrid assay.figf2a, AMBRA1-BECN1 interaction by yeast two-hybrid assay. Yeast cells were co-transfected with the indicated plasmids and plated in medium with (+) or without (-) histidine (H) and adenine (A). AD, activation domain; DBD, DNA-binding domain; G4, Gal4; LAM, lamin. b, AMBRA1-BECN1 interaction in mammalian cells. 2FTGH cells were co-infected with retroviral vectors encoding BECN1 and the indicated Myc-tagged AMBRA1 proteins, or Myc-tagged β-gal as a negative control. CT, C terminal; FL, full length; NT, N terminal. Protein extracts were immunoprecipitated using an anti-Myc-tagged antibody (IP Myc). Purified complexes and corresponding total extracts were analysed by western blot using anti-Myc (WB Myc, upper panels) or anti-BECN1 (WB BECN1, lower panels) antibodies. When multiple bands are present, the upper band has the expected molecular mass. A scheme of the various AMBRA1 mutants is reported. c, AMBRA1 is in a complex that includes BECN1 and Vps34. Protein extracts from 2FTGH cells co-expressing BECN1 and the Myc-tagged AMBRA1 full-length protein (FL) or Myc-tagged β-gal were immunoprecipitated using an anti-Myc-tag antibody (IP Myc). Purified complexes and corresponding total extracts were analysed using anti-BECN1 (WB BECN1) and anti-Vps34 (WB Vps34) antibodies. b, Immunoblot analysis of E14.5embryobrain extracts using antibodies against Ambra1 (left panel) and β-gal (right panel). Specific bands (arrowheads) and unspecific signals (asterisk) are indicated. Molecular weights are measured in kilodaltons (kDa). The fusion protein can be revealed only by means of the more sensitive anti-β-gal antibody. e, BECN1 and AMBRA1 co-localize in mammalian cells. 2FTGH cells co-expressing BECN1 and Myc-tagged AMBRA1 were stained for Myc (green) and BECN1 (red). Scale bars, 8 µm.
Figure 7(A, B) Overview of the cortex immunohistochemically stained for Aβ plaques in 6 months old APP-NL-G-F mice treated with isotype control (A) and 4D9 antibody (B). Scale bar= 100µm. (C) Higher magnification images of cortical Aβ plaques in APP isotype and 4D9 injected mice. Scale bar= 10µm. (D) Quantification of cortical plaque area from stainings shown in C. Mann-Whitney U test, p=0.0082. (E) Quantification of total plaque coverage of Aβ stainings in the cortex shown in C. Mann-Whitney U test, p=0.014. (F) Immunoblot analysis of soluble (DEA fraction, top) and insoluble Aβ levels (FA fraction, bottom) in brains of APP-NL-G-F mice treated with either isotype control or 4D9 antibody. While a reduction in levels of soluble Aβ is evident from the DEA fractions no change in levels of insoluble Aβ could be detected in the FA fractions. Note that the higher background in the DEA immunoblot is due to a much longer exposure time, which was needed to visualize soluble Aβ. (G) MSD ELISA quantification of soluble Aβ42 in DEA extracts as shown in (F). Mann-Whitney U test, p=0.026. (H) MSD ELISA quantification of insoluble Aβ42 in FA extracts as shown in (F). Unpaired t-test (two-tailed) with Welch's correction; p=0.0877.
Figure 2A. A schematic showing the process of the machine learning-based functional screening. B. A heatmap showing changes in indicated cytokines in response to 500 nM treatment of PBMCs with 35 "tool" kinase inhibitors as the training set. Cytokines released in the media were measured by Luminex and normalized to DMSO control. C. Validation of predicted kinases as drivers of cytokine release by siRNAs in THP1 cells. Cytokine release was measured by Luminex and normalized to scrambled siRNA.
Figure 2 A-C Quantitative fluorescence microscopy of LipidTOX- and DAPI-stained female Cited4F/F Lin-Sca1+ progenitor cells transfected with Cre or control mRNA prior to differentiation in the presence of 100 nM Rosi for 8 days (n=5). **P=0.002 in t-test (Cre vs. Ctrl). Scale bar is 10 µm   D, E Ucp1 expression in female Cited4F/F Lin-Sca1+ progenitor cells transfected with Cre or control mRNA prior to differentiation in the presence of 100 nM Rosi for 8 days, as determined by Western blot with VCP as loading control (n=3)   F Cellular respiration in female Lin-Sca1+ progenitor cells differentiated in the presence of 100 nM Rosi for 8 days. The extracellular oxygen consumption rate (OCR) was determined upon injection of the indicated substances and normalized to DNA content. CL: CL-316243   G Normalized CL-stimulated uncoupled respiration of cells in (F). Values represent the means of the 3 time points after CL injection after subtraction of the means of the oligomycin time points (n=9)
a, Mice were infected for 21 days and liver granulomas analysed by transmission electron microscopy (TEM).figf1a, Mice were infected for 21 days and liver granulomas analysed by transmission electron microscopy (TEM). Shown are spacious vacuoles (SLAPs) containing multiple bacteria. Magnification, ×5,200. Region 1 and 2 (white boxes) are enlarged in the lower panels. b, SCID mice were infected as in a and liver sections stained for LAMP1 (green), bacteria (red) and DNA (blue). Shown is a LAMP1+ SLAP. c, The percentage of bacteria in LAMP1+ compartments was quantified and characterized as either spacious or tight-fitting. Mean ± s.e.m. for three mice examined. The image in a (from ref. 2) and the tissue sections were provided by E. Unanue.
68Ga]Pentixafor PET imaging of MM xenografts.Figure 2Real-time PCR analysis of cxcr4 transcript expression levels in HeLa (negative control) and in MM cell lines MM.1S and OPM-2. Shown is the mean relative expression ± SEM (n = 3 independent experiments). Values are normalized to the expression of ubiquitin (Ub). The asterisk indicates statistically significant differences (HeLa vs MM.1S: P < 0.001; HeLa vs OPM-2: P < 0.0001; Student's t-test). CXCR4 protein expression assessed by immunoblotting (one representative blot out of 3 is shown). Binding of [68Ga]Pentixafor to MM.1S and OPM-2 cells after the indicated incubation periods (n = 3 per cell line and time point). Shown is the mean ± SEM. The difference between the OPM-2 groups is statistically significant; *P < 0.0017 (one-way ANOVA). Mean tumor-to-background ratio (TBR) for [18F]FDG (left) and for [68Ga]Pentixafor (right) in MM.1S and OPM-2 xenograft-bearing NOD SCID mice. Shown is the mean ± SEM, n = 8 tumors (4 mice); *P = 0.0111 for [18F]FDG and *P = 0.0113 for [68Ga]Pentixafor (Student's t-test). One-way ANOVA revealed significant differences between the groups; P < 0.0001 (not graphically shown). Representative [68Ga]Pentixafor PET images of three mice bearing MM.1S (right shoulder) and OPM-2 (left shoulder) tumors. Flow cytometric quantification of CXCR4 cell surface expression on resected MM.1S and OPM-2 tumors. Data are the mean ± SEM, n = 4. *P = 0.0286; Mann-Whitney U-test. Correlation of [68Ga]Pentixafor PET mean TBR and CXCR4 cell surface expression assessed by flow cytometry. n = 8 tumors were analyzed. Mice (n = 4) bearing OPM-2 and MM.1S xenografts were coinjected with AMD3100 (right image, one representative mouse) or not pretreated (left image) before undergoing [68Ga]PentixaforPET. The white arrows point to the bladder. Quantification is shown in Supplementary Fig S3A.Source data are available online for this figure.