EMBO/sd-character-level-ner huggingface dataset:
Figure 4(A) Human dermal RDEBF treated daily with 10 μM LY2109761 (LY) (TGF-β Receptor I/II inhibitor‎), 10 μM 1D11 (neutralizing antibody to all TGF-βs) 100 nM Ang-(1-7), or 10 μM losartan (AT1R antagonist). After five days of treatment cell and matrix lysates were extracted and analyzed by western blotting for the indicated proteins, THBS-1, thrombospondin-1, β-tubulin was used as loading control. (B) Densitometric quantification of blots as in A. The abundance was normalized to β-tubulin and then expressed as % of untreated (-). P values are shown; the data were analyzed with one-way ANOVA with Dunnett's correction (n = 5 biological replicates). ( Data information: P values < 0.05 are considered significant and shown, ns = not significant. individual data points, mean ± S.E.M are shown. (C) Western blots of cell lysates from THP-1 monocytes treated daily with 10 μM LY2109761 or 100 nM Ang-(1-7) for two days. The blots were probed with antibodies against the indicated proteins. β-tubulin or β-actin was used as loading control. (D) Densitometric quantification of western blots from THP-1 monocytes treated daily with 10 μM LY2109761 or 100 nM Ang-(1-7) for two days. The abundance was normalized to β-tubulin or β-actin and then expressed as % of untreated (-). P values are shown; data were analyzed with one-way ANOVA with Dunnett's correction (n = 3-8 technical replicates) Data information: P values < 0.05 are considered significant and shown, ns = not significant. individual data points, mean ± S.E.M are shown. (E) Schematic illustration of fibroblast- THP-1 monocyte co-culture assay to assess the sustained effect of Ang-(1-7) and their principal cellular mediators. (F) Western blots of fibroblast lysates from co-cultures as described in E. The blots were probed with antibodies detecting fibronectin, thrombospondin-1 (THBS-1) and pSMAD-2/3. β-tubulin was used as loading control. (G) Densitometric quantification of western blots of fibroblast cell and matrix lysates as shown in F from multiple independent experiments, n = 4 -5 using cells from 4 donors with RDEB. The abundance was normalized to β-tubulin then and then expressed as % of both untreated. The data were analyzed by one-way ANOVA with Tukey's correction. Data information: P values < 0.05 are considered significant and shown, ns = not significant. For B, D and G individual data points, mean ± S.E.M are shown.
Figure 5A,B,F. Expression of Cx3cr1 (A), Spp1 (B) relative to Rps29 was analyzed by qRT-PCR in peritoneal macrophages, resident or inflammatory, treated in vitro with 10 ng/ml of activin A (Act) or untreated (Ctrl, arbitrarily set to 1). Results are combined from 2-5 independent experiments performed with cells pooled from at least 5 wt mice. C,D. Western blot analysis of total ear protein lysates of 12-14 week-old mice for osteopontin (OPN) and GAPDH (C). Each lane represents an individual mouse (n=3). (D) Band intensities were quantified and normalized to GAPDH. The expression level in one of the wt mice was arbitrarily set to 1. E. Ear skin sections from 10 week-old mice stained with anti-CD68 (red) or anti-osteopontin antibody (green); counterstained with Hoechst (blue). Osteopontin-positive macrophages in wt/Act and HPV8/Act mice are indicated with arrows. Representative of N=2 wt/wt and N=3 HPV8/wt, wt/Act and HPV8/Act mice. Boxed areas in top panel are shown at higher magnification in bottom panel. Scale bar top: 100μm, bottom: 20μm. A,B,F. Expression of Arg1 (F) relative to Rps29 was analyzed by qRT-PCR in peritoneal macrophages, resident or inflammatory, treated in vitro with 10 ng/ml of activin A (Act) or untreated (Ctrl, arbitrarily set to 1). Results are combined from 2-5 independent experiments performed with cells pooled from at least 5 wt mice. G. In situ gelatin zymography (top panel) and methylene blue staining (bottom panel) of the ear skin from 10 week-old mice. Representative images of 2-3 mice per genotype are shown. Scale bar = 500μm. H,I. Gelatin zymography of ear skin lysates from 10 week-old mice. Inverted image of a representative gel with 2 (wt/wt) or 3 (HPV8/wt, wt/Act, HPV8/Act) mice per genotype is shown in (H), quantification of the results obtained in 2 independent experiments is shown in (I). N=5 wt/wt, N=6 HPV8/wt, wt/Act and HPV8/Act mice. J. Representative pictures of ear skin from 10 week-old mice stained for MECA32 (red), LYVE1 (green); counterstained with Hoechst (blue). Boxed areas in top panel are shown at higher magnification in bottom panel. Scale bar top: 100μm, bottom: 20μm.K,L. Quantification of MECA32+ area (K) and average distance between MECA32+ vessels and basement membrane (BM) (L).
Inhibition of IIS extends the lifespan of OXPHOS deficient nematodes.Figure 1(A) OCR of wt and gas-1 mutant nematodes (normalized to nematode size as given by the COPAS Biosort (Time of Flight -TOF)). One representative curve shown (mean ± SEM; n=3) ****p value< 0.0001; Kruskal-Wallis Test, Dunn's post hoc correction. Representative curves show the lifespan of wt and gas-1(fc21) nematodes compared to (B) age-1 and age-1; gas-1 mutants animals. Average median lifespans ± SEM from all replicates indicated beneath representative curves. Representative curves show the lifespan of wt and gas-1(fc21) nematodes compared to (C) daf-16 and daf-16; age-1; gas-1 mutant animals. Average median lifespans ± SEM from all replicates indicated beneath representative curves. (D) Volcano plot of differentially phosphorylated proteins in gas-1(fc21) mutants compared to wt. Cutoffs: 20% FDR and |log2(foldchange)| >0.345 (in red). (E) Schematic representation of IIS pathway including results and predictions from IPA analysis of phosphoproteome of gas-1(fc21) compared to wt. Shades of red and green are proportional to fold change, as calculated by IPA. (F) Lifespan analysis of mev-1(kn1) nematodes compared to wt, age-1(hx546) and age-1(hx546); mev-1(kn1) animals. (G) (G) ATP measurements in wt, age-1, gas-1 and age-1; gas-1 mutant nematodes (normalized to wt), n=4. Error bars: Mean ± SEM, *p value <0.05, ***p value =0.001, Kruskal-Wallis Test, Dunn's post hoc correction. (H) OCR of gas-1 and age-1; gas-1 mutant nematodes (normalized to TOF). Left panel: One representative curve (Mean ± SEM). Right panel: Basal respiration in wt, gas-1 and age-1; gas-1 animals, pooled from 3 biological replicates, ****p value <0.0001, ns= non-significant, Kruskal-Wallis Test, Dunn's post hoc correction. (I) STED images of representative mitochondrial morphology in muscle cells of L4 mutant worms. Scale bar: 2 μm. (J) Left panel: Percentage of FRAP. Each point: mean recovery of ~30 bleached regions (2 regions per animal, 15 animals per condition) ± SEM. Right panel: values of the maximum (total) recovery per strain ± SEM, ***p value <0.001, **** p value <0.0001, one-way ANOVA, Tukey's post-hoc correction. (K) Representative lifespan of age-1; gas-1 nematodes fed with empty vector (EV) and fzo-1 RNAi bacteria. Average median lifespans ± SEM from all replicates indicated beneath representative curves. For all panels: wt= wild type, a= age-1, g= gas-1, a; g= age-1; gas-1.
Cytokine treatment of C2C12 myotubes alters the levels of TCA cycle intermediates and activates AMPK in an iNOS-dependent manner.Figure 5Representative immunofluorescence imaging for myoglobin and myosin heavy chain (MyHC) in not treated (NT) controls and IFNγ/TNFα (IT) samples treated with or without GW. Nuclei were visualized with DAPI staining (n=4). Quantification of mean fiber widths (n=4). (top) Western blot analysis for iNOS and tubulin (n=3). (bottom) Media nitrite levels (n=3). Heatmap visualizing mean concentration corresponding to metabolites of IFNγ/TNFα (IT) treated samples relative to not treated (NT) controls (n=3). Red and green indicate an increase or decrease in metabolite levels, respectively. Western blot analysis for pThr172-AMPK (pAMPK), total AMPK (AMPK), pSer79-ACC (pACC), and total ACC (ACC). Quantification of the pAMPK to AMPK ratio (left) and the pACC to ACC ratio (right) relative to the untreated control (n=3). Cellular ATP content quantified as a percentage of the untreated control (n=3). Western blot analysis for pThr389-S6K (pS6K), total S6K (S6K), pSer235/236-S6 (pS6), and total S6 (S6) (n=5). Quantification of the (left) pS6K to S6K ratio and the (right) pS6 to S6 ratio relative to the untreated control (n=5).
Figure 1(A) Heatmap of differentially regulated transcripts between HS and Rosacea as determined by RNA-sequencing of whole skin lesions (P<0.05). HS, skin biopsies from healthy individuals; Rosacea, skin biopsies from patients with rosacea. Blue color denotes low FPKM expression; red, high FPKM expression. (B) Volcano plot of differentially regulated genes between HS and Rosacea. The red dots show the significantly upregulated genes; blue dots, significantly downregulated genes (P<0.05). (C) Top-ranked upregulated KEGG terms related to immune responses in genes that were differentially regulated between Rosacea and HS samples revealed through Gene set enrichment analysis (GSEA). (D) GSEA analysis on RNA-sequencing data from Rosacea versus HS skin samples shows enrichment for mTOR signaling pathway in rosacea. NES, normal enrichment score; FDR, false discovery rate; FDR value < 0.05 was considered significant. Significance was calculated by permutation test. (E) Immunohistochemistry (IHC) of pS6 (Ser235/236) on skin sections from HS and Rosacea. Higher magnified images of boxed areas are shown at the bottom right of lower magnified images for each group. Epi, epidermis; Der, dermis. Scale bar: 50 μm. (F) Correlation of pS6 (Ser235/236) expression (from IHC) in the epidermis of human rosacea lesions (n = 17) with the Clinician's Global Severity (CGS) scores. Sperman's correlation coefﬁcient was used for the correlation analysis (two-tailed). (G) Immunostaining of pS6 (Ser240/244) in skin lesions from control and LL37-induced mice. Bottom right panels, magnified images of boxed areas. DAPI staining (blue) indicates nuclear localization. Scale bar: 50 μm. Right panel, the quantification of relative fluorescence intensity for pS6 (Ser240/244) in control and LL37 group (n=5 for each group). Data represent the mean ± SEM. **P < 0.01. two-tailed unpaired Student's t-test was used. (H) Immunoblotting of pS6 (Ser235/236 and Ser240/244), total S6, pAkt (Ser473) and total Akt in cell lysates from primary human keratinocytes treated with different doses of LL37 (0 - 8 μM) and scramble LL37 (8 μM) for 2 h. Scr, scramble LL37. pS6 and pAkt protein levels were analyzed relative to total S6 and Akt respectively. Tubulin is the loading control. Data (H) are representative of at least 3 independent experiments.
Figure 1Electron microscopy revealed a number of demyelinated or degenerated axons in PLP-150Q mice at the age of 5 months. LAQ (5 mg/kg) treatment at 3 months of age for 2 months improved myelination in PLP-150Q mice. Scale bars: 2μm (Low magnification) and 0.5 μm (High magnification). G-ratios, which were calculated and plotted against axon diameter with linear regression, were shown beneath the micrographs and were significantly decreased in PLP-150Q mice after LAQ treatment (G=0.6418±0.0191), compared with age-matched vehicle group (G=0.7727±0.0203). One-way ANOVA with Tukey's test. *P=0.0166. At least 182 axons per genotype were examined from 3 mice in each group. Western blotting showing that LAQ (5 or 25 mg/kg) treatment for 2 months up-regulates multiple myelin proteins (MBP, MOBP and MOG) in the corpus callosum in PLP-150Q mice, which were treated from 3 months of age. Ratios of MBP, MOBP or MOG to vinculin obtained from 3 independent experiments were presented on the right. One-way ANNOVA followed with Tukey's test. MBP: ***P=0.0009; MOG: ***P=0.0003; MOBP: **P=0.0067. Data are mean ±SEM Quantitative PCR of the transcript expression of myelin associated genes (MBP and MOG) in PLP-150Q mouse corpus callosum at 5 months of age after LAQ (5 or 25 mg/kg) treatment for 2 months. Student's t-test. MBP: ***P=0.0007; MOG: ***P=0.0009. Data are mean ±SEM (n=3). The MBP DNA promoter was inserted into the pGL4.1 luciferase report vector and was co-expressed with N-terminal MYRF (nMYRF) and mHTT to assess its transcription activity via the luciferase assay. MYRF markedly enhanced the MBP promoter activity. N-terminal mutant HTT significantly inhibited the reporter activity, ***P< 0.001; which was reversed by LAQ (5 μM) treatment, **P< 0.01. The ratios were obtained from 3 independent experiments. One-way ANNOVA followed with Tukey's test. Data are mean ±SEM. Immunoprecipitation of transgenic mHTT from 3-month-old PLP-150Q mouse brains revealed that 5 or 25 mg/kg LAQ treatments for 2 months reduced the interaction between transgenic mutant HTT and MYRF. fMYRF: full-length MYRF; nMYRF: N-terminal MYRF. The ratio of immunoprecipitated MYRF to input obtained from 3 independent experiments was shown on the right. One-way ANNOVA followed with Tukey's test. **P=0.009. Data are mean ±SEM.
Figure 6AK2 activity assay with varying AMP concentrations. Pyruvate kinase (PK) and lactate dehydrogenase (LDH) thereby couple the oxidation of NADH to the interconversion of adenine nucleotides. The concentration of ATP is maintained constant, whereas the concentration of AMP is titrated. AK2 activity assay with varying ADP concentrations. Hexokinase (HK) and glucose-6-phosphate dehydrogenase (G6PDH) thereby couple the reduction of NADP+ to the interconversion of adenine nucleotides. The concentration of ADP is titrated. AK2 activity in dependence of its substrate AMP. Heterologously expressed and purified AK2 variants exhibited maximal activity at around 40 µM AMP with an apparent KM of 0.012 mM AMP. The profiles for both AK2C40S,C232S and AK2C40S,C42S,C92S,C232S are superimposable. Reported values are the mean of at least 3 independent experiments; error bars represent ±SD.   AK2 activity in dependence of its substrate ADP. Maximal activity was observed at around 1-2 mM ADP with an apparent KM of 0.12 mM and 0.1 mM ADP for AK2C40S,C232S and AK2C40S,C42S,C92S,C232S, respectively. The profiles for both AK2C40S,C232S and AK2C40S,C42S,C92S,C232S are thus highly similar. Reported values are the mean of at least 3 independent experiments; error bars represent ±SD.   Activity assay for AK2-HA variants isolated from HEK293 cells. Indicated AK2-HA variants were isolated by native immunoprecipitation from HEK293 cells stably expressing these variants. Then, the AK2 activity assay described in (B) was performed. While import-competent AK2 variants exhibited full activity, a cytosolic AK2 variant with the native N-terminus showed very low activity. This activity strongly increased upon incubation with the DPP9 inhibitor 1G244 or upon using DPP9-insensitive cytosolic AK2 variants. The enzymatic inactive K28Q variants served as negative control indicating that only AK2-HA-dependent activity ways measured by our assay. Reported values are the mean of 2 independent experiments; error bars represent ±SD. Model for the cytosolic quality control pathway controlling cellular AK2 level and its localization. Details see text.
Figure 4A Schematic diagram showing organization of mouse major and minor satellite repeats. Major and minor satellite sequences comprise six megabases of a 234 bp- repeats and six hundred kilobases of a 120 bp repeats, respectively. The terms "sense (S)" and "antisense (As)" refer to T- and A-rich transcripts, respectively. The length of major and minor satellite sequences used to perform in vitro transcription is indicated. B RNA pull-down assay using biotinylated major and minor satellite RNA transcripts and whole cell extracts from Miwi heterozygoous (Miwi+/-), knockout (Miwi-/-), slicer activity-deficient mutant (Miwi-/ADH), and Mov10l1 germline knockout (Mov10l1f/-;Stra8-Cre) testes. Mov10l1f/-;Stra8-Cre mice represent a pachytene piRNA-deficient condition. Approximately one repeat of In vitro transcribed major and minor satellite RNAs corresponding to the sequences in A were used as baits. Precipitated proteins were visualized by Western blotting using antibodies against the indicated proteins (MIWI and MILI). The input equals 1/10 to 1/80 of the protein lysate used in the pull-down. Transcripts corresponding to 300 nt and 162 nt of the 18S rRNA sequence served as controls for the antisense (As) and sense (S) sequence of major and minor satellite transcripts, respectively. The blot was re-probed with anti-GAPDH antibody serving as a negative control. C In vitro RNA processing assay with nuage-nuclear fraction from testicular lysates of Miwi knockout (-/-), heterozygous (+/-), and slicer activity-deficient (-/ADH) mutants, and 5'-end radioactively labelled RNA targets corresponding to approximately one and two repeats of major and minor satellite sequences (see panel A for details). Transcripts corresponding to 162, 300, and 542 nt of the 18S rRNA sequence served as controls. Arrow indicates the cleavage products of In vitro transcribed sense major satellite RNAs after reactions. D In vitro RNA processing assay with nuage-nuclear fraction from Mov10l1 heterozygous (Mov10l1f/+;Stra8-Cre) and Mov10l1 germline knockout (Mov10l1f/-;Stra8-Cre) testis lysates, and 5'-end radioactively labelled RNA targets corresponding to approximately one and two repeats of sense and antisense major satellite sequences. Mov10l1f/-;Stra8-Cre mice represent a pachytene piRNA-deficient condition. The arrow indicates the cleavage product of the in vitro transcribed sense major satellite RNA. E Major satellite RNA with respective piRNA target sequences. piR major-a and b target the sense strand of major satellite transcripts at their cleavage sites. The MIWI slicer activity results in 125- and 119-nt cleavage products. RNA-immunoprecipitation was performed using anti-MIWI antibody. RNA was extracted from anti-MIWI immunoprecipitates and subjected to Northern blot analysis using probes specific for potential piRNAs (piR major-a and b), as indicated in panel 20% of input RNA from each group was used as the control. The blot was re-hybridized with piR-A and piR-E complementary probes to demonstrate the efficiency of the RNA-IP. A miR-16 complementary probe served as a negative control.
Figure 5Heatmap showing Th17 signature genes (left) or Th17 regulatory genes (right) up- or downregulated in WT or Stat4-KO LPLs by LIF treatment (n=2). Quantitative mRNA expression analysis of the indicated genes in LPLs from colitis mice (n=3 per group). Quantitative mRNA expression analysis of the indicated genes in LPLs from colitis mice (n=3 per group). Quantitative mRNA expression analysis of the indicated genes in WT or Stat4-KO naïve CD4+ T cells on day 4 of induction into Th17 cell subsets in the absence or presence of LIF. The GAS-luciferase reporter activity assay was performed in Stat3-/- MEFs transfected with empty vector (EV) or STAT4 constructs and then treated with LIF for 8 hours. The GAS-luciferase reporter activity assay was performed in Stat3-/- MEFs transfected with empty vector (EV) or STAT4 constructs and then treated with LIF for 8 hours. Relative SIE-(I) activities in Stat3-/- MEFs transfected with EV or the indicated STAT constructs and treated with LIF for 8 hours. Relative AGG-luciferase reporter activities in Stat3-/- MEFs transfected with EV or the indicated STAT constructs and treated with LIF for 8 hours. The Il17a/Il17f locus, including qPCR primer sites covering SIEs (purple triangles) or AGG elements (green triangles). ChIP analysis of naïve CD4+ T cells obtained from IL-17A-eGFP mice cultured under Th0 or Th17 conditions in the absence or presence of LIF for 3 days. GFP+ cells isolated by flow cytometry were restimulated with IL-6 in the presence or absence of LIF, crosslinked with formaldehyde and immunoprecipitated with anti-STAT3 (top) or anti-STAT4 (bottom) antibodies, followed by the amplification of the immunoprecipitated DNA by quantitative PCR with the p1-p10 primer pairs. The results are presented relative to the amount of input DNA. ChIP analysis of naïve WT or Stat4-KO CD4+ T cells cultured under Th0 or Th17 conditions in the absence or presence of LIF for 3 days. The cells were restimulated with IL-6 or LIF for 30 minutes, crosslinked with formaldehyde and immunoprecipitated with the anti-STAT3 antibody, followed by the amplification of the immunoprecipitated DNA by quantitative PCR with the p1-p10 primer pairs. The results are presented relative to the amount of input DNA. Relative Il17a-SIE- (left) or AGG-luciferase reporter (right) activities in Stat3-/- MEFs transfected with EV, STAT3 or STAT4 or cotransfected with STAT3 and STAT4 and then treated with IL-6 or LIF for 8 hours. Relative Il17a promoter-luciferase reporter activities in Stat3-/- MEFs transfected with the indicated constructs and then treated with LIF for 8 hours.
a) Left: starvation increases the level of RFP–LC3 puncta (arrows) in rat cortical neurons.figf2(a) Left: starvation increases the level of RFP-LC3 puncta (arrows) in rat cortical neurons. Middle: quantification of the percentage of cells with RFP-LC3 puncta. Right: pretreatment with 10 mM 3-MA attenuates the starvation-induced increase in LC3-II level in neurons. Data are means ± s.e.m.; n=3. Samples were run on the same gel and spliced together to create the image shown. (b) Knockdown of EndoB1 abolishes the starvation-induced increase in LC3-II levels (left) and the percentage of cells with RFP-LC3 puncta (right) in neurons. Data are means ± s.e.m.; n=3. (c) Starvation enhances Cdk5 activity (top) and increases the level of Thr 145-phosphorylated EndoB1 in neurons (top and bottom). Histone H1 was used as a Cdk5 substrate. Data are means ± s.e.m.; n=3. (d,e) Pretreatment with Ros (d) or knockdown of Cdk5 (e) attenuates starvation-induced EndoB1 phosphorylation at Thr 145 and autophagy induction in neurons. Data are means ± s.e.m.; n=3 for d and n=8 for e. DMSO, dimethylsulphoxide. (f) Overexpression of EndoB1T145A significantly reduces the starvation-induced increase in LC3-II levels (left) and the percentage of cells with RFP-LC3 puncta (right) in neurons. Data are means ± s.e.m.; n=6. Uncropped images of blots are shown in Supplementary Fig. S8.
Histological findings in neurons and pancreas.Fig 1(A) Swelling of neurons in the vestibular nucleus due to fine vesiculation (arrows) and clear vacuolization (arrowhead) of the cytoplasm. HE, scale bar 100 μm. (B) Clear cytoplasmic vacuolization (arrows) in cerebellar corticalPurkinje cells. HE, scale bar 100 μm. (C) Normal cerebellar cortex of an unaffected dog shows viable Purkinje cells (arrows) and a dense granular cell layer. HE, scale bar 100 μm. (D) Marked neuronal loss is present the cerebellar cortex of an affected dog. The number of neurons in the granular cell layer is reduced and only scattered Purkinje cells remain (arrow). HE, scale bar 100 μm. (E) Axonal spheroids of varying quality were seen in the white matter (arrows) of cerebellum and brainstem. HE, scale bar 100 μm. (F) Diffuse cytoplasmic vacuolization of the exocrine pancreatic acinar cells. HE, scale bar 100 μm. (G) Purkinje cell with numerous single-membrane bound, cytoplasmic vacuoles tethering to each other (arrows, inset). Electronmicrograph, scale bar 2 μm. Inset: scale bar 1 μm. (H) Axonal spheroid containing aggregated degenerated mitochondria, occasional double-membrane-bound autophagosomes (arrows) and free electron dense material, compressed by a peripheral clear vacuolar space. Electronmicrograph, scale bar 0.5 μm. Abbreviations: n, nucleus; ml, molecular layer; pl, Purkinje cell layer; gl, granular cell layer.