EMBO/sd-character-level-ner huggingface dataset:
TSC1 function restricts oxidative phosphorylation and ROS production.Figure 4TSC1 knockdown increases respiration in an mTORC1 and MYC dependent manner. Rate of basal oxygen consumption in P493-6 (-Tet) cells expressing scrambled control sh-RNA or TSC-specific sh-RNA, treated with 20 nM rapamycin for 12 h, or tetracycline to repress MYC for 48 h where indicated TSC1 knockdown increases respiration in an mTORC1 and MYC dependent manner. Rate of basal oxygen consumption in response to 10 μM of the chemical uncoupler DNP to determine maximal respiration or 10 μM of the ATP synthase inhibitor oligomycin where indicated (B) (mean ± st. dev., n=6 biological replicates). Ratio of mitochondrial to genomic DNA determined by qRT-PCR in P493-6 (-Tet) cells expressing either scrambled control sh-RNA or a TSC1-specific sh-RNA, and treated with 20 nM rapamycin for 12 h where indicated (mean ± st.dev., n=3 technical replicates). expression of cytochrome C (CYCS) or ATP5G1 mRNAs (D) determined by qRT-PCR in P493-6 (-Tet) cells expressing either scrambled control sh-RNA or a TSC1-specific sh-RNA, and treated with 20 nM rapamycin for 12 h where indicated (mean ± st.dev., n=3 technical replicates). TSC1 knockdown results in elevated ROS levels in a MYC and mTORC1 dependent manner. FACS analysis of DCF-DA stained P493-6 (-Tet) cells expressing scrambled control sh-RNA or a TSC1-specific sh-RNA to evaluate ROS production and treated with 20 nM rapamycin for 12 h and tetracycline for 48 h where indicated (mean ± st.dev., n=3 biological replicates). TSC1 knockdown results in increased phosphorylation of SAPK/JNK. Immunoblot of SAPK/JNK Thr183/Tyr185-phosphorylation in P493-6 (-Tet) cells expressing scrambled control sh-RNA or a TSC1-specific sh-RNA. α-tubulin expression serves as loading control. Antioxidant treatment rescues cells from death caused by TSC1 knockdown. Relative viable cell number counts of P493-6 (-Tet) cells expressing scrambled control shRNA or TSC1-specific sh-RNA 3 days after seeding equal number of viable cells (Trypan blue exclusion), in the presence of 10 μM of the antioxidant BHA where indicated (mean ± st.dev., n=3 biological replicates).
Figure 5A. Quantitative RT-PCR detection of CAV1, CAV2 and CAVIN1 in primary human umbilical venous endothelial cells (HUVECs) upon Notch blockade (dnMAML) and induction (NICD). n=3, data represent mean ± SEM, unpaired t-test. B. Representative western blot of CAV1 expression in HUVECs upon Notch manipulation. C. Densitometric analysis of western blot in B. Data represented as fold change over GFP control. n=3, data represent mean ± SEM, unpaired t-test. D. Representative western blot of CAVIN1 expression in HUVECs upon Notch induction. E. Densitometric analysis of western blot in D. Data represented as fold change over LacZ control. n=5, data represent mean ± SEM, unpaired t-test. F. Expression of CAV1 in HUVECs upon Notch manipulation and HEY1 induction. n=3, data represent mean ± SEM, unpaired t-test. G. Expression of Cav1, Cav2, Cavin1 in primary microvascular ECs freshly isolated from skeletal muscle of RbpjiΔEC mice compared to control mice. n=5, data represent mean ± SEM, unpaired t-test. H. Expression of Cav1, Cav2, Cavin1 in primary microvascular ECs freshly isolated from skeletal muscle of NICDiOE-EC mice compared to control mice. n=4, data represent mean ± SEM, unpaired t-test. I. Expression of Cav1, Cav2, Cavin1 in microvascular endothelial cells isolated from skeletal muscle of mice kept on control diet (CD, 10% fat, 70% carbohydrates) or high-fat diet (HFD, 60% fat, 20% carbohydrates) or high-fat and sugar diet (HFS, 60% fat, 20% carbohydrates and 42 g/l sucrose in drinking water). n=4, data represent mean ± SEM, unpaired t-test. J, K. Quantification of number of caveolae attached to apical membrane in EC of cardiac capillaries from NICDiOE-EC mice (6 segments of 1000 nm length from 3 hearts per genotype) or RbpjiΔEC (6 segments of 1000 nm length from 8 hearts per genotype) based on electron microscopy. Box plot depicts median and percentiles (10th, 25th, 75th, 90th), unpaired t-test, * means p<0.05. Representative immune-histochemistry of Cav1 staining in heart sections from control and RbpjiΔEC mice. n=3, scale bar 50 µm. Representative confocal images of Cav1 staining in heart sections from control and RbpjiΔEC mice. n=3, scale bar 50 µm.
SseL reduces autophagic flux and can promote bacterial replication in macrophages.Figure 8(A) Lipidated LC3B (LC3-II) levels in RAW264.7 macrophages infected with the indicated strains of S. Typhimurium for 12 h. Cells were left untreated (−) or subjected to 10 mM NH4Cl treatment (+) for 2 h before harvesting. (B) Quantification of relative LC3 II levels in infected cells. Denistometry of bands was analysed using Image J software. The ratio of LC3-II/tubulin signal intensity was normalized to 1 for wild-type untreated infections. The graph shows the mean relative LC3 II/tubulin ratios ± SEM for 3 independent experiments. C) RAW264.7 or (D) murine bone marrow-derived primary macrophages were infected with the indicated strains of S. Typhimurium for (C) 10 h or (D) 16 h and bacterial replication was monitored using flow cytometry. Results are represented as fold replication normalized to wt; values are the means ± SEM of at least 3 independent experiments. * p<0.05; ** p<0.01.
An ATP-induced enteric gliosis in postoperative bowel inflammation A: ATP measurement at the indicated postoperative time points in peritoneal lavages of mice that underwent intestinal manipulation (IM) or were naïve.Figure 3A: ATP measurement at the indicated postoperative time points in peritoneal lavages of mice that underwent intestinal manipulation (IM) or were naïve. B: PCA plot of gene expression from POI mice at different disease stages and naïve mice; n=3 for each group. C: Visual representation of p-value (-log10) against fold enrichment of GO terms associated with enriched genes in mice that underwent IM. D: Heat map of enriched genes connected with GO term gliosis in mice that underwent IM or in naïve animals. E: Venn diagram of gliosis genes expressed in vitro and in vivo. F-H: qPCR analysis of indicated gliosis marker in mice that underwent IM (n=7). I: Histological analysis of EGC proliferation in vivo. Representative confocal images and quantification show Sox10 (green) and Ki67 (violet) positive EGCs (white arrows) in the small bowel muscularis externa at naïve, IM6h and 24h. Scale bar 50µm. J: Experimental setup for glia specific RNA analysis. GFAP-cre+ x Ai14-floxed mice underwent IM and small intestine ME was digested and sorted for tdTomato by FACS to provide glia-specific RNA for qPCR measurements seen in (K-M). K-M: qPCR analysis of indicated gliosis markers in td+ glia cells from naïve mice and mice that underwent IM (n=5-7, POI mice).
OCRL deficiency impairs lysosome positioning leading to mTOR inactivation and abnormal nutrient sensingA Immunoblot analysis of endogenous p-mTOR (Ser2448), total mTOR, p-p70 S6K (Thr389) and total p70 S6K in NHF, Lowe 1676 and Lowe 3265 cells grown in complete media or 5 h serum starvation.Figure 2A Immunoblot analysis of endogenous p-mTOR (Ser2448), total mTOR, p-p70 S6K (Thr389) and total p70 S6K in NHF, Lowe 1676 and Lowe 3265 cells grown in complete media or 5 h serum starvation. p-p70 S6K (Thr389) over total p70 S6K ratios normalized to NHF cells, three independent experiments. B Immunoblot analysis of endogenous p-mTOR (Ser2448), total mTOR , p-p70 S6K (Thr389) and total p70 S6K in WT (Ocrl+/y) and IOB MEFs grown in complete medium or 5 h serum starvation. p-p70 S6K (Thr389) over total p70 S6K ratios normalized to NHF cells, three independent experiments. C Data information: data are presented as mean ± SEM. * ≤ 0.05, ** ≤ 0.01, ***≤ 0.001, ****P ≤ 0.0001, n.s., not significant, calculated by two-tailed Student's t-test unless otherwise stated. C Immunoblot analysis of GFP, p-mTOR (Ser2448), total mTOR , p-p70 S6K (Thr389) and total p70 S6K in Lowe 1676 transfected with or without GFP-OCRL. Data information: , data are presented as mean ± SEM. * ≤ 0.05, ** ≤ 0.01, ***≤ 0.001, ****P ≤ 0.0001, n.s., not significant, calculated by two-tailed Student's t-test unless otherwise stated. D NHF and Lowe 1676 cells were grown in complete media, amino-acid (aa)/FBS starved for 5 h, or starved and then recovered in amino-acid/FBS-containing medium, then immunoblotted using antibodies against endogenous p-mTOR (Ser2448), total mTOR, p-p70 S6K (Thr389) and total p70 S6K. Data information: data are presented as mean ± SEM. * ≤ 0.05, ** ≤ 0.01, ***≤ 0.001, ****P ≤ 0.0001, n.s., not significant, calculated by two-tailed Student's t-test unless otherwise stated. E Quantification of p-mTOR (Ser2448) over total mTOR ratios normalized to NHF cells as shown in D, n = 3. Data information: data are presented as mean ± SEM. * ≤ 0.05, ** ≤ 0.01, ***≤ 0.001, ****P ≤ 0.0001, n.s., not significant, calculated by two-tailed Student's t-test unless otherwise stated. F Quantification of p-p70 S6K (Thr389) over total p70 S6K ratios normalized to NHF cells as shown in D, n = 3. Data information: data are presented as mean ± SEM. * ≤ 0.05, ** ≤ 0.01, ***≤ 0.001, ****P ≤ 0.0001, n.s., not significant, calculated by two-tailed Student's t-test unless otherwise stated. G WT and IOB MEFs were grown in 1% FBS, 15% FBS media with or without 2 μmol MHY1485 for 4 days. Relative cell number is the ratio of cells on day 4 normalized to day 1, three independent experiments. ****, #### P < 0.0001, calculated by two-way ANOVA. H NHF and Lowe 1676 cells were grown in 1% FBS, 15% FBS media with or without 2 μmol MHY1485 for 4 days. Relative cell number is the ratio of cells on day 4 normalized to day 1, three independent experiments. ****, #### P < 0.0001, calculated by two-way ANOVA.
Figure 7Immunofluorescence analysis of lipofuscin (red) and IBA1 (green) in sagittal brain sections. Representative images of the Thalamus (TH). Scale bar indicates 25 µm. Western blot analysis of ubiquitin and the autophagy marker LC3 in RIPA, and p62 in RIPA and urea total brain lysates from 4.5-month-old mice with the indicated genotype. Calnexin used as a loading control. (n = 3 biological replicates per genotype). Quantification of B. Protein expression was normalized to levels in wild type animals. Data represent the mean ± SD. Immunofluorescence analysis of p62 in sagittal brain sections. Representative images of indicated brain regions (CTX = Cortex; TH = Thalamus; CBX = Cerebellum; Medulla = MY). Scale bar indicates 100 µm. Localization of p62 mainly in NeuN positive cells in the cortex (CTX) of 4.5-month-old mice. Minor overlap of aggregates with microglial marker IBA1. Scale bar indicates 15 µm. Complete overlap of co-staining of p62 with NeuN in the cortex (CTX) and not with the reactive astrocycte marker GFAP. Scale bar indicates 15 µm.
Schematic diagram.Figure 8DOCK5 deficiency results in Rac1 inhibition and the activation of mTOR/mTORC1 (raptor)/S6K1 signaling to increase the phosphorylation of IRS-1 Ser1101 and inhibit InsR-Akt phosphorylation cascades. These changes facilitate the hepatic gluconeogenesis and exacerbate both glucose metabolic disorders and IR.
Figure 6(A) Proliferation of WT and EVL-/- MLEC was assessed by BrdU incorporation. The number of BrdU positive cells (red) was normalized to the total number of cells (white). Eight fields of view per condition and genotype for each of 5 independent WT and EVL-/- cell batches; error bars represent SEM; **p<0.01, ***p<0.001, #p=0.171 non-significant vs. Basal EVL-/-, one-way ANOVA with Bonferroni's multi comparison test; scale bar = 50 µm. (C) Sprouting of WT and EVL-/- primary MLEC spheres in the absence (Basal) or presence of VEGF (50 ng/ml); asterisks indicate sprouts; scale bars, 100 µm.
Figure 4(A, B) IL-2Rα ΔSS is stabilized in UBQLN4-knockdown cells. HeLa cells were transfected with three distinct siRNA duplexes for UBQLN4 (UBQLN4 siRNA#1~#3) or control siRNA. At 48 h after siRNA transfection, Flag tagged-IL-2Rα ΔSS was expressed in the cells. At 24 h after IL-2RaSS transfection, the cells were chased with 20 μg/mL CHX and harvested at the indicated time after CHX addition. Anti-Flag signals in the control or UBQLN4 siRNA-treated cells (siRNA#1~#3) were quantified at the indicated time points. The data represent mean ± SEM calculated from 4 independent experiments (n=4). The UBQLN4 signal is marked by an arrowhead, while the band just below it (indicated by an asterisk) is a non-specific band, since this signal was never affected by any of the UBQLN4 siRNAs. Actin was used as a loading control. (C) Polyubiquitin modification of IL-2Rα ΔSS was not diminished, but rather strengthened, in UBQLN4 knockdown cells. At 48 h after transfection of HeLa cells with siRNA for UBQLN4 or BAG6, Flag-tagged IL-2Rα ΔSS and T7-tagged ubiquitin were expressed with (+) or without (-) 10 mM MG-132. From these cells, Flag-tagged IL-2Rα ΔSS was affinity-purified using Hot lysis analysis, and Flag-precipitates were blotted with an anti-T7 antibody to detect polyubiquitination of IL-2Rα ΔSS.
Figure 1A. A family tree for the juvenile ALS patient with L95fs mutation in Sigma 1 receptor (Sig1R). B. Schematic diagram of domain structure of Sig1R, coded by SIGMAR1. Amino acid sequence misplaced by L95fs mutation is shown by a red rectangle. Note that both of ALS-causative mutations (L95fs, E102Q; highlighted with red arrows) are located near the ligand-binding motif. In addition, recently identified mutations that cause distal hereditary motor neuropathy (dHMN), E138Q, E150K and a splice-site mutation resulting in in-frame deletion of 20 amino acids (Δ31-50), are indicated by green arrows.
Multiplexed in vivo base editing.Figure 4Sanger-sequencing chromatograms showing the target region of sgTrp53Q97* in wild-type (WT) and base edited (BE) cells. Arrowheads highlight cytosines of the protospacer that show base editing 5 days after transduction of BE3-expressing NIH3T3 cells with Lenti-sgTrp53Q97 EditR was used to calculate the frequency (%) of C-to-T conversion at C8 of the protospacer targeted by sgTrp53Q97* in BE3-expressing NIH3T3 cells 5 days after transduction with the indicated sgRNA vectors. Overview of the intraductal injections performed in WapCre;Brca1F/F;Trp53F/+;Col1a1invCAG-BE3/+ (Trp53F-het WB1P-BE3) females with high-titer lentiviruses encoding Myc and either a non-targeting sgRNA (Lenti-sgNT-Myc), the sgRNA targeting Trp53 (Lenti-sgTrp53Q97*-Myc) or two arrayed sgRNA cassettes encoding sgPik3caE545K and sgTrp53Q97* (Lenti-sgPik3caE545K/sgTrp53Q97*-Myc). Kaplan-Meier curves showing mammary tumor-specific survival for the different models. WapCre;Brca1F/F;Trp53F/+;Col1a1invCAG-BE3/+ females injected with Lenti-sgPik3caE545K/sgTrp53Q97*-Myc (n=6) showed a reduced mammary tumor-specific survival compared to animals injected with Lenti-sgTrp53Q97*-Myc (n=5) vectors (76 days after injection vs 101 days after injection, P < 0.05 by Mantel-Cox test). Females injected with Lenti-sgNT-Myc (n=11) did not develop any palpable tumors during the 150 days observation period. BE Analyzer was used to assess from next-generation sequencing data the fraction of wild-type Trp53 alleles, base edited alleles or alleles with indels in tumors from WapCre;Brca1F/F;Trp53F/+;Col1a1invCAG-BE3/+ animals injected with Lenti-sgTrp53Q97*-Myc. Tumors from WB1P-BE3 animals injected with Lenti-sgNT-Myc mice were used as control. TIDE analysis showing the spectrum of indels of the targeted Trp53 alleles in two independent representative tumors from WapCre;Brca1F/F;Trp53F/+;Col1a1invCAG-BE3/+ mice injected with Lenti-sgTrp53Q97*-Myc. For the two tumors shown in (F), Sanger-sequencing chromatograms showing the target region of sgTrp53Q97*. Arrowheads highlight cytosines of the protospacer that show base editing. In the lower example the gene was inactivated by a deletion, while in the upper one by Q97* base editing. Of note, the allele with the indel also displays base editing at C6 of the protospacer.
Increasing VEGF doses inhibit activation of endothelial SMAD2/3.Figure 5Relative gene expression of Id1, specifically induced by activated SMAD1/5, and Pai1, specifically induced by activated SMAD2/3, in total muscle tissues 1 week after implantation with V Low-, V Med- and V High-expressing myoblast clones or control cells (Ctrl), expressed as fold-change versus the Ctrl group. Data represent individual values, with mean ± SEM (n = 3 or 4, as indicated); **P < 0.01 and ***P < 0.001 by one-way ANOVA with Bonferroni multiple comparisons test, after data normalization by logarithmic transformation; V Low versus Ctrl P = 0.0002; V Low versus V Med P = 0.005; V Low versus V High P = 0.0016. Immunofluorescence staining of endothelium (CD31, in red), nuclei (DAPI, in blue), and phosphorylated SMAD2/3 (in cyan) on cryosections of similarly injected muscles confirmed that endothelial SMAD2/3 activation was inhibited by increasing VEGF doses. Scale bar = 10 μm; n = 3.
Differential gene expression of HSCs during lymphopoietic compensation.Figure 5 A Number of genes that are differentially expressed in WT HSCs co-transplanted with NSG or uMT-/- HSCs as compared to WT HSCs co-transplanted with WT HSCs in the control group. Gene lists are generated by Partek Flow. Thresholds are defined as p<0.05 and fold change <-2 or >2  B Biological functions that are expected to increase or decrease based on the observed gene expression changes generated by the Ingenuity Pathway Analysis (IPA). Representative genes involved in each biological function are shown on the left. HSPC is an abbreviation for hematopoietic stem and progenitor cells. NK is an abbreviation for natural killer cells. Shown are the functions with Fisher"s exact test p-value<0.05 Data information: Ingenuity Pathway Analysis (IPA) was used to perform gene ontology analysis on differentially expressed genes. Data were collected at month 7 post transplantation for the uMT-/- group and month 8 post-transplantation for the NSG group. n = 3 mice for each group, except for the NSG control group where n=2 mice  C Top genes whose expressions are both up regulated or both down regulated in the NSG and uMT-/- co-transplantation groups. Shown are the genes whose expression passes the threshold p<0.05 and fold change <-2 or >2 Data information: Ingenuity Pathway Analysis (IPA) was used to perform gene ontology analysis on differentially expressed genes. Data were collected at month 7 post transplantation for the uMT-/- group and month 8 post-transplantation for the NSG group. n = 3 mice for each group, except for the NSG control group where n=2 mice  D Predicted upstream regulators based on the observed gene expression changes and the known gene regulatory relationships in the IPA data base Data information: Ingenuity Pathway Analysis (IPA) was used to perform gene ontology analysis on differentially expressed genes. Data were collected at month 7 post transplantation for the uMT-/- group and month 8 post-transplantation for the NSG group. n = 3 mice for each group, except for the NSG control group where n=2 mice  E Regulatory genes of key hematopoiesis steps that are significantly changed in the NSG co-transplantation group. Common myeloid progenitor (CMP), megakaryocyte/erythrocyte (MEP), granulocyte/macrophage progenitor (GMP), Erythrocytes (Ery), granulocytes (Gr), megakaryocytes (Meg), monocytes (Mon), and macrophages (Mac), and common lymphoid progenitors (CLP)
Figure 5(A) Western blot detection of p-CHK2 Thr68, CHK2, p-AMPKα Thr172, AMPKα, p-Beclin 1 Ser90, p-Beclin 1 Ser93 and Beclin 1 in H1299 cells transfected with the indicated shRNA in normal medium or after glucose starvation. (B) Western blot detection of p-CHK2 Thr68, CHK2, p-Beclin 1 Ser90, p-Beclin 1 Ser93 and Beclin 1 in H1299 cells transfected with the indicated shRNA in normal medium or after hypoxia. (C) Western blot detection of ATM, p-CHK2 Thr68, CHK2, p-Beclin 1 Ser90, p-Beclin 1 Ser93 and Beclin 1 in H1299 cells transfected with the indicated shRNA in normal medium or after glucose starvation. (D) Western blot detection of p-ATM Ser1981, ATM, p-CHK2 Thr68, CHK2, p-Beclin 1 Ser90, p-Beclin 1 Ser93 and Beclin 1 in H1299 cells transfected with the indicated shRNA in normal medium or after hypoxia. (E) P-ATM Ser1981, ATM, p-CHK2 Thr68, CHK2, p-Beclin 1 Ser90, p-Beclin 1 Ser93 and Beclin 1 were analyzed in H1299 cells, and the cells were subjected to hypoxia with or without pretreatment with NAC. (F) P-ATM Ser1981, ATM, p-CHK2 Thr68, CHK2, p-AMPKα Thr172, AMPKα, p-Beclin 1 Ser90, p-Beclin 1 Ser93 and Beclin 1 were analyzed in H1299 cells, and the cells were subjected to glucose starvation with or without pretreatment with NAC. (G) Western blot detection of p-ATM Ser1981, ATM, p-CHK2 Thr68, CHK2, p-AMPKα Thr172, and AMPKα in H1299 cells in normal medium or after H2O2 (25μM) stimulation. (H) Western blot detection of p-ATM Ser1981, ATM, p-CHK2 Thr68, CHK2, p-AMPKα Thr172 and AMPKα in H1299 cells in normal medium or after 2DG (5mM) and oligomycin (2.5μM) treatment.
RNA metabolic labeling defines tumor cell trajectories in patient-derived organoids.Figure 3A Phenotypes of patient-derived organoid lines P009T and P013T. B UMAPs of organoid single cell transcriptomes. Organoid lines and medium conditions as indicated. LGR5-ISC stem cell, enterocyte and Goblet cell signatures are visualized. C Schematic representation of SLAM-Seq workflow to infer RNA dynamics ("RNA velocity"). In short, organoids were passaged and assigned to different medium conditions. After three days, nascent RNA was metabolically labelled for 2 h using 4sU. Organoids were harvested, dissociated, and fixed. RNA in single cells was alkylated, and cells were subjected to single cell sequencing. Reads were assigned to nascent or old RNA status, depending on diagnostic T-C mutational status. D Developmental trajectories inferred from RNA metabolic labelling. Bold and thin arrows indicate high versus low directionality of RNA velocity. Colors below RNA velocity show latent time (yellow: early latent time; blue: late latent time). E Activities of Wnt/β-catenin and MAPK target genes in organoid single cell transcriptomes, ordered along latent time. F Activities of TFF3, FABP1, MKI67 and MMP7 in organoid single cell transcriptomes, ordered along latent time.
For all growth regimes tested, the glucose uptake rate shows a biphasic response curve to increasing ATPase levels.Figure 3For all growth regimes tested, the glucose uptake rate shows a biphasic response curve to increasing ATPase levels. Data information: The means for n = 3 biologically independent samples are shown. The error bars represent ± SD.