EMBO/sd-character-level-ner huggingface dataset:
Figure 3Signalling downstream of VEGF-A/VEGFR2 assessed in HDMEC, untreated (NT, non-transfected), transfected with non-targeting siRNA (c/cntrl), or with two different PALD1-targeting siRNAs (KD#1 and KD#2), and treated with VEGF-A for 0-10 and 0-20 min, respectively. Immunoblotting of cell lysates for phosphorylated (p) VEGFR2 (pY1175), total VEGFR2 Actin served as loading control (a PALD1 knockdown was verified by blotting for Paladin. Quantification of pVEGFR2 (normalized to total VEGFR2) (b) Mean±SEM, two-way ANOVA, n=3 biological replicates. Data information: *p < 0.05, **p < 0.01. Signalling downstream of VEGF-A/VEGFR2 assessed in HDMEC, untreated (NT, non-transfected), transfected with non-targeting siRNA (c/cntrl), or with two different PALD1-targeting siRNAs (KD#1 and KD#2), and treated with VEGF-A for 0-10 and 0-20 min, respectively. Immunoblotting of cell lysates for phosphorylated Erk1/2 (pT202 and pY204), and total Erk. Actin served as loading control c). PALD1 knockdown was verified by blotting for Paladin. Quantification of pErk1/2 (normalized to total Erk1/2) (d). Mean±SEM, two-way ANOVA, n=3 biological replicates. Data information: *p < 0.05, **p < 0.01. (e-f) Immunoblotting on total heart lysates from adult Pald1+/+ and Pald1-/- mice, tail-vein injected with VEGF-A (0.25µg/g body weight) or PBS for the indicated time points, for Paladin, phosphorylated and total levels of VEGFR2, Erk1/2 and β2-microglobulin (β 2-MG, loading control). (f) Quantification of pVEGFR pY1173 normalized to total VEGFR2 (n=3), total VEGFR2 levels normalized to total loading control (n=4), pT202/pY204 Erk1/2 normalized to Erk1/2 (n=5). Mean±SEM, multiple t-test with Holm-Sidak correction (total VEGFR2), two-way ANOVA (others). Data information: *p < 0.05, **p < 0.01.
Figure 6A-B Relative enrichment of H3K4me3 and H3K27me3 at the SNCA promoter/intron 1 region was evaluated from six iPSC lines of two control subjects and five iPSC lines derived from two sporadic PD (sPD) cases by ChIP. The same genomic region as shown in Fig 1 and Appendix Figure S2C was evaluated for enrichment of H3K4me3 and H3K27me3. Mouse IgG (mIgG) was used as control for the target antibody. The relative intensity of the target band was normalized by respective input. Data information: Three independent repeats were performed for ChIP experiments. *P < 0.05; ****P<0.0001. Data were analyzed using non-parametric t-test followed by Mann-Whitney post-hoc corrections. One-tailed p-values were calculated for ChIP analysis data are presented as mean ± SEM. C ChIP on H3K4me3 at the SNCA promoter between control and sPD1-1 lines revealed a marginally higher enrichment in sPD lines as compared to the controls. Data information: Three independent repeats were performed for ChIP experiments. *P < 0.05; ****P<0.0001. Data were analyzed using non-parametric t-test followed by Mann-Whitney post-hoc corrections. One-tailed p-values were calculated for ChIP analysis D Relative comparison of H3K27me3 enrichment between control and sPD lines revealed no significant difference between the two groups. E ChIP on H3K4me3 at the SNCA promoter between sPD1-1 lines after locus-specific epigenomic modulation. Differentiated cells were transiently transfected with either sgA-dCas9 5xGCN4-scFV-JARID1A or sg A-dCas9 5xGCN4-scFV-empty backbone vectors. A significant reduction in H3K4me3 at the SNCA promoter was observed in cells transfected with JARID1A. Data information: Three independent repeats were performed for both ChIP experiments. *P < 0.05; ****P<0.0001. Data were analyzed using non-parametric t-test followed by Mann-Whitney post-hoc corrections. One-tailed p-values were calculated for ChIP analysis F Western blot analysis of α-synuclein (α-SYN) levels in the cells under the same conditions as analyzed in (E). A significant decrease (56-66%) in α-SYN levels was observed in cells transfected with JARID1A. The normalized and relative expression of α-SYN in JARID1A transfected cells are shown as a percentage of control (transfected by empty backbone vector). Data information: Three independent repeats were performed for western blot experiments. *P < 0.05; ****P<0.0001. Data were analyzed using non-parametric t-test followed by Mann-Whitney post-hoc corrections. two-tailed p-value was calculated for western blot experiments. All data are presented as mean ± SEM.
Figure 1A PTAR1 protein expression in rat tissues. Proteins extracted from various rat tissues (15 μg each) were analyzed by immunoblotting with antibodies against PTAR1. B Identification of RabGGTβ as a binding protein of PTAR1. Anti-FLAG immunoprecipitates from control HeLa S3 cells or FLAG-PTAR1-expressing HeLa S3 cells were analyzed by SDS-PAGE and silver staining. The 35 kDa protein was identified as RabGGTβ by mass spectrometry. The asterisk denotes a degradation product of FLAG-PTAR1. C Co-immunoprecipitation of endogenous PTAR1 and RabGGTβ from rat brain and thymus cytosol. D Fractionation of rat brain cytosol by Mono Q anion exchange column chromatography. Column fractions were analyzed by immunoblotting with antibodies against RabGGTα, PTAR1, and RabGGTβ. RabGGTβ eluted in two peaks (peak A and peak B). E Co-immunodepletion of PTAR1 and RabGGTβ by anti-PTAR1 IgG. PTAR1 was immunodepleted from the Mono Q peak A fraction using anti-PTAR1 IgG-bound protein A beads, and the supernatant was analyzed by immunoblotting with antibodies against PTAR1 and RabGGTβ. Normal rabbit IgG and anti-PTAR1 IgG preabsorbed with the antigen peptide were used as controls. F Fractionation of the Mono Q peak A fraction by Superdex 200 gel filtration chromatography. Column fractions were analyzed by immunoblotting with antibodies against PTAR1 and RabGGTβ. G Gel filtration chromatography of the recombinant PTAR1-RabGGTβ complex purified from Sf9 insect cells on a Superdex 200 column. Inset shows SDS-PAGE and Coomassie staining analysis of the purified complex. H Schematic diagram of the protein prenyltransferase family. FTase and GGTase-I share the same α subunit. RabGGTase and the PTAR1-RabGGTβ complex (GGTase-III) share the same β subunit.
Fine morphology of Δypt7 and Δapg8 cells and proteinase-K accession to proAPI in these mutants.Figure 10Fine morphology of Δypt7 and Δapg8 cells and proteinase-K accession to proAPI in these mutants. A, EM image of Δypt7 cell starved in SD(−N) medium for four hours. Autophagosomes (AP, arrows) were accumulated. B, EM image of the starved Δapg8 cell. C-E, The representatives of the membrane structures detected in the starved Δapg8 cells. C, Autophagosome-like structure indistinguishable from autophagosome. D and E, Aberrant multivesicular structures. N, nucleus; V, vacuole. F, Proteinase K-sensitivity of proAPI. Cell lysates were prepared from Δypt7 and Δapg8 cells starved in SD(−N) medium for 4.5 h. The lysates were treated with 100 μg/ml proteinase K in the presence or absence of Triton X-100. Asterisk shows mature API and digestion product of proAPI by proteinase K.
Evidence for the enrichment of PS1-dependent γ-secretase activity in AVs.Figure 7.(A and B) Ultrastructure of AVs (A1 and A2) and lysosomes in subcellular fractions from serum-deprived L/APP cells (A) and Western blot analysis (B) for LC3-II, rab24, APP, βCTF, PS1 (PS1 amino-terminal fragment), and nicastrin (NCT) in L/APP subcellular fractions. AVs, A1 and A2; L, lysosomes; E, tubulovesicular compartments (Golgi/ER/endosomes); P, postnuclear pellet; C, cytosol; M, mitochondria. Empty lanes in the original blot have been removed from the figure and noted with a white line. (C) Rates of cleavage of the fluorogenic substrate in subcellular fractions from L/APP cells grown in −serum media (left) or −Leu/−His media (right). PNP, postnuclear pellet. (D and E) Proportions of the total recovered cell γ-secretase activity in different subcellular fractions after serum deprivation (D) or in uninduced (+serum) or induced (−Leu/−His) cells (E). Comparison of +serum versus −Leu and −His conditions shows the redistribution of γ-secretase activity from the tubulovesicular to AV fractions after macroautophagic induction. G, Golgi; A, AV. (F) Liquid chromatography mass spectrometry analysis of the fluorogenic substrate after incubation with purified AVs. Selected ion chromatogram corresponding to different cleavage products is displayed. Numbers in graph refer to the cleavage/amino acid site from the Aβ peptide. (G) γ-Secretase activity in AV and lysosome fractions from mice blastocysts in which the PS1 and PS2 genes were deleted (PS KO; BD8) or in which human PS1 was stably transfected into the PS KO blastocysts (hPS1; BD8/hPS1). Numbers on x axis are in minutes. (H) Aβ40 and Aβ42 levels in medium from these cells as detected by sandwich ELISA. Values are given as means ± SEM (error bars).
Figure 4(A) The experimental set-up to study the androgen response in kidney. (B) PCA on the renal transcriptomes of WT ORX, WT ORX + T, ARLmon/Y ORX and ARLmon/Y ORX + T. (C) Heatmap for the genes that are differentially expressed in kidney between WT ORX and WT ORX + T (q < 0.05; FC > 1.5) and corresponding levels in ARLmon/Y ORX and ARLmon/Y ORX + T. mRNA expression levels extracted from RNA-seq data for Fkbp5 (D) in kidneys used for RNA-seq. Expression levels are normalized to WT ORX. mRNA expression levels extracted from RNA-seq data for , Odc1 (E) and Kap (F) in kidneys used for RNA-seq. Expression levels are normalized to WT ORX.
Figure 3Two top-ranked SplashRNA-designed shRNAmirs targeting human or mouse surface protein FAS were cloned into Venus-expressing miR-AB lentiviral vectors which harbor different promoters to drive the shRNAmir expression. The packaged lentiviruses were used to infect the indicated human or mouse cell lines. A shRNAmir targeting human or mouse CD4 driven under hCMV promoter was used as control. After >96h of infection, FAS protein levels in the transduced Venus-positive cells were determined by surface staining and shown in histograms (top panels) and quantified by MFI of APC (bottom panels).
Ectopic centriolar protein complexes can be the sites of microtubule nucleation.Figure 5A Schematic of microtubule regrowth assay during mitosis in RBM14-depleted U2OS cells. B-E Control U2OS cells or U2OS cells treated with siRNA targeting RBM14 were cold-treated for 30 min, followed by 30-60 min incubation at 37°C and stained with antibodies against centrin (magenta in (B) and (D)) or γ-tubulin (magenta in (E)) as well as α-tubulin (green). DNA is shown in blue. Insets show approximately twofold magnified views of fluorescent foci around the centrosome. Scale bar, 5 μm. Histograms represent the percentages of mitotic cells showing the indicated phenotype at each time point. Values are mean percentages ± standard error of mean (SEM) from three independent experiments (n = 30 for each condition). *P < 0.05, **P < 0.01 (one-tailed t-test).
Fig.Figure 7(A) Colocalized RGC inputs and PSVue signals in P6 control and CKO. White circles indicate PS+ RGC inputs. Arrows pointing to the RGC inputs enlarged in the insets. (B) The percentage of PS+ RGC inputs in total inputs in CKO and controls. (C) Representative images of engulfed PS+ and PS- RGC inputs by microglia in control and CKO. (D) Quantification of engulfed PS+ RGC inputs by microglia, which is calculated as: Volume of engulfed PS+ RGC inputs / Volume of microglia cell. (E) Quantification of engulfed PS- RGC inputs in control and CKO.
Cardiomyocyte SASP induces fibrosis and reduces proliferation in fibroblasts and induces hypertrophy in cardiomyocytes.Figure 5(Above) RNA-sequencing of purified CMs from 4 mice per age group reveal age-dependent increased expression of 3 secreted proteins Edn3, Tgfb2, and Gdf15; the colour intensity represents column Z-score, where red indicates highly and blue lowly expressed. (Below) mRNA expression of Edn3, Tgfb2, and Gdf15 was independently validated by RT-PCR in young and old isolated adult CMs. Data are mean ±S.E.M of n=8 mice per group. CMs were isolated by Langendorff heart perfusion, purified and cultured for 48h. Conditioned medium (CM) was collected and added to cultures of neonatal fibroblasts in the presence of 10μM EdU. Representative images of immunofluorescent staining against α-SMA and EdU in neonatal fibroblasts cultured in the presence of CM from young and old CMs. Quantification of % of EdU incorporation (D) in neonatal fibroblasts after treatment for 48h with CM from young and old CM. Data are mean ±S.E.M of n=3-4 mice per age group; >200 cells were quantified per condition. % of a-SMA positive cells (E) in neonatal fibroblasts after treatment for 48h with CM from young and old CM. Data are mean ±S.E.M of n=3-4 mice per age group; >200 cells were quantified per condition. Representative micrographs of neonatal fibroblasts and CMs treated with recombinant proteins: Tgfb2, Edn3, and Gdf15 for 48h and immunostained against α-SMA and EdU (fibroblasts) and α-actinin (CMs). Quantification of α-SMA (G) in neonatal CMs following treatment with the indicated recombinant proteins. Data are mean ±S.E.M of n=3-4 independent experiments. EdU positive neonatal fibroblasts (H) in neonatal CMs following treatment with the indicated recombinant proteins. Data are mean ±S.E.M of n=3-4 independent experiments. surface area (μm2) (I) in neonatal CMs following treatment with the indicated recombinant proteins. Data are mean ±S.E.M of n=3-4 independent experiments.
/2 is required for phosphorylation of ATG16L1 and xenophagy induction (A) Wild-type, ULK1/2 double knockout (dKO), or IKKα KO mouse embryonic fibroblasts (MEFs) were incubated with either complete medium, amino acid-deficient DMEM, or HBSS for 1 hour.Figure 2(A) Wild-type, ULK1/2 double knockout (dKO), or IKKα KO mouse embryonic fibroblasts (MEFs) were incubated with either complete medium, amino acid-deficient DMEM, or HBSS for 1 hour. Samples were immunoblotted using the indicated antibodies. Data information: Unless otherwise indicated experiments were performed three times. Data are represented as mean ± standard deviation and p values were determined by Student's T-Test. (B) Wild-type, ULK1/2 dKO, or IKKα KO MEFs were infected with log phase Salmonella for 2 hours; bacteria-containing media was then removed and cells were incubated with gentamycin (50 µg/mL)-containing DMEM for 2 hours. Samples were immunoblotted using the indicated antibodies. Data information: Unless otherwise indicated experiments were performed three times. Data are represented as mean ± standard deviation and p values were determined by Student's T-Test. (C) Wild-type, ULK1/2 dKO, or IKKα KO MEFs cells were infected with Salmonella for 1 hours. Autophagic capture of Salmonella was analyzed by immunostaining for LPS and LC3B. Representative images are shown (scale bars, 10 μm and 3 μm). Quantification was generated from 8 fields of view from a representative experiment. The experiments were repeated twice. Data information: Unless otherwise indicated experiments were performed three times. Data are represented as mean ± standard deviation and p values were determined by Student's T-Test. (D) Wild-type, ULK1/2 dKO and IKKα KO MEFs were infected with Salmonella for 1 hour. Xenophagy rates were examined through Colony Forming Unit (CFU) assays. Quantification of infection rates by immunofluorescence is demonstrated in the right panel. Data information: Unless otherwise indicated experiments were performed three times. Data are represented as mean ± standard deviation and p values were determined by Student's T-Test.
Figure 3Confocal z-projections of brains expressing MiMIC mediated Dpr12GFSTF (Dpr12-GFP; A-D) and DIP-δGFSTF (DIP-δ-GFP; E-H) fusion proteins at the indicated time points. See Figures EV 1 and 4 for more details on the fusion protein structure. (A-D) Dpr12-GFP is localized to the distal part of the γ-lobe at L3 (A; n=10/10), 48hr APF (C; n=12/12) and the adult stage (D; n=20/20), where it colocalizes with the γ4>γ1γ2 MBON (γ4; labeled by GMR18H09-Gal4 driving the expression of CD4-tdT). At 24hr APF (B; n=10/10), Dpr12-GFP appears diffuse. (E-H) DIP-δ-GFP is localized to the distal part of the γ-lobe throughout development: L3 (E; n=16/16), 24hr APF (F; n=10/10), 48hr APF (G; n=12/12) and adult (H; n=24/24). At the adult stage, DIP-δ-GFP is colocalized with the γ4>γ1γ2 MBON (γ4).
Figure 1A. Targeted mutagenesis of the Rnaseh2b gene. Top: A 7 kb region of the Rnaseh2b genomic locus; black boxes, exons 6 (ex6) and 7 (ex7). Middle: NotI/SalI restriction fragment of the targeting construct, comprising 4.5 kb of genomic DNA and a Neomycin selection cassette (Neo) flanked by loxP sites (triangles). (Bottom) Targeted locus containing exon 7 with the c.520G>A mutation (ex7*). Red arrowheads, primers used to confirm correct targeting. Red bar, 400 bp probe for Southern blotting. B. Southern blotting confirms successful targeting. Introduction of an additional EcoRI site results in a 4.1 kb restriction fragment detectable by Southern for targeted ES cells (A174T/+) but not for parental DNA (+/+). C. Capillary sequencing for Rnaseh2b+/+, Rnaseh2bA174T/+, and Rnaseh2bA174T/A174T DNA confirmed the presence of the introduced missense mutation. D. Mouse genotyping by multiplex PCR. Top: A 221 bp PCR product is present in wild-type mice (+/+); the Rnaseh2bA174T allele (also) give a 460 bp product. Bottom: Position of forward (x) and reverse primers (y, z). E. Immunoblotting demonstrates depletion of all three RNase H2 protein subunits in Rnaseh2bA174T/A174T MEFs and RNASEH2BA177T/A177T LCLs. Representative of three independent experiments. F. Schematic showing enzyme activities attributed to RNase H1 and RNase H2 (DNA blue, RNA red), G, H. RNase H2 enzyme activity is reduced in mouse and patient cells. (G) Enzyme activity for Rnaseh2bA174T/A174T MEFs and passage-matched Rnaseh2b+/+ controls, against RNase H substrate (RNA:DNA heteroduplex) and RNase H2-specific substrate, double stranded DNA with a single embedded ribonucleotide (DRD:DNA). Mean activity for three independent cell lines, error bars, SEM. Enzymatic activity expressed relative to the average value of control MEFs. *** = p<0.001, two-tailed t-test (n=3 Rnaseh2bA174T/A174T and n=3 Rnaseh2b+/+ control MEF lines). (H) RNase H2 activity in LCLs from two independent healthy controls and an AGS patient homozygous for the RNASEH2B-A177T mutation. Enzyme activity normalised to average activity of control lines. Three independent experiments, error bars SEM. *** = p<0.001 versus either control, two-tailed t-test.
Schematic showing Ago1x-mediated negative feedback regulation of miRNA pathway Mammalian AGO1 transcript has two in-frame stop codons.Figure 9Mammalian AGO1 transcript has two in-frame stop codons. Translation termination at the first stop codon generates canonical Ago1 isoform. Ago1 can load miRNAs on target mRNAs and repress their expression. Translation readthrough at the first stop codon and termination at the downstream in-frame stop codon generates Ago1x. let-7a miRNA promotes this process. Ago1x can load miRNAs on target mRNAs, but it cannot bring down their expression because of its inability to recruit GW182. Thus, it can act as competitive inhibitor and de-repress gene expression.
Mitochondrial functions altered by HCV infection are associated with Parkin-mediated mitophagy.Figure 7(A) Rescue effect of 3-MA and BafA1 on reduction of mitochondrial complex I enzyme activity caused by HCV infection. Huh7 cells infected with HCVcc were treated with 3-MA (10 mM) or BafA1 (100 nM) for 12 h before harvest. At 3 days post-infection, the activity of mitochondrial complex I enzyme was measured according to manufacturer's instructions (mean ± SD; n = 3; *p<0.01). . (B) Rescue effect of Parkin knockdown on reduction of mitochondrial complex I enzyme activity caused by HCV infection. NT-KD and P-KD cells infected with HCVcc were harvested on day 3 post-infection and used for analysis of the activity of mitochondrial complex I enzyme (mean ± SD; n = 3; *p<0.05). (C) Western blot analysis of mitochondrial respiratory chain complex enzyme expression. NT-KD and P-KD cells were infected with HCVcc and at 3 days post-infection, the expression levels of complex I and IV enzyme were analyzed by immunoblotting with anti-Hu total OxPhos complex antibody. Huh7 cells treated with CCCP (10 µM) for 12 h were also analyzed as a control. β-actin was used as an internal loading control. P values were calculated by using an unpaired Student's t-test. (D) Effect of Parkin silencing on depletion of mitochondrial DNA caused by HCV infection. Huh7 cells transfected with NT or Parkin-specific siRNA pools were infected with HCVcc. At day 3 post-infection, mitochondrial DNA levels of ND-2 and COX-2 were analyzed by real-time qPCR. GAPDH was used to normalize changes in ND-2 and COX-2 gene expression (mean ± SD; n = 3; *p<0.01). (A, B, and D) P values were calculated by using an unpaired Student's t-test.
Figure 4A. Phos-tag band-shift analyses of osmostress-induced Hog1 phosphorylation. The yeast strain FP4 (hog1Δ) was transformed with the single-copy expression plasmid pRS416-Hog1-ΔL16, and was stimulated with the indicated concentrations of NaCl for 5 min. B. Averages of three independent experiments from A were plotted. Results for Hog1-WT (from Fig 1I) are included for comparison. Data information: Representative results from three independent experiments. Error bars are SEM (n=3). C-D. the yeast strain KT259 (ste11Δ hog1Δ) was used. Data information: Representative results from three independent experiments. Error bars are SEM (n=3). E-F. the yeast strain KY523 (ssk2/22Δ hog1Δ) was used. Data information: Representative results from three independent experiments. Error bars are SEM (n=3).
Disturbed deuterosome formation and impaired centriole biogenesis in Ccno‐deficient MCCsDouble IF analysis of mTEC cultures at early stage of MCC differentiation and centriole amplification (ALI day 3) using antibodies for the deuterosome protein DEUP1 and the early centriole marker SAS‐6.Figure 5Double IF analysis of mTEC cultures at early stage of MCC differentiation and centriole amplification (ALI day 3) using antibodies for the deuterosome protein DEUP1 and the early centriole marker SAS‐6. Deuterosome numbers are decreased and deuterosome size enlarged in Ccno‐deficient MCCs as quantified in (C). Deuterosome‐dependent centriole biogenesis is severely reduced in Ccno‐deficient MCCs as shown by reduced staining for the early centriole marker SAS‐6. IFstaining for DEUP1 reveals a further size increase of deuterosomes of Ccno‐deficient MCCs at later stages of centriole biogenesis (ALI day 7). Deuterosome number and size were quantified in (C). IF using a Centrin antibody shows globally reduced presence of centrioles at ALI day 7, which stay localized below the level of deuterosomes and fail to dock to the apical cellmembrane in Ccno‐deficient MCCs as seen in z‐projection. IFstaining for DEUP1 in (A, B) was analysed by Imaris 7.7.2 software to calculate average deuterosome number of wild‐type (n = 17) and Ccno‐deficient (n = 19) MCCs at ALI day 3, and wild‐type (n = 12) and Ccno‐deficient (n = 14) MCCs at ALI day 7. Deuterosome size was measured using the spot measurement tool in n = 10 MCCs for both genotypes at ALI day 3, and in wild‐type (n = 12) and Ccno‐deficient (n = 14) MCCs at ALI day 7.