EMBO/sd-character-level-ner huggingface dataset:
Figure 1qRT-PCR for INHBA relative to RPL27 using RNA from SCC13 cells transduced with a lentiviral vector allowing expression of INHBA in a doxycycline (DOX)-inducible manner (SCC13 Act clone 1 and 2) or empty vector (EV) (N=3). SCC13 Act and EV cell lines were generated from lentivirally transduced cells upon single cell clonal expansion. Western blot of cell lysate and conditioned media (CM; bottom) of transduced SCC13 cells for the activin βA subunit and GAPDH (loading control for cell lysate) under reducing conditions. The higher molecular weight of recombinant activin βA results from the HA-epitope tag. Ponceau S staining of the membrane was used as a loading control for the CM. Representative pictures of 5 week-old tumors (indicated by arrows) formed in NOD/SCID mice upon intradermal injection of SCC13 Act (clone 2) or SCC13 EV cells and tumor volume at different time points of tumor development. N=3-6 tumors. Representative images of H&E stainings of tumors formed by SCC13 EV and Act cells (clone 2) at day 35 (asterisk indicates cartilage, arrow indicates site of invasion). An inset panel indicates the area where tumor cells invade into the cartilage. Tumor sections were evaluated for invasion through the basement membrane. N=6 (EV), N=6 Act (3 tumors from clone 1 and 3 tumors from clone 2). Tumors from Act mice were pooled in the graph). Tumor weight at endpoint (5 weeks after injection). N=3-6 tumors. Herovici staining and quantification of percentage of collagen-positive (dark blue) area per field of view. N=3 tumors, n=3-4 histological sections. Representative images of sections from tumors formed by SCC13 EV and SCC13 Act (clone 2) stained for Ki67 (red), PDGFR-α (green) and counterstained with Hoechst (blue). Quantification of Ki67/PDGFR-α positive cells is shown in the graph. N=3 tumors, n=3-4 histological sections. Representative images of sections from 3D cultures of primary human skin fibroblasts with either SCC13 EV or SCC13 Act (clones 1 and 2) cells stained with either H&E or Herovici. Quantification of Ki67-positive cells in sections from SCC13 EV and SCC13 Act (clone 2) organotypic cultures (co-stained with E-cadherin to indicate epithelial cells). N=3 independent cultures, n=3 histological sections. Representative images of sections from SCC13 EV and SCC13 Act (clone 2) organotypic cultures stained for K14 (red) and collagen IV (green) combined with Hoechst staining (blue) and quantification of the K14-positive area below the basement membrane (BM). N=3 independent cultures, n=4 histological sections. The area indicated with a white rectangle in the left picture is shown at higher magnification in the right picture.
Hsp42 and Btn2 are compartment‐specific aggregases A, B Cellular localizations of Hsp42 and Btn2 were determined at the indicated temperature by immunofluorescence.Figure 7A, B Cellular localizations of Hsp42 and Btn2 were determined at the indicated temperature by immunofluorescence microscopy. DNA was stained by either Htb1‐mCherry or DAPI. Scale bars, 2 μm. In case of Hsp42 immunofluorescence, a line intensity plot of a deconvoluted widefield image is given. C S. cerevisiae btn2Δ hsp42Δ Hsp42‐NLS cells expressing GFP‐VHL (green) were grown at 30°C and heat‐shocked to 37°C for 30 min in the presence of MG132. Changes in protein localizations were recorded. DNA was stained by DAPI (blue). The total number of GFP‐VHL foci per cell and frequencies (%) of INQ formation were determined. Scale bars, 2 μm. D S. cerevisiaebtn2Δ hsp42Δ Hsp42‐NLS cells expressing mCherry‐VHL (red) were treated as described in (C). The nuclear membrane was visualized by Nsp1 immunofluorescence (green) and colocalization of mCherry‐VHL foci with Hsp42 was probed by Hsp42 (green) immunofluorescence microscopy. DNA was stained with DAPI (blue). Scale bars, 2 μm. E Hsp42 and Btn2 levels were determined prior and post‐heat shock at the indicated time points by Western blot. The numbers of molecules/cell were calculated using purified proteins as standard.
Figure 1A Schematic of spheroid differentiation method. L-WRN CM, conditioned medium from an L cell line engineered to secrete Wnt3a, R spondin 3 and Noggin. Y-27632, ROCK inhibitor. EGF, epidermal growth factor. B-F Jejunal spheroids were cultured in differentiation medium containing 0-1,000 nM dmPGE2 or Iloprost. (B) Representative bright-field images of spheroids treated with dmPGE2. Scale bars, 200 µm. B-F Jejunal spheroids were cultured in differentiation medium containing 0-1,000 nM dmPGE2 or Iloprost. (C) Quantification of average spheroid area ± s.e.m. relative to spheroids treated with 0 nM dmPGE2 (average area was 3761 µm2 for 0 nM group; n = 4 images from two independent experiments). B-F Jejunal spheroids were cultured in differentiation medium containing 0-1,000 nM dmPGE2 or Iloprost. (D) Representative histological sections of spheroids treated with 1 µM dmPGE2 or an equivalent volume of DMSO and stained for hematoxylin. Scale bars, 100 µm. B-F Jejunal spheroids were cultured in differentiation medium containing 0-1,000 nM dmPGE2 or Iloprost. (E) Quantification of the average expression ± s.e.m. of Cldn4mRNA relative to the 0 nM treatment group (n = 3 independent experiments). B-F Jejunal spheroids were cultured in differentiation medium containing 0-1,000 nM dmPGE2 or Iloprost. (F) Quantification of average spheroid area ± s.e.m. relative to spheroids treated with 0 nM Iloprost (n = 4 images from two independent experiments).
Figure 1Western blot analysis of nephrin expression in immortalized podocytes cultured in normal (5 mM) or high glucose (HG; 30 mM) or high mannitol (Mann; 5 mM glucose plus 25 mM mannitol) for 24, 48 and 72 hours. *P < 0.05 versus normal controls for the indicated time points (Wilcoxon two-sample test; n = 3). B Analysis of the methylation status at nephrin gene promoter in podocytes cultured in normal, high glucose or high mannitol at the indicated time points. U, unmethylated-specific primers; M, methylated-specific primers. C Effect of pargyline hydrochloride, a pan-KDM inhibitor, on high glucose-mediated nephrin expression. Immortalized mouse podocytes were cultured in the indicated cultured media in the presence or absence of pargyline hydrochloride (1 μM) for 48 hours. Protein lysates from the treated podocytes were analyzed for nephrin expression by immunoblotting. *P < 0.05 versus normal controls, #P < 0.05 versus untreated HG-incubated cells (Wilcoxon two-sample test; n = 3). D Effect of pargyline hydrochloride on high glucose-mediated DNA methylation at nephrin gene promoter. Genomic DNA samples from the treated podocytes were analyzed for methylation status of nephrin gene promoter by methylation-specific PCR. E Quantitative RT-PCR analyses of different KDMs in immortalized podocytes exposed to high glucose or high mannitol for 48 hours. *P < 0.05 versus normal controls (Wilcoxon two-sample test; n = 3). F Western blot analysis of KDM6A expression in podocytes cultured in normal, high glucose or high mannitol conditions for 24, 48 ad 72 hours. *P < 0.05 versus normal controls for the indicated time points (Wilcoxon two-sample test; n = 3). G Western blot analysis of H3K27me1, H3K27me2, H3K27me3 and pan-methyl H3K9 in podocytes cultured in normal, high glucose or high mannitol conditions for 9 and 24 hours. *P < 0.05 versus normal controls (Wilcoxon two-sample test; n = 3).
Figure 4(A) RT-PCR was used to detect any USH1C transcripts (green arrows) and for discriminating USH1C_a and _c splice variants (blue arrows) from USH1C_b3 (orange arrows). (B) USH1C transcripts were consistently detected in all tested organs of WT and USH1C pigs, except in the heart and cerebellum. RT-PCR for GAPDH was used as control (Ctrl.). (C) USH1C_a or _c splice variants were detected in the retina, kidney and intestine (blue arrows). Electropherogram from the duodenum, confirm alternative splicing of exon 13, but not exon 11. USH1C_b variants were also detected in all examined organs, with presumably two splice variants (orange arrows). Electropherogram from the colon, indicates alternative splicing of exon 20 as presumable reason for the double band. Exon 27 was not detected in any amplicon of USH1C_b RT-PCR (Figure S10D, E). Sequencing was done with a forward primer. (D) Representative Western blot analysis proved lack of harmonin protein expression in retina and duodenum of USH1C pigs at the age of 3 weeks (3w) (1 piglet, 1 retina, 1 duodenum) and 1 year (1y) (2 pigs, 1 retina each, 3 TRs). Arrow indicates the expected size of harmonin isoform a (72kDa). (E) Representative immunofluorescence staining of harmonin (green) of longitudinal cryosection through WT and USH1C pig retinas (2 pigs, 1y, 1 retina each, 3 TRs, scale bar 10µm) demonstrated the absence of harmonin staining in the outer segment (OS) of photoreceptor cells and in the other layers below the outer limiting membrane (OLM, arrow) of the USH1C pig retina. (F) Representative Western blot analysis of retinal protein extracts, indicating that the abundance of the USH proteins SANS (USH1G), whirlin (USH2D) or myosin7a (USH1B) is unaffected (1 piglet, 3w, 1 retina each, 2 TRs).
Figure 3Normalized Hi-C contact maps for ΔrsaAvan::promoterless-rsaA, ΔrsaAvan::PrsaA-rsaA (ATG→TAA), and ΔrsaAvan::PrsaA-rsaA (anti-termination + ATG→TAA) cells. Only the region of the genome containing the van locus (dashed line) is shown. Above each Hi-C contact map is a schematic of the corresponding rsaA construct. The transcription anti-terminator is shown as a red hairpin, the start codon mutated to TAA is in red font, and the transcript produced is shown as a wavy red line. Below each map is the corresponding directional preference plot.
Figure 6Ki67 and cleaved caspase3 staining of representative colons obtained from mice on day10 of colitis induction as described in Figure 2A. The number of proliferating cells per crypt in (A) (upper panel) or apoptotic cells per field in (B) was determined (n=5 per group, 5 crypts were counted per mouse). Scale bar, 50μm. Quantitative mRNA expression analysis of the indicated genes in colon tissues obtained from WT or Stat4-KO colitis mice treated Immunoblot analysis of the expression and modification of the indicated proteins in DLD-1 cells treated with LIF (20 ng/ml) for different durations. Immunoblot analysis of YAP expression in WT or Stat4-KO IECs treated with LIF (20 ng/ml) for different durations; β-actin was used as a loading control (top). The statistical analysis of YAP protein intensity is shown in the bottom panel. The human and mouse Yap promoter locus contains multiple AGG elements, as indicated. Relative YAP promoter-luciferase reporter activities in HEK293T cells transfected with EV, STAT3 or STAT4 and then treated with IL-6 or LIF for 8 hours. RNA sequencing analysis of the gene expression profile in IECs treated with or without LIF (50 ng/ml) for 6 hours. Statistical analysis of the average fragments per kilobase of transcript per million mapped reads (J). Heatmap showing the expression profile of cell proliferation-related genes in LIF-treated or control IECs (Ctrl) (K).
Figure 2Time-lapse confocal images of Dictyostelium AX3 cell co-expressing tPH-CynA-KikGR (green) and LimE-RFP (red). Front and back of the cell are shown. Box shows the vesicle. Time-lapse confocal images of Dictyostelium AX3 cell co-expressing tPH-CynA-GFP (green) and PH-Crac-RFP (red). Box shows the vesicle. Relative intensities of tPH-CynA-KikGR on macropinosomes and LimE-RFP on macropinosomes in experiments in Fig 3A. Mean ± SEM is shown for n=5 cells. Relative intensities of tPH-CynA-KikGR on macropinosomes and PH-Crac-RFP on macropinosomes in experiment in Fig 3B. Mean ± SEM is shown for n=3 cells. Time-lapse confocal images of AX3 cell co-expressing PH-Crac-GFP (green) and another established PI(3,4) P2 biosensor tPH-TAPP1-RFP (red). Box shows the vesicle. Time-lapse confocal images of tPH-CynA-KikGR (green) expressing wild-type AX3 cells. FM4-64 (red) was added at time 0 during live cell imaging. Front and back of the cell are shown. Box shows the vesicle.
Therapeutic enzyme is trapped in the area of autophagic build-up in PD fibres.Figure 2Confocal microscopy of live unstained fibres from a GFP-LC3:WT mouse (WT) and from untreated (GAA−/−) or ERT-treated (GAA−/−; +ERT) GFP-LC3:GAA−/− mice. Images show typical centrally located autophagic build-up with multiple clusters of LC3-positive autophagosomes. Structures like these are never seen in WT muscle. Autophagic build-up is present virtually in all fibres derived from EDL or gastrocnemiusmuscles (not shown), but only in 60-70% of FDBfibres. Labelled recombinant human GAA (red) was administered i.v. into 3-4 month-old GFP-LC3:GAA−/− mice (n = 5) at a dose of 100 mg/kg twice with a 24 h interval. Mice were sacrificed the next day, and live fibres (shown for EDLmuscle) were analysed by confocal microscopy. The labelled recombinant GAA was detected almost exclusively in the autophagic vesicles. At least 500 fibres were analysed for each condition. Bar: 10 µm.
Amino-acid starvation inactivates TORC1, de-repressing ULK1.figf8Amino-acid starvation inactivates TORC1, de-repressing ULK1. ULK1 is recruited to VPS34-Beclin-1 complexes through binding to ATG14L, phosphorylating Beclin-1, and activating the VPS34 kinase and PtdIns(3) P production at the nascent autophagosome. In addition, UVRAG-bound Beclin-1 is phosphorylated by ULK1, which may promote autophagosome maturation.
Figure 4Representative Western blot for Fam134 proteins in single knockout mouse embryonic fibroblasts (MEFs). Western blot for Fam134 proteins in indicated MEFs, expressing the ER-phagy reporter ssRFP-GFP-KDEL and being reconstituted with the respective wild-type or ∆LIR mutant Fam134 protein. ER-phagy flux in ssRFP-GFP-KDEL Fam134 knockout and reconstituted MEFs, represented by the RFP/GFP ratio (total fluorescent intensities). Data is representative for three biological replicates and presents averaged data obtained from three individual wells (technical replicates) via the IncuCyte® S3, each view containing >150 cells. C) ER-phagy flux in basal (DMSO) or autophagy induced (Torin1) conditions. D,E) ER-phagy flux in Thapsigargin (Tg) treated MEFs.
Ubiquitylation of Rpl25 at K74 regulates 60S ribophagy.Figure 3.(A) Indicated strains were transformed with plasmids encoding either Rpl25-GFP (Rpl25) or rpl25 K74,75R-GFP (rpl25KR) before the deletion of a genomic copy of RPL25. (top) Comparable expression levels of Rpl25-GFP or rpl25 K74,75R-GFP in whole cell lysates were confirmed with anti-GFP blotting. Purified 6His-ubiquitin-conjugated forms of Rpl25-GFP or rpl25 K74,75R-GFP (middle) and 6His-ubiquitin expression (bottom) were analyzed as in Fig. 2. Ub, ubiquitin. (B) Wild-type and ubp3Δ cells expressing Rpl25-GFP or rpl25 K74,75R-GFP were starved for the indicated period of time. (C) Degradation of GFP-tagged proteins was analyzed by anti-GFP blot of whole cell extracts, and the ratio between cleaved GFP and full-length protein was quantified for all time points in four independent experiments. a.u., arbitrary units. (D) Localization of GFP-tagged proteins. C, cytoplasmic localization; V, vacuolar accumulation; C + V, localization in both cytoplasm and vacuole (E) Degradation of Rpl3 was analyzed at 24 h upon starvation by Western blotting of whole cell extracts using anti-Rpl3 (gift from V. Albanese, Institut Jacques Monod, Paris, France) or anti-Mex67 (as a control) antibodies. Rpl3 and Mex67 expression was quantified and normalized to the expression level at time 0. Significance of the differences observed for the ribophagy efficiency was evaluated using Student's t test. *, P = 0.01-0.05; **, P = 0.001-0.01; ***, P < 0.001. White lanes indicate that intervening lanes have been spliced out. The errors bars correspond to standard deviations.
Figure 8A) HEK293A and VPS15 KOs were treated with vehicle or VPS34-IN1 (1μM; IN1), alone or with MRT68921 (1μM; MRT), for 18 hrs before fixation and visualisation of ULK1 (blue), p62 (green) and ATG9A (red). Dashed boxes show magnified regions of interest. B) HEK293A were transfected with siRNAs targeting ATG14, UVRAG or p62, or with non-targeting siRNA (RISC-free, RF) and cells were treated with VPS34-IN1 (IN1; 1μM) for 0, 4 or 18 hrs as indicated. Quantification of ATG13 pS318/Total ATG13, mean +/- SEM, n=3. Identical samples were loaded onto 2 separate SDS PAGE gels for Western blot analysis, indicated by dashed line. C) Cells transfected as in B were treated with IN1 for 18 hours as indicated, fixed and labelled for ubiquitin (green), FIP200 (red) and p62 (blue). FIP200-positive bodies were quantified from 4 independent experiments with mean +/- SEM plotted. Cells transfected with ATG14 siRNA generated significantly more FIP200 bodies per cell than all other conditions tested.
Figure 4hCDC14A, not the inactive hCDC14AC278S, dephosphorylates the serine residue 142 of DBN1 in vivo. RPE1 cells expressing GFP, hCDC14A-GFP, and hCDC14AC278S-GFP under TetON promoter were serum starved in presence or absence of Dox for 48 h prior to fixation for immunofluorescence. DBN1pS142 was detected with a phospho-specific antibody. DNA was stained with DAPI. Scale Bar = 10 μm Immunoblot analysis of samples from (A). The DBN1pS142/DBN1 ratio was densitometrically measured from the Dox treated samples and represented in the graph next to the immunoblot. In this experiment two GAPDH blots were used to normalize protein levels because DBN1 and DBN1pS142 were analyzed in separated blots. Three independent experiments. Mean ± SD. ** P≤ 0.01, *** P≤ 0.001 DBN1S142 was hyper-phosphorylated in hCDC14APD cell lines during ciliogenesis compared to the WT control. In this experiment we sequentially analyzed DBN1pS142 and DBN1 on the same blot. The specificity of the DBN1pS142 antibody was validated by siRNA mediated knock-down of DBN1 in both hCDC14APD cells and GFP/hCDC14A-GFP/hCDC14AC278S-GFP stable cell lines. Three independent experiments. Mean ± SD. ** P≤ 0.01, *** P≤ 0.001 In vitro dephosphorylation of DBN1 by purified hCDC14A. DBN1-GFP was transfected into HEK293T cells and expressed for 48 h. DBN1-GFP was immunoprecipitated from cell lysates with GFP binder. The immunoprecipitated phospho-DBN1 was then incubated with and without recombinant hCDC14A in vitro. The DBN1pS142/DBN1 ratio was densitometrically measured. Three independent experiments. Mean ± SD. **** P≤ 0.0001
Figure 6(A) Overlap among H4K5 hyperacetylated regions in the arid2/3/4 and epcr1/2 mutants relative to the wild type. The overlap is significant (P=4.410-78) as determined by hypergeometric test. (B) Metaplot of histone H4K5 acetylation levels of TEs in wild type, arid2/3/4, and epcr1/2 plants. Histone H4K5 acetylation levels are represented by normalized reads that obtained in the H4K5Ac ChIP-seq analysis. TSS represents transcription start site and TTS represents transcription termination site. (C) Boxplot of histone H4K5 acetylation levels of TEs in wild type, arid2/3/4, and epcr1/2 plants. Asterisks indicate that H4K5Ac levels is significantly higher in the arid2/3/4 (P=2.4210-13) and epcr1/2 (P=1.5210-8) mutants than in the wild type as determined by Welch's Two-Sample t-test. Horizontal lines represent the median, and the bottom and top of the box represent the 25th and 75th percentile. The whiskers represent data range within 1.5× of the interquantile range. (D) The nuclei with condensed, intermediate, or decondensed heterochromatin status, as marked by DAPI staining and H3K27me signals. (E) Percentages of nuclei with condensed, intermediate, or decondensed heterochromatin signals in the wild type and the arid2/3/4 mutant. n=113. (F) Abundance of Pol IV-dependent siRNAs in the epcr1/2, arid2/3/4, nrpd1, nrpe1, and hda6 mutants relative to the wild type [(Mut-WT)/(Mut+WT)] from chromosome 3 of Arabidopsis. (G) Venn diagrams showing the numbers of differentially expressed Pol IV-dependent 24-nt siRNA clusters (P4-siRNAs) in arid2/3/4, epcr1/2, and hda6 relative to the wild type. All the overlaps are highly significant (P→0) as determined by hypergeometric test. (H) Boxplot showing levels of Pol IV-dependent siRNAs that are up-regulated in the hda6 mutant. Asterisks indicate that siRNA levels in the mutants are significantly (P→0) up-regulated compared to the wild type as determined by paired student t-test. Horizontal lines represent the median, and the bottom and top of the box represent the 25th and 75th percentile. The whiskers represent data range within 1.5× of the interquantile range. (I) Distribution of the Pol IV-dependent siRNA regions in which siRNAs are up-regulated in the mutants relative to the wild type. The pericentromere refers to 6 million base pairs of a pericentromeric region on each chromosome.
Figure 1A Scheme of H1-GFPFRAP in heterochromatin foci (with HP1a-mCherry) and euchromatin (without HP1a-mCherry). The relative FRAP curves of euchromatin (Eu) and heterochromatin (Het) are shown divided into two parts-the mobile fraction (MF) and the immobile fraction (IF) after bleaching recovery. (ROI, region of interest) B FRAP curves of H1-GFP in reprogramming with SKO. The mean values of relative fluorescence recovery were shown in the curves. For heterochromatin, the recovery ratio is higher in cells transfected with reprogramming factors than that in the control on day 3. Changes are significantly different. More than 16 cells were analyzed for each group. (*p≤0.05)C The ratio of MF at 120 s post-bleaching in FRAP was compared in the reprogramming stages. For heterochromatin, cells transfected with SKO showed much more rapid recovery than control cells on day 3. More than 16 cells were analyzed for each group. (*p≤0.05) D DNA FISH images showing the localizations of endogenous Oct4 locus and HP1a foci in MEFs infected with SKO or Flag control. More than 72 cells were analyzed for each group. Scale bar: 5 μm.E Summary of percentage of co-localizations at the Oct4 locus and HP1a foci in SKO mediated reprogramming. More than 72 cells were analyzed for each group.
OCRL deficiency causes failed microtubule nucleationA Representative images of NHF, Lowe 1676 and Lowe 3265 cells immunostained with anti-α-tubulin (red) and anti-γ-tubulin (green) antibodies.Figure 3A Representative images of NHF, Lowe 1676 and Lowe 3265 cells immunostained with anti-α-tubulin (red) and anti-γ-tubulin (green) antibodies. Scale bar, 10 μm. B Quantitative analyses of focused microtubule arrays numbers at γ-tubulin (microtubule organizing center MTOC) measured in the experiments shown in A. n = 3 cells per group, three independent experiments. C Data information: data are presented as mean ± SEM. * ≤ 0.05, ** ≤ 0.01, calculated by two-way ANOVA, multiple comparisons. C WT (Ocrl+/y), OCRL knockout (Ocrl-/-) and IOB MEFs derived from the corresponding mice were costained with anti-α-tubulin and anti- γ-tubulin antibodies. Nuclei were stained with DAPI (blue). Scale bar, 10 μm. D Quantitative analyses of focused microtubule arrays numbers at γ-tubulin (microtubule organizing center MTOC) measured in the experiments shown in C. n = 3 per group in which 30 cells were counted per condition per experiment. E Data information: data are presented as mean ± SEM. * ≤ 0.05, ** ≤ 0.01, calculated by two-way ANOVA, multiple comparisons. E Microtubule regrowth assay was performed on NHF, Lowe 1676 and Lowe 3265 cells with 20 μM nocodazole for 2 h on ice, followed by rinse with PBS, and addition of pre-warmed medium to induce microtubule regrowth. Cells were then immunostained with anti-α-tubulin and anti-γ-tubulin antibodies at the indicated time point after addition of pre-warmed medium. The magnified view shows the cytoplasmic microtubules organization center. Scale bar, 10 μm. F Quantitative analyses of focused microtubule arrays numbers at γ-tubulin (microtubule organizing center, MTOC) measured in the experiments shown in E. n = 3 per group in which 30 cells were counted per condition per experiment. D Data information: data are presented as mean ± SEM. * ≤ 0.05, ** ≤ 0.01, calculated by two-way ANOVA, multiple comparisons.