EMBO/sd-character-level-ner huggingface dataset:
Figure 3A) Differential expression analysis of transposable elements in rpb-9 (mj261) mutants versus wild type (polyA-selected RNA-seq libraries). [p<=0.01, log2(fold change)>=1, DESeq2 analysis] B) Table showing the numbers of matching piRNAs (with 0, 1, 2 and 3 mismatches respectively) for each of the upregulated transposon families. C) Genome-wide differential gene expression analysis of rpb-9 (mj261) mutants versus wild-type (polyA-selected RNA-seq libraries). [p<=0.01, log2(fold change)>1 or <-1, DESeq2 analysis]. D) Differential expression analysis of germline-specific and soma-specific genes, as classified in (Reinke et al, 2004). "All genes" is shown for comparison. (PolyA-selected RNA-seq libraries, n=3). Pairwise correlation plots of rpb-9 (mj261) and prg-1 (n4357) (E), hrde-1 (tm1200) transcriptomes. Differentially expressed genes in rpb-9 (mj261) are shown in black, shared upregulated genes in red, shared downregulated genes in blue. The piRNA sensor transcript is highlighted in green. (Total RNA "Ribo-Zero" RNA-seq libraries). Pairwise correlation plots of gonad-dissected prg-1 (n4357) transcriptomes. Differentially expressed genes in rpb-9 (mj261) are shown in black, shared upregulated genes in red, shared downregulated genes in blue. The piRNA sensor transcript is highlighted in green. (Total RNA "Ribo-Zero" RNA-seq libraries).
ΔPrPs lacking in addition the N-terminal region still exert efficient DNI.Figure 7. B. Δ31-160 variants are expressed at comparable levels and have EndoH-sensitive and -resistant N-glycans. Immunoblot comparing EndoH-digested and non-digested samples from N2a cells transiently transfected with Δ31-160 variants, probed with anti-PrP mAb C16-S raised against the C-terminal portion of H3. Non-Tf, samples prepared from N2a cells without transfection. Arrowhead denotes deglycosylated Δ159 (lane 11), bracket deglycosylated fragments, square bracket EndoH-resistant fragments. Asterisks indicate non-specific bands. Δ31-160 variants are not completely deglycosylated by EndoH, resulting in EndoH-resistant fragments (square bracket) which are observed also in reducing conditions (right panel; +/− dithiothreitol (DTT) treatment). Note that endogenous wild-type PrPc was not detected under used conditions. C. DNI of Δ31-160 variants is similarly inversely related to size of deletion and dependent on intact C-terminal H1-H2 portion. Immunoblot probed with mAb 3F4 showing PK-resistant (3F4) MoPrP co-transfected with indicated constructs into 22L-ScN2a cells (left panel). Δ31-160 was slightly more effective than Δ159. Right panel shows quantification of results as obtained from a triplicate experiment, using densitometry on ImageJ. Bars illustrate mean ± standard deviation. *, p<0.05. D. Δ31-160 forms spontaneous aggregates with moderate chymotrypsin (Chy) resistance. Immunoblots probed with mAbs C16-S (upper panel) or 3F4 (lower panel) for comparing Chy-resistant fragments of Δ31-160 with that of Δ159. Lysates from N2a cells expressing Δ159 or Δ31-160 were digested with indicated concentrations of Chy for 30 minutes and then digested with PNGaseF. A sample from non-transfected N2a cells is shown in the lane 5. Asterisks indicate non-specific C16-S bands. Arrowhead denotes deglycoform of Δ159, bracket deglycoform of Δ31-160.
Figure 1 A Ex vivo flow cytometry on LN cells of mice 7 days after two s.c. immunizations (14 days apart) with PBS + DMSO (Veh), KLH + DMSO (KLH), KLH + IL-2, KLH + RA, KLH + RA + IL-2. Results are representative FACS plots of LAG3+CD4+ or CD25+Foxp3+ cells after gating on live single CD3+CD4+CD49b+Foxp3- and live single CD3+CD4+ lymphocytes respectively.C Expression of Tr1 markers PD-1, CD226, ICOS, CD25 and CTLA-4 on CD49b+ LAG-3+ Foxp3-CD4+ T cells or on CD49b-CD4+ T cells. D IL-10, IL-17 and IFN-γ production by ELISA on supernatants of spleen cells from mice immunized with PBS + DMSO (Veh), KLH + DMSO (KLH), KLH + RA, KLH + IL-2, KLH + RA + IL-2 or KLH + LP-GMP (LP1569 + c-di-GMP) and cultured for 5 days with increasing concentrations of KLH. Bars are mean + SEM (n=4 or 5). **p<0.01, ***p<0.001, KLH + IL-2 + RA vs KLH, ##p<0.01, ###p <0.001 KLH + IL-2 + RA vs KLH + RA, †††p<0.001 KLH + IL-2 + RA vs KLH + IL-2 by two-way ANOVA and Tukey post hoc test.
Figure 4Hypoxia induced YAP stabilisation via Ser397 dephosphorylated was abrogated in GPRC5A depleted cells. Hypoxia-induced nuclear localisation of YAP was attenuated in GPRC5A depleted cells. Hypoxia activated TEAD activity (8x GTIIC-luc reporter) but this was reduced by GPRC5A depletion. A representative triplicate experiment is shown (n=3). Hypoxia reduced LATS activity and expression but this was prevented by GPRC5A depletion. Constitutively active RhoA (G14V) expression restored YAP stabilisation (Ser397 dephosphorylation) by hypoxia in GPRC5A depleted cells. The YAP/TEAD inhibitor verteporfin selectively inhibited cancer cell survival in hypoxia by crystal violet assay (n=3 independent experiments). YAP knockdown was sufficient to induce caspase-3 activation/PARP cleavage in hypoxia and was not further enhanced by GPRC5A depletion. Crystal violet assays show that YAP was required downstream of GPRC5A to promote cell survival. GPRC5A depleted cells were rescued by expression of an siRNA-resistant GPRC5A cDNA (GPRC5Asi1R) but this was prevented by co-depletion of YAP (n=3 independent experiments). Expression of an siRNA-resistant GPRC5A rescued the critical phenotypes of GPRC5A depletion. GPRC5Asi1R expression prevented PARP cleavage in hypoxia as well as restoring hypoxia-induced YAP stabilisation (Ser397 dephosphorylation); these phenotypes were reversed by YAP depletion. GPRC5A depletion attenuated hypoxia-induced BCL-XL expression. Constitutively active YAP (S127A) expression induced BCL-XL expression and prevents caspase-3 activation/PARP cleavage by GPRC5A depletion in hypoxia. GPRC5Asi1R expression restored BCL-XL expression and prevents the appearance of cleaved caspase-3 induced by GPRC5A depletion in hypoxia.
Figure 6(A-C) Schematic of the CLP model treated with rRv2626c-CA or its mutants (upper). The survival of mice was monitored for 10 days; mortality was measured for n=25 mice per group (lower). Statistical differences compared with the rVector-treated mice are indicated (log-rank test). (D) Serum cytokine levels (E) representative hematoxylin and eosin (H&E) staining of the lung, liver and spleen (left) from 10 mice per group. Histopathology scores were obtained from H&E stained were determined at 20 h in CLP mice were treated with rRv2626c-CA or its mutants. Scale bar, 100 μm. (F) Splenocytes were used for IP with αHis or αTRAF6, followed by IB with αHis or αUb. WCLs were used for IB with αTRAF6, αHis or αActin. (G) In vivo imaging using IVIS spectrum-chromatography CT system. Intraperitoneal administration of rRv2626c-CA/Cy5.5 was carried out at intervals of 0, 6, 12, and 18 h in a CLP-induced sepsis model. Pharmacokinetics and biodistribution were observed in spleen, liver, lung, and kidney tissues. (H) The percentage of F4/80+ macrophages cells and rRv2626c-CA-His were found in the spleen, liver and lung using Fluorescence-activated cell sorting analysis in the background of CLP-induced sepsis.
Figure 4. p53-dependent cell viability and telomere protection in response to DNA damage. (A) Western blot of total p53, γH2AX, and GAPDH in HCT116 p53-/- or p53+/+ cells treated with 1 µM or 50 µM etoposide for 24 hrs. s. (B) Cell cycle analysis of HCT116 p53-/- or p53+/+ cells untreated or treated with 50 µM etoposide for 24 hrs. Cell cycle was determined by FACS analysis of propidium iodide stained cells. (C) Cell clonal survival assay for HCT116 p53-/- or p53+/+ cells treated with DMSO or 1, 1.5 or 2 µM etoposide for 24 hr prior to replating. . (D) Quantification of clonal survival assay relative total cell numbers (top panel) or normalized to DMSO controls (lower panel). E) TIF assay for HCT116 p53+/+ or p53-/- cells treated with 1 µM etoposide for 3 hrs. Telomere DNA FISH with TelC probe (red), γH2AX (green), Dapi (blue). (F) Quantification of TIF assay for % cells with > 4 TIFs per cell for p53+/+ (black) or p53-/- (red) cell. G) Telomere length assay showing p53-/- or p53+/+ cells treated with either DMS0, 1 or 50 µM etoposide. Ethidium bromide (EtBr) stained agarose gels (left panel), Southern blot hybridized with Tel C probe (middle panel) or with α Satellite DNA promote (right panel). Quantification of telomereDNA signal relative to invariant bands in α Sat. Error bars represent SD and * indicates p values <.05 determined by student t-test (TLA), or chi-square (TIF assay). (H) Two-dimensional agarose gel analysis of telomeric DNA from HCT p53+/+ or or p53-/- treated with 50 µM etoposide for 24 hrs. Ethidium gel (top) and Southern blot probed with TelG probe (lower panel). Single stranded telomere DNA (red arrow) and recombination/ replication-associated structures (blue arrow) are indicated.
Figure 7(A) CD45+ immune cells infiltrating 99LN-BrM (n=5 for isotype, n=4 for the other groups). (B) Flow cytometric analysis of dendritic cell (CD45+CD11c+CD83+) infiltration (n=4 for isotype, n=3 for all other groups). (C) Quantitative analysis of IHC CD3+ T cells at trial end point. (n=10 for isotype, n=7 for WBRT, n=10 for PD-1, and n=8 for WBRT+αPD-1 group). (D) Flow cytometric analysis of T cell subpopulations in 99LN-BrM (n=4 for isotype, n=3 for all other groups). (E) Quantitative analysis of IHC FoxP3+ T cells at trial end point. (n=10 for isotype, n=7 for WBRT, n=10 for PD-1, and n=8 for WBRT+αPD-1 group). (F) Flow cytometric analysis of PD-1 expression on T cells in response to different treatments (control n=4, all other groups n=3). (G) Composition of the myeloid compartment in 99LN-BrM in the different treatment groups (n=5 for isotype, all other groups n=4). (H) Relative abundance (%) of PD-L1+ myeloid cell types in BrM samples of the four treatment groups (n=5 for isotype, all other groups n=4). (I) Experimental design of the in vitro T cell activation assay. (J) Relative CD69 protein level on T cells cultivated with different cell types, unstimulated or stimulated with tumor conditioned media (Cond.) (n=3). (K) Relative Gzmb and IFNγ protein level on CD4+ and CD8+ T cells cultivated with different cell types, with or without aPD-1 and unstimulated or stimulated with tumor conditioned media (Cond.) (n=4 for conditions including BMDM, n=5 for all other conditions).
Figure 5(A) Representative 5-day-old seedlings of wild-type Arabidopsis thaliana Col-0 and TF mutants gata12 and wri3 grown in the dark. Scale bar, 1 mm. (B) Heat map indicates which TFs significantly affects hypocotyl length, root length and the composite trait of root to total length in dark-grown seedlings and whether the effects of TFs are dependent on condition. TFs are hierarchically clustered using Euclidean distance. (C) Heat map of the average relative effects of TF mutant alleles on hypocotyl length, root length and the ratio of root to total length reveal that TF lesions significantly perturbed TCA cycle-dependent growth. Mutant alleles are listed in rows and traits in columns. Cells of TF mutant alleles in heat map are colored if the AGI or AGI:TCA Metabolite linear model terms for each trait are statistically significant (P < 0.05, two-way ANOVA, 16-20 seedlings per genotype per condition across two experiments per genotype). Mutant alleles are hierarchically clustered using Euclidean distance. (D-F) Hypocotyl length, root length, and root to total length is dependent on TF and exogenous TCA cycle metabolites. Radar plots presents mutant phenotypes relative to Col-0 (black). Ratio of mutant:WT traits were determined using the estimated marginal means of each genotype calculated from 16-20 seedlings per genotype per condition across two experiments.
Figure 3(A) The schematic diagram for cell-cell fusion and syncytia formation. (B) HEK293 and H1975 cells were transduced with full-length ACE2 by lentivirus. Protein extracts were immunoblotted with the indicated antibodies. O/E represents overexpression. (C) GFP and full-length Spike co-transfected HEK293T cells were used as effector cells (293-S). HEK293/ACE2 (HEK293T-overexpressing ACE2) (left panel) or H1975/ACE2 (H1975-overexpressing ACE2) (right panel) cells were used as target cells. The 293-S cells were preincubated with normal human IgG or ACE2-Fc at 37°C for 1 hr. After that, the mixtures were added to target cells for 4 hr (cell-cell fusion) or 24 hr (syncytia formation). The fluorescent areas were measured by inverted fluorescence microscopy and Metamorph software (Metamorphosis). Scale bars equal to 50 µm. The white arrows indicate the cell-cell fusion or syncytia formation. (D) The inhibition of cell-cell fusion and syncytia formation by ACE2-Fc in HEK293/ACE2 and H1975/ACE2 was determined with the formula described in the Methods section. Error bars represent the standard deviation (SD), n=6. Statistical analysis was performed by unpaired two tail t-test. ** P < 0.01, *** P < 0.001.
Figure 4Representative immunofluorescence images of pancreatic acinar cells after vehicle or 24h cerulein (CER) treatment stained for RAB7 (red) and Cathepsin D (CTSD, green, lysosomes). Top row is wild-type, bottom row is Atf3−/−. Yellow arrowhead highlights RAB7 and CTSD co-expression. White arrowhead points to an area of RAB7 expression only. Scale bar, 20μm. Representative immunofluorescence images of gastric chief cells after vehicle or 24h high-dose tamoxifen (HDT) treatment stained for RAB7 (red) and Cathepsin D (CTSD, green, lysosomes). Top row is wild-type, bottom row is Atf3−/−. Yellow arrowhead highlights RAB7 and CTSD co-expression. White arrowhead points to an area of RAB7 expression only. Scale bars 20μm. Stomach unit base outlined by dashed white line. Representative immunofluorescence images of gastric chief cells after vehicle or 24h HDT treatment stained for Pepsinogen C (PGC, red, granules) and MAP1LC3B (green, autophagy marker). Top row is wild-type, bottom row is Atf3−/−. Scale bar, 50μm. Stomach unit base outlined by dashed white line. Transmission electron micrograph images of zymogenic chief cells (ZCs) after 24h vehicle or HDT treatment in WT (top) and Atf3−/− mice (bottom). Scale bar, 1μm. Arrowheads indicate cell components being degraded. Quantification of degradation events per cell counted from EM images of ZCs after 24h HDT treatment in WT and Atf3−/−mice as shown in panel 3D. Each data point=total granules in a cell in a single TEM section with cells from 2 separate mice plotted together (data point colors indicate cells from a single mouse). Red line indicates mean across all the mice.
Figure 5At 4 DIV, cultured cortical neurons were transfected with indicated plasmids. Three days later, neurons were treated with 10 μg/ml poly(I:C) for 24 h. Neuronal morphology was monitored by GFP signals at 8 DIV. Mean values SEM of representatives of three independent experiments are shown. The numbers of analyzed neurons in the representative experiments are indicated in each column. Data were analyzed by two-way ANOVA with Bonferroni's multiple comparison test. Scale bar, 20 μm. *** P < 0.0001, ns: non-significant.
Figure 1U2OS cells modified to expressed endogenous GFP-tagged ATG2A (green) were grown in complete media (CM) or starved (EBSS) for 2 h before fixation and immunostaining with antibodies against ATG2B (red) and LC3B (magenta) and analysed by confocal microscopy. Arrows mark ATG2A/LC3B/ATG2B positive structures. Scale bar 10µm. (B-D) Cells were treated as in (A) and stained with anti-WIPI2 (red) or (C) anti-ATG16L1 (red) or (D) GABARAP-L1 (red). Arrows mark structures of interest. Scale bar 10µm. U2OS WT or U2OS GFP-ATG2A knock-in (KI) cells were grown in CM or starvation media for 2 h, lysed and incubated with anti-GFP nanobody beads coupled to agarose to immunoprecipitate (IP) GFP-ATG2A. IP samples and 2% input lysates were run on 4-12% gradient gel and processed for western blotting. Anti-ATG2A, anti-WIPI4 and anti-LC3B, anti-GABARAP (pan) were used to probe for the presence/absence of autophagy proteins in the immunoprecipitated samples. p-p70S6K (T389) was used as a marker for starved cells and total p70S6K as loading control.
Model.Figure 6 Figure 6: Model. In absence of HH, Smaug is associated, likely as S-bodies (in red) to inactive SMO present on intracellular vesicles and leads to the repression (blue line) of its target mRNA(s) (called here xmRNA) via the recruitement of proteins (not represented here) that promotes their decay. Upon HH reception, activated and phosphorylated (yellow P circles) SMO accumulates at the plasma membrane where it relocates both the kinase FU and Smaug. This lead to FU activation which in turn phosphorylates (green full circle) SMO and Smaug. This phosphorylation of Smaug would reduce its negative effect (dashed blue line) favoring the release of its target mRNAs - possibly via the dissolution of the S-foci - and leading to their accumulation. Finally, when the highest levels of SMO phosphorylation are reached (red P circle), phosphorylated Smaug is released, leading to attenuation of the effects of HH/SMO/FU on Smaug. EV Figure Legends
Figure 2A Photo of the individual with 2.1 years. B Photo of the individual with 4 years. C Photo of the individual with 5.9 years. D Magnetic resonance imaging (MRI) of the affected individual at the age of 5.9 years. E Growth charts / anthropometry of the patient before and after treatment with fucose
Overall abundance changes of mitochondrial proteins.Figure 1(A, B) Relative change in total abundances between control and ρ0 cells averaged over four independent migration profiles (Dataset EV1). A, all mitochondrial proteins detected. Grey area indicates proteins that changed less than 2-fold in both directions. Subunits of selected mitochondrial machineries are highligted as follows: magenta, pyruate dehydrogenase complex (PDH); blue, mitochondial carriers (Carrier); brown, translocase of the outer membrane (TOM); yellow, translocase of the inner membrane (TIM); green, mitochondrial contact site and cristae organizing system (MICOS); yellow-green, mitochondrial fusion-fission system (Dynamics); red, porin or voltage dependent anion channel (VDAC); B, subunits of OXPHOS complexes (Complex I-V) and mitoribosomes (Ribosome). Gene names of the subunit with the highest abundances in ρ0 cells are indicated for each complex. For the full gene name, add NDU (complex I), SDH (complex II), UQC (R10), COX (complex IV), ATP (5L, 5J2), MRP (Ribosome) in front of the abbreviated gene names given in the figure.
HCV induces complete mitophagolysosome formation.Figure 5Confocal microscopy showing the formation of mitophagolysosome in HCV-infected cells. (A) Huh7 cells transiently expressing GFP-LC3 protein were infected with HCVcc in the absence or presence of 3-MA (10 mM) and BafA1 (100 nM), respectively, for 12 h before fixation. At 3 days post-infection, cells prestained with LysoTracker (red) were immunostained with antibodies against TOM20 (blue) and HCV core (cyan). Nuclei are demarcated with white dot circles. In the zoomed images, the arrows indicate the colocalization of GFP-LC3 puncta (green), lysosome, and mitochondria in HCV-infected cells (white puncta). (B) Ultrastructure of HCV-infected cells showing the formation of mitophagolysosome. HCV FLR-JFH1 replicon cells were examined by electron microscopy. In the zoomed image, the formation of mitophagolysosome in HCV-infected cells is shown. Organelle marker: ML, mitophagolysosome. Scale bar = 0.1 µM. (C and D) Quantitative analyses of the colocalization of GFP-LC3 containing lysosomes with mitochondria (C) or Parkin (D) in the panels (A) and Figure S5 (mean ± SEM; n≥10 cells; *p<0.001, **p<0.05). (E) ImageJ quantitative analysis of the number of mitochondria (mean ± SEM; n≥10 cells; *p<0.001, **p<0.05). P values were calculated by using an unpaired Student's t-test.
Phospho-mimic (S3D) cofilin efficiently binds and severs oxidized F-actin.Figure 2A. Time-lapse images of eGFP-S3D-cofilin-1 (blue) binding and severing of an oxidized actin filament (yellow). B. Global quantification of the severing events on oxidized and non-oxidized filaments exposed to unlabelled S3D-cofilin1 from time t=0 onwards. From top to bottom, N = 50 (from 1 experiment), 50 (1 experiment), 100 (2 experiments) filaments. Data information All lines are survival fractions calculated from the experimental data 95% confidence intervals are shown as shaded surfaces. C. Growth rate of single cofilin domains, normalized by the cofilin concentration. N = 20 domains for WT mCherry-cofilin-1 (1 experiment) and eGFP-S3D-cofilin-1 (2 experiments). The growth rate was measured at 10 nM WT cofilin-1, and 500 nM and 1 µM S3D-cofilin-1 (Appendix Fig S4C). Bars: mean and S.D. P-value = 8.10-9 (t-test). D. Filament severing at single cofilin domains. Time t=0 is defined for every domain as the frame on which they nucleate. N = 60 (1 experiment) and 75 (2 experiments) domains of WT and S3D-cofilin-1,respectively. The severing dynamic was measured at 10 nM WT eGFP-cofilin-1 and 1 µM eGFP-S3D-cofilin-1. P-value = 0.98 (log-rank test). Data information: All lines are survival fractions calculated from the experimental data 95% confidence intervals are shown as shaded surfaces.
Figure 4Principal-component analysis of differential expressed genes between 45-day-old wildtype H9 hCOs and RGCC-KO hCOs #23 based on RNA-seq. Each plot represented an individual organoid (n=3 for each groups from 1 experiment). Heatmap representing hierarchical cluster of 6716 DEGs (adjusted absolute log2(FC)>0.5, padj <0.05). Heatmap representing DEGs of NSC markers and neuronal markers from RNA-seq analysis (padj <0.05). , GO, Reactome and DisGeNET enrichment analysis of DEGs between WT H9 and RGCC KO.
Atg16L1 interacts with the ULK1–Atg13–FIP200–Atg101 complex.Figure 1(A) Yeast two‐hybrid screening for Atg16L1‐interacting proteins. The Atg16L1‐interacting FIP200 clones are indicated. (B,C) HEK293T cells were co‐transfected with the indicated constructs. Cell lysates were subjected to IP using anti‐FLAG antibodies. The resulting precipitates were examined by immunoblot analysis with anti‐FLAG and anti‐HA antibodies. (D) HEK293T cells were cultured in regular or starvation medium for 2 h. Cells were harvested and treated with DSP. (E) HEK293T cells were harvested and treated with DSP. (F) HEK293T cells were cultured in regular DMEM or starvation medium in the presence or absence of 200 nM wortmannin for 2 h. After treatment with DSP, cell lysates were subjected to IP analysis. (G) Atg14F/F (undeleted) or Atg14Δ/Δ (deleted) MEFs were harvested and treated with DSP. * (E) and ** (G) indicate the positions of the immunoglobulin light and heavy chains, respectively. aa, amino acid; DSP, dithiobis(succinimidyl propionate); IP, immunoprecipitation; MEFs, mouse embryonic fibroblasts.
VAMP3 Regulates Autophagy.Figure 5(A) HeLa cells were transfected with siRNAs for different R-SNAREs, and we assessed the levels of the autophagy substrate p62 versus actin. (B) HeLa cells transfected with control or VAMP3 siRNA (oligo A) for 4 days were treated during the last 16 hr with Bafilomycin A1 (Baf A1) or DMSO (basal) and processed for LC3-II western blots. (C) Endogenous LC3 immunocytochemistry in HeLa cells transfected with control or VAMP3 siRNA for 4 days. The histogram shows the number of vesicles (LC3 positive) in a minimum of 30 cells. Error bar, SEM. ∗∗∗p < 0.001. (D) HeLa cells were transfected with VAMP3-HA for 20 hr and stained for EEA1 or RAB11. Histogram shows colocalization between VAMP3-HA and EEA1 or VAMP3-HA and RAB11 (Manders' coefficient). Error bar, SEM. (E) HeLa cells were transfected for 20 hr with VAMP3-HA, incubated for 1 hr at 37°C or 18°C, fixed, and labeled for HA and mATG9. Histogram shows the percentage of colocalization between VAMP3 with mATG9 (Manders' coefficient). Error bar, SEM. NS, not significant.