EMBO/sd-character-level-ner huggingface dataset:
Figure 4A. HumanHEK293T cells were transfected with plasmids expressing different 3xHA-tagged rhomboid proteases: S. pomberbd2, S. pomberbd2 recoded for human expression, humanRHBDL2, D. melanogaster Rho1, and P. falciparumROM4. Whole cell lysates were analyzed by western blot to detect the HA-tagged rhomboid [asterisks indicate loading control bands detected with antibody ab179726 (abcam)]. B. HumanHEK293T cells were transfected with plasmids expressing the indicated rhomboid substrates and either recoded S. pombe rbd2, D. melanogaster Rho1, or P. falciparumROM4. Conditioned media and cell lysates were analyzed by western blot. P and C denote precursor and cleaved products, respectively. C. HumanHEK293T cells were transfected with plasmids expressing the GFP-TatA-Flag substrate and the indicated rhomboid proteases. Whole cell lysates were analyzed by western blot. P, CN, CC denote precursor, N-terminal cleaved and C-terminal cleaved products, respectively. D. HumanHEK293T cells were transfected with plasmids expressing the indicated GFP-TatA-Flag substrates and either wild-type or mutant recoded S. pomberbd2. Whole cell lysates were analyzed by western blot. E. HumanHEK293T cells were transfected with plasmids expressing the indicated GFP-TatA-Flag substrates and different rhomboid proteases. Whole cell lysates were analyzed by western blot. F. HumanHEK293T cells were transfected with plasmids expressing the GFP-TatA (A8L)-Flag and recoded S. pomberbd2. C-terminal cleavage products were purified using anti-Flag antibodies and purified protein was analyzed by mass spectrometry.
The DNA methylation inhibitor 5-AzadC induces HIV-1 expression in latently-infected T cells.Figure 1A-D J-Lat 8.4 cells were mock-treated or treated with increasing concentrations of 5-AzadC or 5-AzaC. At 72 h post-treatment, viral production was measured by quantifying p24 antigen production in culture supernatants (A) A-D J-Lat 8.4 cells were mock-treated or treated with increasing concentrations of 5-AzadC or 5-AzaC. At 72 h post-treatment, metabolic activity was assessed by a WST-1 assay (B) A-D J-Lat 8.4 cells were mock-treated or treated with increasing concentrations of 5-AzadC or 5-AzaC. At 72 h post-treatment viral protein expression was analyzed by FACS (C) A-D J-Lat 8.4 cells were mock-treated or treated with increasing concentrations of 5-AzadC or 5-AzaC. At 72 h post-treatment; and initiated (primers TAR) or elongated (primers tat) transcripts were quantified by RT-qPCR (D). E J-Lat 8.4 cells were mock-treated or treated with 5-AzadC (400nM) or TNFα (10ng/ml) as positive control. At 24 h, 48 h or 72 h post-treatment, initiated (primers TAR) or elongated (primers tat) transcripts were quantified by RT-qPCR.
Fig.Figure 2Illustrations show the two major SVs associated with filament/s in the wild-type RA-SV pool at rest; synaptic ribbon (R, red), SVs (dark green) with filament/s (blue). Not drawn to scale. (A,B) Ri-attached SVs (Ri.) and (C,D) interconnected SVs (In.) harbor single (A,C) or multiple (B,D) filaments. Example virtual section from resting wild-type; single Ri. (A',A'') and In. (C',C''), multiple Ri. (B',B'') and In. (D',D''). Magnification 12,000x; scale bar, 40 nm. Structures are color-coded similarly to the illustrations, the number of ribbon-attached filaments per SV are indicated with red arrowhead/s and interconnectors per SV with purple arrowhead/s in A'', B'', C'' and D''. Magnification 12,000x; scale bar, 40 nm. The graph is representing the percentage of RA-SVs around the whole ribbon at wild-type under inhib, rest and stim. Upon stimulation, the percentage of SVs without filaments and ribbon-attached SVs are significantly reduced, while interconnected SVs increased significantly. Simplified illustration summarizing the findings in the RA-pool. Not drawn to scale; SVs with filaments (dark green, blue filaments) and without filaments (light green).
Figure 5(A) Amino acid sequence of the last 50 residues of FtsA (MPN316), FtsZ (MPN317) and DnaB (MPN525) proteins and their mutant derivatives. Hydrophobic motifs that can potentially act as Lon degrons are highlighted in bold. Mutations performed in these putative Lon degrons are shown in red. (B) Protein stability assessment of Lon substrates containing multiple or single mutations in putative Lon degrons. N-terminal FLAG-tagged derivatives with mutations shown in panel A were expressed in the ΔIndLon mutant. Protein levels were then determined by immunoblot using anti-Lon and anti-FLAG antibodies comparing inducing (+) or depleting (-) conditions (48h of depletion). LC, loading control.
Figure 4A) Schematic illustration of HP1 mutants used in this study. V23M and I161E mutants lose the ability to bind H3K9me3 and to form dimers, respectively. B, Elucidation of protein-protein interaction between HP1 and H3K9 MTs by co-immunoprecipitation analysis. Whole-cell extracts of HEK293T cells co-expressing HA-HP1β mutants and epitope-tagged proteins of interest were subjected to immunoprecipitation with the anti-HA tag antibody. The immunocomplex was analyzed by immunoblotting with the indicated antibodies. The intensities were quantified using ImageJ software, normalized, and shown on the top. C) Elucidation of protein-protein interaction between HP1 and H3K9 MTs by co-immunoprecipitation analysis. Whole-cell extracts of HEK293T cells co-expressing HA-HP1β mutants and epitope-tagged proteins of interest were subjected to immunoprecipitation with the anti-HA tag antibody. The immunocomplex was analyzed by immunoblotting with the indicated antibodies. The intensities were quantified using ImageJ software, normalized, and shown on the top. D) Strategy to establish mESCs solely expressing mutated HP1 (left). We introduced expression constructs for 3xFlag-hHP1β (wild-type or mutants) into HP1-cTKO cells and then deleted the formerly introduced wild-type Myc-hHP1β transgene by 4OHT treatment. E) Evaluation of HP1β mutants for maintaining H3K9me2/3 and ensuring protein stability of H3K9 MTs. H3K9me2/3 levels and protein levels of H3K9 MTs in HP1-cTKO lines expressing 3xFlag-hHP1β mutants were compared before and after 4OHT treatment. The intensities were quantified with ImageJ software and are summarized on the right. Data represented as mean ± SD (n > 3, biological replicates). *p < 0.05, **p < 0.01 (Welch's test).
Figure 6Schematic diagram describing the generation of mixed bone marrow chimeras. Lineage-depleted bone marrow of B cell-deficient JHT Prdm1+/+ mice was mixed at ratio of 1:15 with lineage-depleted bone marrow of T-cell deficient Eβ-/- Prdm1ihCd2/+ or control Eβ-/- Prdm1+/+ mice prior to injection into lethally irradiated Rag2-/- recipient mice and subsequent analysis 4 months after transplantation Flow cytometric analysis of total B cells and different B cell subsets in the bone marrow of non-immunized Eβ-/- Prdm1ihCd2/+ (red) and Eβ-/- Prdm1+/+ (gray) chimeric mice. Bar graphs indicate absolute numbers of the different B cell types in these chimeric mice Flow cytometric analysis of total B cells and different B cell subsets in th splee of non-immunized Eβ-/- Prdm1ihCd2/+ (red) and Eβ-/- Prdm1+/+ (gray) chimeric mice. Bar graphs indicate absolute numbers of the different B cell types in these chimeric mice Histograms display the expression of hCD2 (Blimp1) in splenic FO B, GC B and naïve CD4 T cells from the chimeric mice of the indicated genotypes
Figure 1(A) Schematic of the ARS306 leading-strand lesion template showing the origin of replication (Ori) and leading- (red) and lagging-strand (blue) replication products. The SmaI restriction site is shown; cleavage is used to reduce heterogeneity in product sizes for denaturing gel analysis. (B) Schematic of the uncoupled fork and uncoupled product generated by persistent stalling of leading-strand synthesis. (C, D) Standard replication assays on undamaged (UD), thymine glycol (Tg), and cyclobutane pyrimidine dimer (CPD) templates in the presence (C) and absence (D) of Pol η, Pol ζ, Rev1, Rad6-Rad18, Uba1, and ubiquitin (TLS machinery). Reactions contained 2.5 nM Pol δ.
a) MDCK/pGFP–LC3 cells infected with a wild-type or ΔactA2 strain of L.figf1(a) MDCK/pGFP-LC3 cells infected with a wild-type or ΔactA2 strain of L. monocytogenes were stained with an anti-Listeria antibody (red) and rhodamine phalloidin (stains F-actin; light blue). Scale bars, 10 μm. Arrowheads indicate GFP-LC3-positive bacteria. (b) High magnification of framed images in a, and graphs show intensity of the fluoresence signal along the arrow. (c) Quantification of the number of GFP-LC3-positive bacteria shown in a. Data are mean ± s.e.m. At least 500 bacteria were counted in each experiment (n = 7 and 6 experiments at 2 and 4 h, respectively). (d) LC3 levels in infected MDCK cells. MDCK cells were infected with wild-type or ΔactA2 bacteria for the indicated times. EBSS (Earle's balanced salt solution, a starvation medium) and rapamycin (100 μg ml−1) were used as controls. (e-f) Intracellular survival of wild-type or ΔactA2 bacteria in MDCK cells (e; n = 8), MDCK cells treated with 10 mM 3-MA (f; n = 4), (e-h) Intracellular survival of wild-type or ΔactA2 bacteria in atg5+/+ MEF cells (g; n = 8 and 7 for wild-type and ΔactA2, respectively) or atg5−/− MEF cells (h; n = 4 and 3 for wild-type and ΔactA2, respectively). Data are presented as the number of colony-forming units (CFU) relative to that at 30 min after infection, and are mean ± s.e.m. WT, wild type.
a) Western blot analysis of HeLa cells treated with siRNA for 84 h.figf1(a) Western blot analysis of HeLa cells treated with siRNA for 84 h. (b) Western blot analysis of HeLa cells treated with a second independent siRNA targeting ATG5, ATG7 or NDP52. GAPDH and total protein stained with Coomassie blue serve as loading controls. (c) Western blot analysis of HeLa cells cultured in Hank's buffered salt solution (Starved) or DMEM+ 10% FBS (Serum). (d) Western blot analysis of HeLa cells treated (48 h) with an inhibitor of mTORC1 (RAP, 20 nM) or control (DMSO). (e) Western blot analysis comparing the levels of DICER in MDA-231, T47D and MDA-435 cells. (f) Western blot analysis of MDA-231, T47D and MDA-435 cells treated (48 h) with inhibitors of mTORC1 (RAP, 20 nM), mTORC1 and 2 (PP242, 100 nM), or DMSO (control). (g) Western blot analysis of HeLa cells treated with CQ or DMSO for 12 h. (h) Western blot analysis of HeLa cells treated with BAF (200 nM, 12 h), RAP (20 nM, 24 h), or RAP and BAF. The results in a-h are representative of three experiments and use α-tubulin (TUBA) as a loading control. (i) Western blot analysis of DICER (monoclonal antibody 13D6) or control (EEF1A1) in HeLa cells treated with control siRNA or siRNA targeting DICER. (j) Confocal analysis of the signal from an anti-DICER monoclonal antibody (clone 13D6) or an isotype control monoclonal antibody in HeLa cells. Scale bar, 10 μm. (k,l) RT-qPCR analysis of AGO1, AGO2 and DICER mRNA in HeLa cells treated for 12 h with DMSO, BAF or CQ (k) or siRNA for 84 h (l). Error bars show s.e.m. Uncropped images of blots are shown in Supplementary Fig. S3.
Figure 2(A) Respiratory chain super-complex levels are slightly restored by over-expression of Mcp3. Mitochondria isolated from the indicated strains were lysed in 1% digitonin and subjected to a 4-8% BN-PAGE. Proteins were analysed by immunodecoration with antibodies against either Cor1 of complex III or Cox2 of complex IV. (B) Assembly defects in TOM and TOB complexes in mdm10 cells are partially restored by Mcp3 over-expression. Mitochondria of the indicated cells were lysed in 1% digitonin and subjected to a 6-13% BN-PAGE and immunoblotting with antibodies against Tom40 (left) or Tob55 (right). The assembled TOM complex and an unassembled Tom40 species at ca. 100 kDa (arrowhead) are indicated as well as the TOB complex and an additional higher molecular weight species of the TOB complex (asterisk). (C) Over-expression of Mcp3 partially rescues the alterations in lipid composition of cells lacking Mdm10. Lipids were extracted from highly pure mitochondria isolated from the indicated cells and then analysed by mass spectrometry analysis. The level of each phospholipid species in wild-type mitochondria was set to 100% and the ERG/PL ratio to 1. Relative changes in mitochondria from other cells are presented. The mean with standard deviations of three biological repeats with two technical repeats for each (n=6; SD; *, p < 0.05) is given. (D) Mcp3 rescues loss of Mdm12. Wild-type (WT) or mdm12Δ cells transformed with the empty plasmid pYX142 () or mdm12Δ cells over-expressing either Mcp3 or Mdm12 (as a control) were analysed at 30°C or 37°C by drop dilution assay on rich medium containing glycerol (YPG). (E) Mcp3 rescues loss of Mmm1. WT and mmm1Δ cells were transformed and analysed as described in (D). (F) Mcp3 rescues loss of Mdm34. WT and mdm34Δ cells were transformed and analysed as described in (D).
a,b) E1-charging assay for Uba1 and Atg7 with either Atg8a (a) or ubiquitin (Ub; b),.figf7(a,b) E1-charging assay for Uba1 and Atg7 with either Atg8a (a) or ubiquitin (Ub; b), n = 2 experiments with independently isolated proteins and analyses. Flag-tagged baculoviral-expressed E1s were incubated with either His-tagged Atg8a or Ub in the presence or absence of β-mercaptoethanol, separated by electrophoresis and blotted. Band shift was detected with anti-Flag antibody. Relative molecular mass (Mr K) ladders are indicated. Representative images are shown. Relative molecular masses: Flag-Uba1, 135K; Flag-Atg7, 81K; His-Atg8a, 29K; Ub, 9K. (c) Midgut protein extracts at puparium formation from wild-type, Atg7 mutant (Atg7d77/d14) and Atg8 (Atg8KG07569) mutant animals blotted with anti-Atg8 and anti-Tubulin, n = 3 independent biological experiments. Representative images are shown. (d) Midguts from control Atg310/wild-type (Atg3/+) and Atg310/Df(3L) cat mutant (Atg3/Df) animals at puparium formation analysed by differential interference contrast microscopy. Representative images are shown. (e) Cell size quantification (μm2) from d, n = 11 (Atg3/+) and n = 9 (Atg3/Df) animal intestines per genotype with 5 cells measured per intestine. (f) Midguts dissected from early third instar larvae that mis-express Atg1GS10797 (Atg1) and Atg3IR only in the DsRed-marked cell clone and analysed by fluorescence and differential interference contrast (DIC) microscopy. Representative images are shown. (g) Midguts dissected from animals expressing Atg4DN specifically in DsRed-marked clones of cells at puparium formation and analysed by fluorescence and differential interference contrast microscopy. Representative images are shown. (h) Cell size quantification (μm2) from g, n = 10 animal intestines per genotype with 1-5 cells measured per intestine. (i) Midguts dissected from animals expressing mCherry-Atg8a in all cells, and expressing UbIR specifically in GFP-marked clones of cells at puparium formation. Representative images are shown. (j) Midguts dissected from animals expressing UbIR specifically in GFP-marked clones of cells at puparium formation and analysed by fluorescence and differential interference contrast microscopy. Representative images are shown. (k) Cell size quantification (μm2) from (j), n = 12 animal intestines per genotype with 1-5 cells measured per intestine. (l,m) Midguts dissected from early third instar larvae that exhibit Atg1GS10797 (Atg1) mis-expression in either a wild-type GFP-positive cell clone (l) or a Uba1H33 loss-of-function MARCM mutant cell clone (GFP-positive; m) and analysed by fluorescence and differential interference contrast microscopy. Control wild-type and heterozygous Uba1H33/wild-type cells have no GFP. Representative images are shown. (n) Quantification (μm2) from l and m, n = 22 (control), n = 16 (Atg1) and n = 6 (Atg1 +Uba1H33) animal intestines per genotype with 1-5 cells measured per intestine. Quantification data for control cells (l,m) were pooled because they are genetically identical. Quantification is shown as mean ± s.d. NS, not significant. Scale bars, 20 μm.
Decreased chromatin-bound RNAPII levels triggered by RBP3-TAP tag rescue mec1-S1991A defects on HU.Figure 6(A) Preparations of chromatin-bound proteins were prepared in triplicate and subjected to SDS-PAGE and immunoblotting with the indicated antibodies in a strain that either does or does not express Rpb3-TAP Quantification of chromatin-bound Rpb1-S2P and Rpb1-S5P is shown on the right. Mcm2 is used as a loading control. (B) G1 synchronized cells were released into S phase with 0.2M HU and total protein extracts were collected at the indicated time points (in min), samples were subjected to SDS-PAGE followed by immunoblotting with Rpb1-S2P, Rpb1-S5P and Tubulin antibodies. (C) Enrichment of the RNAPII subunit Rpb1 on chromatin in G1 synchronized cells was assessed by ChIP-qPCR at indicated genes. ChIP signal of Rpb3-TAP strain was normalized to the corresponding untagged strain. The dashed line indicates the amount of Rpb1 in the untagged strain. FIG2 is a mating pheromone-induced gene used as a control to compare inducible gene and constitutively expressed genes (PYK1, YEF3, PMA1, PDC1 and snR13). (D) mRNA levels measured by RT-qPCR in G1 synchronized cells in the indicated strains. Expression is normalized to either snR13 or FIG2. Data are expressed as a ratio of Rpb3-TAP/WT in percentage. (E) Analysis of replication fork progression (BrdU track lengths) at the single-molecule level by DNA combing in the indicated strains. Box, Whiskers and Median p value < 10-3 ; ns= p value > 0.05, by Mann-Whitney rank sum test using RPB3-TAP as a reference. RPB3-TAP 90 min (n=367) and 180 min (n=490), mec1-S1991A 90 min (n=309) and 180 min (n=477), mec1-S1991A RPB3-TAP 90 min (n=490) and 180 min (n=691). (F,G) A 10-fold dilution series of cells from exponential SC cultures of the indicated strains were spotted on SC +/- 200mM of HU. A high level of HU was used to be able to demonstrate robust suppression of the mec1-S1991A phenotype. (G) Histogram presents quantification of 2 independent HU sensitivity assays with mean and individual data point values indicated for each yeast dilution.
Generation of ICL in MEFs by Cre recombination.Figure 1(A) Schematic of the inverted lox P (iLoxP) site, flanked by sortable GFP and hCD2 markers. (B) Model of reciprocal recombination yielding parental configurations 1/2' and 1'/2 and dicentric/acentric configurations as a result of inverted recombination yielding configurations 1/1' and 2/2'. (C) Representative FACS plot showing loss of hCD2 marker expression of Ch14 ICL MEFs after treatment with 4-hydroxy tamoxifen (4OHT) for 14 days to induce Cre, compared to Ethanol treated control cells (See also Appendix figure S1A). (D) Shallow whole genome sequencing (copy number profiles) of large T antigen immortalized, MEFs after exposure to Cre recombinase and sorted for control (hCD2 plus) and ICL (hCD2 Minus) cells for chromosomes 12, 14 and 9 and 10, without any in vitro culturing, post FACS sort.
Structure and biochemical characterization of the C-terminus of SPIN90.Figure 1 A. Domain organization of SPIN90 and SPIN90 constructs used in this study (SH3 - Src homology 3, PRD - proline rich domain, MS - middle segment, AR - armadillo repeat  B. Ribbon diagram of SPIN90(351-722). Helices are colored according to their position within the repeat (1-yellow, 2-red, 3-cyan). Armadillo repeats are numbered 1-7. Disordered residues are indicated with dotted lines  C. Surface representation of SPIN90(351-722) showing conservation scores derived from an alignment of WDS family proteins from 31 diverse species (see methods). Conservation score is indicated by color, with dark red > pink > light pink > grey > light cyan > cyan, and higher scores meaning greater conservation  D. Time course of pyrene actin polymerization showing the influence of different SPIN90 constructs in reactions containing 50 nM Bos taurus Arp2/3 complex and 3 μM 15% pyrene labeled actin. E. Plot of maximum polymerization rate versus concentration of SPIN90 for reactions shown in (D)  F. Plot of maximum polymerization rate versus concentration of SPIN90 for pyrene actin polymerization assays containing 2.5 μM SPIN90, 500 nM Arp2/3 complex, 3 μM actin and the indicated concentration of the SPIN90 ARM domain construct  G. Time courses of pyrene actin polymerization as described in panel (D), except the influence of the SPIN90 middle segment is tested on its own or in the presence of the 351-722 construct
Figure 1A Cytokine levels (TNFα (24h)), IL-6, TGF-ß and IL-10 (72h)) in supernatants collected from BMDC and AM culture in presence or not (Ctrl) of LPS (50ng/ml), CVF, CLys and CSN (10µg/ml). B FACS assessment of BMDC maturation pre-exposed or not (Med Ctrl) to CVF, CLys and CSN for 72h (left panel) before stimulation with LPS for 24h (right panel). A representative histogram of each condition is shown. C Induction of Tregs (CD4+CD25hiFoxP3+ cells) from spleen cells incubated with CVF, CLys and CSN for 72h. D Induction of Tregs in vivo after adoptive transfer of BMDCs pre-treated for 48h with CVF or CLys and pulsed for 24h with OVA (20 µg/ml) into recipient mice, which had received sorted naïve CD45.1-OT-II cells. On day 7, the lymph nodes were collected and end points analysis was performed.
Figure 5Number of alternative splicing events that are sensitive to a slow elongation rate, including cassette exons, microexons, alternative 3' or 5' splice sites and retained introns in ESCs and at different stages of neural differentiation. UP and DOWN refers to increased or decreased levels of a splicing event in slow/slow cells relative to WT cells with dPSI (percent spliced in) ≥10% and FDR<0.05 when comparing regulated events to all detected events and retained (Total). Splicing was quantified using VAST-TOOLS. RT-PCR analysis validation of selected alternatively spliced exons. RT-PCR was performed on total RNA from WT or slow/slow ESCs, NPCs or neurons. PCR products were visualized and quantified by Bioanalyzer (Agilent). Images are representative of experiments performed in triplicate. Themean ± SEMis plotted with *p-value<0.05, **p<0.005, ***<0.0001 as determined by t-test.
Fig.Figure 1(A) A schematic drawing of experimental procedure where CTB488 is intraocularly injected, followed by intracranial injection of PSvue550 to dLGN border. (B) A schematic diagram shows the timeline of procedures for ex vivo imaging and engulfment analysis. (C) Top panel: Representative images show PS labeling in the WT dLGN at P6 and P13. RGC inputs were labeled with CTB488. Bottom panel: Enlarged images of the boxed region in top panels. Circles indicate PS+ RGC inputs. Arrows point to the enlarged PS+ RGC inputs in the insets. (D) Quantification of the percentage of PS+ RGC inputs in total RGC inputs at P6 and P13. (E) Co-labeling of vGlut2 and PSVue in the dLGN at P6. Circles indicate colocalized vGlut2 and PSVue. (F) Co-labeling of Homer1 and PSVue in the dLGN at P6. Circles indicate colocalized Homer1 and PSVue. (G) Orthogonal sections showed a triple positive PS+/CTB488+/CD68+ RGC inputs inside of microglia. (H) Orthogonal sections showed a triple positive PS+/vGlut2+/CD68+ synapse inside of microglia. (I) A representative image of microglia (upper-left) is surface rendered (bottom-left). The white arrow points to cells which might be apoptotic cells labeled by PSVue. RGC inputs and PSVue outside or inside of microglia are shown in the two right panels. Scare bar, 20 µm.(J) Quantification of the percentage of PS+ and PS- RGC inputs outside of microglia in total inputs. N = 4. (K) Quantification of the percentage of engulfed PS+ and PS- RGC inputs in total engulfed inputs. N=4.