EMBO/sd-character-level-ner huggingface dataset:
High expression of FBN2 and placensin in human, but not mouse, placenta.Figure 2A FBN2 mRNA levels in multiple human tissues. Human tissue cDNAs were used as templates for PCR of FBN2, followed by agarose gel analyses. GAPDH levels served as loading controls (biological replicates, n=2). B FBN2 mRNA levels in mouse tissues based on quantitative RT-PCR. Data were normalized based on levels in adipose tissues. C Comparison of FBN2 transcript levels between human and mouse placentas. Common primers for FBN2 and GAPDH in both species were used for RT-PCR analyses (biological replicates, n=3) . D Immuno-histochemical detection of placensin in human placental villi obtained from women at 9 and 38 weeks of pregnancy. During early pregnancy, placensin immunostaining was found in cytotrophoblasts (c), whereas syncytiotrophoblasts (s) showed weak staining. At 38 weeks of pregnancy, strong placensin signals were detected in both syncytiotrophoblasts and cytotrophoblasts (arrows). Staining using E-cadherin antibodies confirmed the identity of cytotrophoblasts (Inset showing higher magnification E-cadherin (E-Cad) staining). Lower panels depict sections stained with nonimmune IgG as negative controls. Scale bars, 100 μm. E Immunoblotting of placensin in human placental villi (term pregnancy). Samples from three individual patients were processed with or without N-glycosidase pre-treatment to reveal untreated and deglycosylated placensin. F For prokaryotic cell expression, a cDNA fragment corresponding to residues 2779 to 2912 of the human FBN2 coding region was appended with GST and 6-histidine tags and sub-cloned into the PGEX-6P-1 vector for expression in E. coli. Left panel: Coomassie blue staining of proteins in the SDS-PAGE gel before and after digestion with the PreScission protease. 1: Flow through; 2: Washes; 3: Before PreScission treatment; 4: After PreScission treatment. Right panel: Immunoblotting using placensin antibodies. G Immunoblotting of recombinant placensin generated in E. coli. and CHO cells. For CHO cell expression, placensin cDNA sequence was subcloned into an adenoviral vector pAV[Exp]-CMV downstream of the IgK signal peptide under the control of the CMV promoter for infection of cells before immunoblotting of media 2 days later. Recombinant placensin proteins secreted from CHO cells (b) showed higher molecular weight than those from bacteria (a). Following treatment with N-glycosidase to remove N-linked carbohydrate side chains, prokaryotic- and eukaryotic cell-derived placensin proteins showed similar sizes.
Figure 4A. Teratomagrowth curves of trisomic and wild-type ES cells after subcutaneous injection into SCIDmice. 1×106 cells were injected per site (6 sites/per group). Error bars, ±S.D. * P < 0.05. B. Four-week teratomas derived from wild-type and trisomic (Ts8 or Ts11) ES cell lines. 5×105 cells were injected per site (8 sites/per group). C. Representative images of undifferentiated regions in Ts8 ES cell derived teratomas (middle, top and bottom) and the primitive neural rosettes in the teratomas derived from Ts15 ES cells (right, top and bottom). The images of mature neuroectoderm from wild-type ES cell formed teratomas (left, top and bottom) were shown. The bottom panels were the enlarged views of the white dotted boxes of the top panels. Hematoxylin and eosin staining. Scale bars, 50 μm. D. Immunohistochemistry analysis of Oct4 expression in wild-type, Homo-11, Ts6, Ts8, Ts11, or Ts15 ES cellteratomas produced via the injection of 5×105 ES cells. Scale bar, 100 μm. E. AP staining of the primary cultured teratomacells derived from trisomic and wild-type ES cells. Ts6, Ts8, or Ts11 ESC teratomacells grew ES-like colonies that were stained positive for AP. Scale bar, 200 μm. F. The expression of marker genes in cultured teratoma cells (p3). Error bars, ±S.D. n=3. ** P < 0.01.
Figure 4(A) The overall structure of the scFv FD20 (cartoon) in complex with SARS-CoV-2 RBD (green cartoon in semi-transparent surface). VH and VL denote heavy and light chain variable domains, respectively. The VH CDRs and VL CDRs are colored as indicated. The RBD structure resembles a highchair that has a 'seat' region and a short 'backrest' region. (B) Detailed interactions between residues from the RBD (green) and scFD20 involve VH CDR3 (magenta), VL CDR1 (orange), and VL CDR2 (cyan). FD20 residues are labeled black and RBD residues are labeled grey. A prime symbol indicates residues from VL CDRs. (C) scFD20 (dark red) binds the RBD (white) at a site distal to that of ACE2 (light blue). (D) Modest perturbation of scFD20 for RBD-ACE2 interaction. A sensor with RBD immobilized was soaked in 200 nM of scFD20 before being further soaked in scFD20-containing buffer with (red) or without (blue) 25 nM of ACE2 for BLI signal recording. As a control, the ACE2-RBD interaction was monitored in the absence of scFD20 (black). (E) The FD20 epitope (red) is conserved between SARS-CoV-2, RaTG13, and SARS-CoV. Dots indicate identical residues and dashes indicate gaps. Brackets indicate sequence identity between the aligned RBD and SARS-CoV-2 RBD. NCBI accession codes for the sequences are as follows. RaTG13, QHR63300.2; SARS-CoV, NC_004718.3. (F) The FD20 epitope (Cα in black sphere) is similarly organized in three-dimension among the coronaviruses that contain an RBD-equivalent region. The PDB accession codes are: SARS-CoV-2, 7CYV (this work); SARS-CoV, 2G75 (Prabakaran et al, 2006); MERS, 5X59 (Yuan et al, 2017); hCoV-HKU1, 5GNB (Ou et al, 2017); and hCoV-OC43, 6OHW (Tortorici et al, 2019). (G) The predicted 'ideal' epitope (red + magenta) is mostly contained in the FD20 binding site (red + blue). The three CDRs (green for heavy chain and cyan for light chain) that are in close contact with RBD (white) are shown. A green arrow indicates Glu465 which is at the center of the predicted 'ideal' epitope (Starr et al, 2020).
a) Proliferation of B cells purified from Atg5f/fCD19-Cre and Atg5f/f control mice and activated for 3 d with LPS, assessed by flow cytometry analysis of dilution of the membrane dye CFSE.figf2(a) Proliferation of B cells purified from Atg5f/fCD19-Cre and Atg5f/f control mice and activated for 3 d with LPS, assessed by flow cytometry analysis of dilution of the membrane dye CFSE. (b) Size of B cells purified as in a and activated for various times (horizontal axis) with LPS, assessed by the intensity of forward scatter (FSC) by flow cytometry. (c) CD138 expression of B cells purified and activated as in a. APC, allophycocyanin; PE, phycoerythrin. (d) Immunoblot analysis of ER proteins in lysates of Atg5f/f and Atg5f/fCD19-Cre B cells 3 d after stimulation with LPS (left), and quantification of band intensity (right; as in Fig. 1c). IgM-H, IgM heavy chain. NS, not significant; *P 0.05 and **P 0.01 (Student's t-test).
Figure 7Under ambient temperatures, CBF1 transcript and protein levels are induced by phyA and phyB in the light. CBF1 promotes PIF4 and PIF5 protein accumulation in the light by directly binding to PIF4 and PIF5 promoters to induce their expression, and by inhibiting phyB interaction with PIF4 and PIF5. Thus, CBF1 promotes hypocotyl elongation under ambient temperatures by positively regulating PIF4 and PIF5 protein accumulation in the light through transcriptional and post-translational mechanisms. Lower levels of PIF4 and PIF5 proteins accumulate in the cbf1 mutants in the light, therefore cbf1 mutants develop shorter hypocotyls under light conditions.
An intact C-terminus facilitates nuclear export of patient FMRP.Figure 4Transfected HEK293 cells expressing GFP-tagged wild-type FMRP fused to the patient NLS motif (GFP-FMR1wt+NLS). Unlike the patient FMRP, GFP-FMR1wt+NLS protein is predominantly cytoplasmic and does not aggregate in the nucleus. However, treatment of HEK293 cells expressing GFP-FMR1wt+NLS with leptomycin B-an inhibitor of nuclear export-resulted in the appearance of nucleolar inclusions. This suggests that the presence of a full-length C-terminus facilitates the nuclear export of FMRP in this context.Transfected HEK293 cells expressing a modified version of GFP-tagged patient FMRP, in which the truncation of the C-terminus is reverted by restoring the open reading frame (GFP-FMR1G-ins.[Ct restored]). The patient protein is not detected in the nucleus when the C-terminus is intact; however, nucleolar retention of the protein is observed upon leptomycin B treatment of the transfected cells. In line with results from (A), this suggests that an intact C-terminus enables nuclear export of the FMR1 protein.Data information: Scale bar represents 10 μm.
Figure 4(A) lncPSCA predominantly locates in the nuclear fraction of gastric cancer cells. In detail, the cytosolic and nuclear fractions of HGC-27 or MGC80-3 cells were separately isolated. RNA was isolated from either the cytosolic fraction or the nuclear fraction. Expression of lncPSCA in the cytosolc and nuclear RNA was measured. Data show one representative example of three biological replicates. (B) Silver staining of lncPSCA pull-down nuclear proteins of gastric cancer cells. (C) lncPSCA pull-down followed by Western blot validated its interaction with DDX5 and other candidate proteins identified by Mass spectrometry. GAPDH served as the control. (D) RIP assays show association of DDX5 with lncPSCA in MGC80-3 and HGC-27 cells. Relative enrichment (means ± SD) represents RNA levels associated with DDX5 relative to an input control from three independent experiments. IgG served as the control. HOTTIP was used as the negative control and SRA as the positive control during RIP. Data show one representative example of three biological replicates. (E) Western blot analyses of DDX5 protein in gastric cancer cells with the enforced expression of lncPSCA. (F) Gastric cancer cells after lncPSCA-knockout or stably overexpressing lncPSCA and control cells were treated with cycloheximide (CHX) or vehicle for the indicated periods of time. DDX5 levels were analyzed by Western blot. (G) Western blot to detect the ubiquitination of DDX5 in HEK293T cells cotransfected with pcDNA-lncPSCA, HA-Ubiquitin and Flag-DDX5 (left panel) or in HGC-27 cells cotransfected with pcDNA-lncPSCA and HA-Ubiquitin (right panel). (H) Expression of DDX5 was compared between normal and cancer samples in TCGA cohort (left), Shandong Discovery cohort (middle) and Shandong Validation cohort (right). (I) Knockdown of DDX5 with siRNA (siD-1 and siD-2) substantially reduced the proliferation of gastric cancer cells. Data show one representative example of three biological replicates.(K) Effects of DDX5 knockdown or DDX5 overexpression on the colony formation of gastric cancer cells. HGC-27 or MGC80-3 were seeded into a 6-well cell culture plate and transfected with DDX5 siRNAs (siD-1 and siD-2). The HGC-27 or MGC80-3 cells stably transfected with the DDX5 shRNA (shD) were seeded into a 6-well culture plate. When colonies were visible after 14 days, cells were washed with cold PBS twice and fixed with the fixation fluid. Cells were stained with crystal violet and photos were taken.
Figure 3Quantitative real-time PCR (RNU1A1 and RNU5G- normalized) showing blockade of microRNA transfer from CD4+ T cells pre-treated with siRNA targeting the exosome secretion mediator Rab27. Bar charts represent the mean values ± SEM of at least 4 independent experiments. Quantitative real-time PCR showing mRNA target down-modulation after IS formation between DICER-KO B cells and CD4+ T cells nucleofected with either siRab27 or scrambled control siRNA, with normalization to GAPDH. Bar charts represent the mean values ± SEM of at least 4 independent experiments. Representative dot-plots, showing IgG1 expression of gated B220+ B cells after IS formation as in (G); the bar chart shows the mean fluorescence Intensity ± SEM and quantification of 3 independent experiments is shown. Experimental design. Rag1KO-mice were adoptively transferred with WT B lymphocytes and either Rab27-KO T cells (top) or WT T cells. Thereafter mice were inoculated with sheep red blood cells, and spleens and serum were harvested after 7 days. Representative plot (left) and quantification (right) of the reconstitution of the B cell and T cell compartments after adoptive transfer. The graph shows the mean percentage of CD19 and CD4 positive cells from n ≥ 5 reconstituted mice ± SEM. Representative flow cytometry plot (left) and quantification (right) of GL7 and CD95/Fas expression in B cells. The graph shows the mean percentage of GL7+Fas+ cells from n ≥ 5 reconstituted mice ± SEM. Representative dot-plot analysis showing CXCR4 and CD86 expression on B cells for Dark zone (DZ) and Light zone (LZ) analysis (gated on GC GL7+ Fas+ B cells). The graph shows the mean percentage of DZ (CXCR4+CD86-) and LZ (CXCR4-CD86+) populations from n ≥ 5 reconstituted mice ± SEM. ELISA quantification of IgM (H) and IgG (I) in serum from immunized adoptively transferred mice. The graph shows the mean serum concentration of IgM and IgG from n ≥ 5 reconstituted mice, respectively ± SEM. Proposed model of exosomal microRNA transfer during cognate B and T cell interactions.
TMEM41B localizes to the ER.Figure 5 A H4 Cas9 cells stably expressing N-terminally (Myc-TMEM41B) or C-terminally Myc-tagged TMEM41B (TMEM41B-Myc) were probed by calnexin (CANX) and Myc tag immunostaining and imaged with an automated CV7000 confocal microscope. Scale bar: 20 μm   B-D Endogenous TMEM41B locus was tagged with C-terminal Myc using CRISPR-directed homologous recombination in HeLa cells. HeLa knock-in (KI) cells were compared to parental HeLa (-) and HeLa cells stably transduced with a TMEM41B-Myc over-expression (OE) construct. (B) Confocal micrographs of cells stained with antibodies against CANX and Myc confirming ER localization of both KI and OE TMEM41B-Myc. Scale bar represents 20 μm  μm. (C) Immunoblot analysis confirming expression of both KI and OE TMEM41B-Myc (D) PCR analysis with primers binding to TMEM41B last intron and to Myc tag confirming successful targeting of the endogenous TMEM41B locus with Myc (band labeled with asterisk). M=size marker  E H4 Cas9 cells and H4 Cas9 cells stably expressing Myc-TMEM41B or TMEM41B-Myc were lysed and subjected to anti-Myc IP. Eluates were analyzed by mass spectrometry. Enrichment of proteins in IPs from Myc-TMEM41B or TMEM41B-Myc cells versus H4 Cas9 cells is depicted as log2 fold changes. Entire data is reported in Dataset EV2
Noc–membrane association requires spreading on DNA A–F Localisation of ParB‐box mutants.Figure 4A-F Localisation of ParB‐box mutants. Cellular localisation of (A) Noc‐mYFP (DWA206), (B) NocQ68R‐mYFP (DWA285), (C) NocG86S‐mYFP (DWA286), (D) NocR88A‐mYFP (DWA545), (E) NocR89A‐mYFP (DWA546) and (F) NocR91A‐mYFP (DWA547). Insets show the corresponding phase contrast images. Scale bar, 5 μm. G Ability of ParB‐box mutants to rescue the growth defect of Δnoc ΔminCD. Strains DWA564 (Pxyl‐noc‐myfp), 590 (Pxyl‐nocQ68R‐myfp), 568 (Pxyl‐nocG86S‐myfp), 598 (Pxyl‐nocR88A‐myfp), 600 (Pxyl‐nocR89A‐myfp) and 602 (Pxyl‐nocR91A‐myfp) were streaked on plates containing 0.5% w/v xylose and incubated for 18 h at either 30 or 39°C, as indicated, before being photographed. H-O ParB‐box mutants are dominant‐negative. Cells of strains DWA362 (ΔminCD, Pspac(hy)‐noc), 363 (ΔminCD, Pspac(hy)‐nocNΔ10), 364 (ΔminCD, Pspac(hy)‐nocQ68R) and 365 (ΔminCD, Pspac(hy)‐nocG86S) were examined after growth for 2 h at 42°C with either no additions (NA) (H-K) or in the presence of 1 mM IPTG (L-O), as indicated. Cell membranes were stained with FM5‐95. Insets show the corresponding phase contrast images. Scale bar, 5 μm.
Figure 9A. Western blot of phosphatase-treated whole cell lysates from yeast strains carrying rbd2-Flag-APEX2 (rbd2-F), rbd2-G246R-Flag-APEX2 (G246R) or rbd2-S130A-Flag-APEX2 (S130A) plasmid in rbd2Δ background and sre1Δ yeast was probed with anti-Sre1 or anti-Flag IgG. P and N denote Sre1 precursor and cleaved nuclear forms, respectively. B. Indicated yeast strains (200 cells) containing chromosomal Flag-tagged rbd2, rbd2-G246R or rbd2-S130A in rbd2Δ background (lane 3-5) or yeast strains (200 cells) carrying rbd2-Flag-APEX2, rbd2-G246R-Flag-APEX2 or rbd2-S130A-Flag-APEX2 plasmid in rbd2Δ background (lane 6-8) were grown on rich medium in the absence or presence of cobalt chloride (CoCl2). C. Comparison of Rbd2 protein levels from overexpression system to endogenous expression. Western blot of whole cell lysates from yeast strains containing chromosomal Flag-tagged rbd2, rbd2-G246R or rbd2-S130A in rbd2Δ background and yeast strains carrying rbd2-Flag-APEX2, rbd2-G246R-Flag-APEX2 or rbd2-S130A-Flag-APEX2 plasmid in rbd2Δ background was probed with anti-FlagIgG. For quantification, the intensity of Flag band in each lane was normalized to the non-specific band (asterisk). D. rbd2Δ cells expressing indicated plasmids of GFP-Anp1 or GFP-Sre2MS together with rbd2-Flag-APEX2, rbd2-S130A-Flag-APEX2 or rbd2-S130A/G246R-Flag-APEX2 were treated with Bz at a concentration of 1 mM for 2 hr prior to biotin-labeling reaction. After labeling termination, cells were lysed and denatured as described in Materials and Methods. Biotinylated proteins were then enriched using streptavidin magnetic beads. Lysates (1x input) and 100x-enriched eluates were analyzed by western blot with IRDye 800CW Streptavidin, anti-Flag IgG, anti-Cdc48 serum, and anti-GFP IgG. Intensity of GFP band in enriched sample was normalized to that in the corresponding input sample, then normalized to the Flag intensity in enriched sample. Relative intensities of biotinylated GFP level are shown in bar graph. Error bars denote standard deviation from three biological replicates.
Figure 3A: Flow cytometric analysis of GFP expression in SSEA-1+ E-GFPd1 ESCs cultured in N2B27/LIF/2i or GMEMβ/LIF/FCS compared to wild-type E14Tg2a ESCs. B: SSEA-1+ E-GFPd1 ESCs sorted into EsrrbHi, EsrrbMed or EsrrbNeg subpopulations. Purity of the sorted populations, assessed by flow cytometry, is indicated above the sorting gates. C: Quantitative Esrrb mRNA expression analysis of populations sorted Error bars: standard deviation of gene expression values measured in four independent experiments. D: Populations sorted were replated at clonal density in GMEMβ/LIF/FCS (top) or N2B27/LIF/2i (bottom) and scored for alkaline phosphatase after 6d. Error bars: standard deviation of the number of colonies observed in two independent experiments. E: Quantitative mRNA expression analysis of populations sorted Error bars: standard deviation of gene expression values measured in four independent experiments. F: Microarray analysis of populations sorted represented as a heatmap ordered by expression change between EsrrbHi and EsrrbNeg. Expression levels were measured from three independent experiments and all genes expressed in at least one sample are shown (n = 14,553). The central line plot summarises the average fold change per cell population compared to EsrrbHi cells (log2 scale) with the colour coding indicated below the heat map. Previously identified Nanog and Esrrb targets are indicated on the right. G: Pearson correlation coefficients of the gene expression profiles of EsrrbH (H), EsrrbM (M), Esrrb- (Neg) E-GFPd1 ESCs and embryo derived (late bud stage) TNG E-2a-tdT EpiSCs (EpiSC) compared to those of naïve ESC and EpiLC H: Pearson correlation coefficients in gene expression obtained comparing EsrrbHi (H), EsrrbMed (M), EsrrbNeg (Neg) E-GFPd1 ESCs and embryo derived (late bud stage) TNG E-2a-tdT EpiSCs (EpiSC) to epiblast/ectoderm cells from embryos dissected at successive developmental stages (CAV, cavity; PS, prestreak; ES, early-streak; MS, midstreak; LMS, late midstreak; LS, late streak; OB/EB, no bud/early bud; LB, late bud). This analysis was restricted to the top 500 differentially expressed gene identified at successive developmental stages sorting by Hotelling T² score
a–e) SH-SY5Y cells expressing control (Ctrl) or scramblase-3 siRNA #508 (PLS3 siRNA) for 72 h were stained with MitoTracker Green FM, a transmembrane potential-independent dye, and treated with rotenone (Rot; 1 μM).figf3(a-e) SH-SY5Y cells expressing control (Ctrl) or scramblase-3 siRNA #508 (PLS3 siRNA) for 72 h were stained with MitoTracker Green FM, a transmembrane potential-independent dye, and treated with rotenone (Rot; 1 μM). Veh, vehicle. (a) Anionic phospholipids exposed to the outer surface of isolated mitochondria were quantified by flow cytometry for Alexa 647-annexin V binding. (a-e) SH-SY5Y cells expressing control (Ctrl) or scramblase-3 siRNA #508 (PLS3 siRNA) for 72 h were stained with MitoTracker Green FM, a transmembrane potential-independent dye, and treated with rotenone (Rot; 1 μM). Veh, vehicle. (b-d) PLS3 knockdown had no effect on rotenone-induced autophagy (b), but decreased mitophagy in SH-SY5Y cells treated with rotenone (c; 1 μM) or 6-OHDA (d; 120 μM). (a-e) SH-SY5Y cells expressing control (Ctrl) or scramblase-3 siRNA #508 (PLS3 siRNA) for 72 h were stained with MitoTracker Green FM, a transmembrane potential-independent dye, and treated with rotenone (Rot; 1 μM). Veh, vehicle. (d,e) The effects of PLS3 siRNA were recapitulated using a second siRNA #433 (d, Supplementary Fig. S3b,g), and reversed by transfection with an RNAi-resistant mouse PLS3 vector (e). Inset: PLS3 overexpression. (f-i) Primary neurons transfected for 72 h with CLS siRNA or scrambled control siRNA were treated with rotenone (250 nM) and analysed for autophagy (Supplementary Fig. S1b) or mitophagy by either co-localization analysis (f) (f-i) Primary neurons transfected for 72 h with CLS siRNA or scrambled control siRNA were treated with rotenone (250 nM) and analysed for autophagy (Supplementary Fig. S1b) or mitophagy by either co-localization analysis (f), or with mitochondrial protein immunoblotting (g) and densitometry (h,i and Supplementary Fig. S4c). Knockdown efficiencies are shown in Supplementary Figs S3 and S4. Mean ± s.d. of n = 4 independent experiments for b,c, and n = 3 independent experiments for a,d-f,h-i (see Supplementary Table S4 for statistics source data); *P0.05 versus vehicle; †P0.05 versus toxin-treated control siRNA; **P0.05 versus Rot/PLS3 siRNA. See Supplementary Fig. S7 for uncropped blots.
DOR stimulates autophagy.Figure 3(A) DOR gain‐of‐function accelerates the degradation of proteins of middle-long half‐life. Untreated HeLa cells and those transiently transfected with DOR were incubated in DMEM or HBSS. *Significant effects of starvation, P0.01; τsignificant effects caused by DOR overexpression, P0.01. (B) DOR overexpression enhances autophagosomal formation. LC3‐GFP‐positive vacuoles were counted in 100 transfected HeLa cells (with GFP‐LC3 and TRα1, or GFP‐LC3 and DOR). *Significant effects of starvation, P0.001; τsignificant effects of DOR overexpression, P0.001. (C,D) HeLa cells transiently transfected with empty pCDNA3 or DOR were incubated in DMEM (basal) or in HBSS (starvation). Cells were processed by transmission electron microscopy. Arrows indicate autophagosomes. Insets are autophagosomes (diameter, 407±63 nm). Scale bars, 1 μm. Autophagosomes were counted in 50 randomly chosen transfected cells. *Significant effects of starvation, P0.05; τsignificant effects of DOR overexpression, P0.05. DMEM; Dulbecco's modified Eagle medium; DOR, diabetes‐ and obesity‐regulated gene; GFP, green fluorescent protein; HBSS; Hank's balanced salt solution; LC3, microtubule‐associated protein 1A/1B‐light chain 3.