EMBO/sd-character-level-ner huggingface dataset:
Figure 1(a) Primary siRNA screening analysis. MDA-MB-231 cells reverse transfected with siRNA pools against MT1-MMP, TKS5, or each human RAB GTPase were plated on fluorescein-labeled-gelatin coated optical microplates. Degradation activity was measured by counting the number of cells degrading gelatin identified as dark black foci (similar results were obtained by measuring the area of degradation/number of cells). The inset shows representative images of degraded Oregon green gelatin (upper panels) and merged images with TRITC-phalloidin (red) and DAPI (blue) in scrambled siRNA (NC), or siRNA against RAB28 or RAB42 transfected cells (lower panel). Data are means ± SEM of 3 independent experiments. Bar, 50 µm. (b) Secondary siRNA screening analysis was performed on candidate RAB GTPases selected from the primary screen using 3 individual deconvoluted siRNAs. Data are mean ± SD (n> 5 different fields/experiment). (c-e) Tertiary siRNA screening. Pooled siRNAs against candidate RAB GTPases identified from the secondary screen or TKS5, used as control, were transfected into MCF10.DCIS.com cells. Cells plated onto fluorescently conjugated gelatin (red) were stimulated with 100 ng/ml HGF overnight in the presence or absence of GM6001, followed by staining with FITC-phalloidin (green) and DAPI (blue) (c). Data are the mean ± SEM (d, error bars; n > 100 cells/experiment in at least 4 independent ones). (c-e) Tertiary siRNA screening. Pooled siRNAs against candidate RAB GTPases identified from the secondary screen or TKS5, used as control, were transfected into MCF10.DCIS.com cells. Efficacy of targeted gene silencing was verified by qPCR (e). Bar, 50 µm. (f-h) Inverse invasion assay. An equal number of MDA-MB-231 cells was reverse transfected with a mixture of three individual siRNA oligonucleotides targeting interest genes. Invasive activity is shown as the average number of cells/field that migrate into fibronectin-supplemented (25 µg/ml) Matrigel further than 20 µm (f, g). Data are mean ± SD from three different fields from one representative experiments out of 5 that were performed. (f-h) Inverse invasion assay. An equal number of MDA-MB-231 cells was reverse transfected with a mixture of three individual siRNA oligonucleotides targeting interest genes. Efficacy of targeted gene silencing was verified by qPCR (h). Bar, 250 µm. *P < 0.05. ***, P < 0.001.
Sodium butyrate induced endoplasmic reticulum stress in colorectal cancer cells.Fig 5  HCT-116 (A) and HT-29 (B) cells were treated with the indicated concentrations of sodium butyrate (NaB) for 24 h. Representative Western blots show the expression of IRE-1a, BIP, PDI, and CHOP. GAPDH was used as the loading control.
Figure 2The graph shows the mitotic numbers of trunks during different time points of regeneration in the starved condition for cct3A RNAi and controls. Error bars are s.d. from the mean and asterisks indicate P < 0.001 (three asterisks), P < 0.01 (two asterisks) and n.s. indicates not significant using two-tailed Student's test with equal variance. n ≥ 9 planarians per time point. Also shown are maximum projections of representative trunks labelled with anti-H3P at different time points of regeneration in the starved condition. On the bottom, the number of planarians with the phenotype shown from the total is displayed. Note that at 50dR planarians show few (a4) or no mitoses (a5). The dashed line delimits the body of the planarian. The graph shows the mitotic numbers during 72 hours of regeneration in feeding conditions for cct3A RNAi and controls. Error bars are s.d. from the mean and n.s. indicates not significant using two-tailed Student's test with equal sample variance; n ≥ 8 planarian per time point. Maximum projections of representative trunks after FISH for smedwi-1 at different time points of regeneration under starved and feeding conditions. On the bottom, the number of planarians with the phenotype shown from the total is displayed. At 15dR the expression levels are similar. At 30dR some planarians have almost no expression (c5) and at 50dR almost all planarians show no expression (c10 and c11) in starved conditions. Under the feeding condition the expression levels at 30dS are similar. On the bottom, the number of planarians with the phenotype shown from the total is displayed. The dashed line delimits the body of the planarian. Relative expression of smedwi-1 at 13 and 50 days of regeneration after either cct3A or gfp RNAi under feeding and starved conditions. Error bars are s.d. from the mean and asterisks indicate P < 0.001 (three asterisks), P < 0.05 (one asterisk) and n.s. indicates not significant using two-tailed Student's test with equal variance. n = 3 replicates (5 planarians each) per time point.
Figure 4A Schematic of AdVs used for in vivo genome editing. AdvCas9: humanized spCas9 is fused to mCherry through 2A peptide. AdvG/T: an 800 bp corrective template and sgRNA targeting the mouseF9 Y381D mutation was inserted into an AdV containing GFP. B In vivo infection efficiency 4 days after Adv injection. Inserts: negative control from untreated mouse liver tissue. Scale bar: 100 μm. C T7E1 assay was performed on liver DNA obtained from samples in (B), and the frequency of Cas9-induced indels is indicated as '%' below the lane. D Measurement of clotting activity by aPTTs and PTs 8 weeks after tail vein injection with indicated vectors. Data represent means ± SE. E In vivo genome editing efficiency of whole liver tissues from mice treated with Adv(Cas9+G/T). (Left) HDR donor used for correction of Y381D mutation, and Illumina sequencing reads (rn) are presented. Red text indicates the correction of the F9 mutation, whereas blue text indicates the intended synonymous mutations. (Right) The percentage of indel or HDR corrections obtained through sequencing of 235 individual TA-clones (low dose) or Illumina sequencing (high dose) of treated mouseliver tissues.
NlpI localizes along the entire cell envelope and associates with PG machineries A.Figure 4A. Phase contrast image and corresponding fluorescence SIM image of BW25113 cells that have been grown in LB at 37°C and immunolabelled with specific antibodies against NlpI. Scale bar equals 2 µm. B. Genetic interactions of NlpI with PG machineries. An example of a 384 plate is shown. Bar plots shows the averaged values of 2 experiments (i=2, n=192). Error bars denote the 95% confidence interval of the mean. C. NlpI deletion exacerbates the morphological defects of the PBP1B/LpoB-mutant strains. The graph shows the cell width of single and double deletions strains (800 < n < 2000 cells). The box has a medium between 25 and 75%. The whiskers with the upper and lower vertical line indicating the 95 and 5%. The dots are individual points outside the 5 and 95% range. Representative images of the strains are shown above the graph. The scale bar equals 2 µm. Gene encoding protein legend: nlpI encodes NlpI, mrcA encodes PBP1A, lpoA encodes LpoA, mrcB encodes PBP1B, lpoB encodes LpoB. D. Dissociation constants for interactions between PBP1A and LpoA with NlpI, MepM, MepS, PBP4 and PBP7 as determined by MST. The values are mean ± standard deviation of three independent experiments. 1PBP1A was used as fluorescently labelled protein in all assays. 2LpoA was used as unlabelled ligand in all combinations, except with MepM and PBP4.
Figure 6Schematic showing the methodology utilized for preparation of plasma membrane sheets from MDCK cells expressing GFP-HRASWT or GFP-HRASG12V. After preparation of plasma membrane sheets, gold conjugated antibodies are used to visualize clusters of GFP-tagged proteins in electron micrographs. Weighted mean univariate K-function curves of immunogold labeled GFP-HRASG12V under indicated conditions, n ≥ 15 for each condition. The gold point patterns were analyzed with univariate K-functions expressed as L(r)-r. The bar graph on the right shows the peak value Lmax of the L(r)-r curve, which represents the extent of GFP-HRASG12V nanoclustering. Values are normalized to DMSO average and represent mean±SEM (n ≥ 15 for each condition). Statistical significances of differences were evaluated in bootstrap tests. Bar graph showing GFP-HRASG12V content in the inner leaflet of the plasma membrane is quantified as the number of gold particles per 1μm2 region from electron micrographs. Values are normalized to DMSO average and represent mean±SEM, n ≥ 15 for each condition. Statistical test employed was Student's t-test with Bonferroni post hoc correction to account for multiple pairwise comparisons. The extent of GFP-HRASG12V nanoclustering quantified by Lmax values in cells exposed to the indicated perturbations. Values are normalized to DMSO average and represent mean±SEM (n ≥ 15 for each condition). Statistical significances of differences were evaluated in bootstrap tests. Bar graph showing GFP-HRASG12V content in the inner leaflet of the plasma membrane is quantified as the number of gold particles per 1μm2 region from electron micrographs. but in cells exposed to the indicated perturbations. Values shown are normalized to DMSO average. Representative Western blots generated using extracts from HN31 cells stably expressing the MCOLN1 shRNA. Samples treated with exogenous cholesterol for 3 hours are indicated at the bottom. Primary antibodies used are indicated on the right. Representative Western blots generated using extracts from HN31 cells stably expressing the MCOLN1 shRNA. Samples treated with exogenous cholesterol for 3 hours are indicated at the bottom. Bar graph showing quantification of the Western blots in (F). All values represent mean±SEM. Data points represent values from biological replicates. Statistical test employed was ANOVA. Schematic summarizing the effects of decreased TRPML1 activity on HRASWT and HRASG12V. Our data indicate that TRPML1 inhibition diminished HRASG12V nanoclustering and plasma membrane levels. Addition of cholesterol restored these parameters. Only the nanoclustering of HRASWT was affected by ML-SI1.
a, Western blot analysis of human breast carcinoma cell lines.figf4a, Western blot analysis of human breast carcinoma cell lines. Results are from 8 representative cell lines out of a total of 11 analysed. b, Western blot analysis of matched normal breast and breast carcinoma tissue from patients with sporadic invasive breast carcinoma. Results are from 7 representative cases out of a total of 17 analysed. N, normal breast tissue; T, tumour tissue; Mr, relative molecular mass. c, Immunohistochemical analysis of paraffin-embedded sections of breast tissue from patient 441 shown in b. Adjacent sections are stained with pre-immune 843 rabbit serum (left column), 843 anti-Beclin 1serum (middle column) and anti-cytokeratin (right column). Top row shows low power view of region of breast containing tumour (T) and normal mammary epithelial duct (N). Middle row shows higher power view of normal mammary epithelial duct. Bottom row shows higher power view of tumour. Scale bars, 1 mm.
cAMP inhibits S.Figure 8(A) GFP-LC3 CHO cells were preincubated with 1 mM dybutiryl cAMP (+dbcAMP) for 30 min and then were infected for 4 h in the presence of db cAMP with the wt strain of S. aureus (wt), the mutant deficient for α-hemolysin (Hla−), or the Hla(−) mutant expressing an α-hemolysin plasmid (Hla(−) +pHla). Cells without any treatment were used as control (−dbcAMP). The nucleus and the bacteria were labeled with TOPRO as indicated in Materials and Methods, and immediately visualized by confocal microscopy. Images are representative of two independent experiments. . (B) Quantification of the percentage of cells presenting LC3 puncta (i.e., stimulated cells) upon incubation in the different conditions. * p<0.05, ** p<0.01 (paired Student's t-test, n = 50 cells/condition). These data are representative of two independent experiments. (C) Quantification of the number of CFU/ml of CHO cells infected for 3 h with the wt strain of S. aureus, the mutant deficient for α-hemolysin (Hla−), or the Hla(−) mutant expressing an α-hemolysin plasmid in the absence or presence of cAMP. * p<0.05 (paired Student's t-test). These data are representative of two independent experiments.
Figure 6A-B U2OS GFP-LC3 cells were transfected with the indicated siRNAs for 72 hrs before treatment with complete media or EBSS for 6 hrs. Cells were then fixed, imaged, and the number of GFP-LC3 puncta were quantitated per cell. Representative images (A) and quantitation of >50 cells from three independent experiments (B) are shown. Scale bar indicates 50 µm. Data information: Asterisks indicate * p<0.05; ** p<0.01; *** p<0.001. ns indicates p>0.05 using student t-test. C-D A375 parental or DCAF12 KO cells were transfected with the indicated siRNAs for 72 hrs before treatment with complete media or EBSS for 6 hrs. Cells were then fixed, endogenous LC3 was immunostained, and the number of LC3 puncta were quantitated per cell. Representative images (C) and quantitation of >50 cells from three independent experiments (D) are shown. Scale bar indicates 20 µm. Data information: Asterisks indicate * p<0.05; ** p<0.01; *** p<0.001. ns indicates p>0.05 using student t-test. E A375 parental or DCAF12 KO cells were treated Cell lysates were collected and immunoblotted for the indicated antibodies to confirm MAGE-A3/6 depletion and monitor the autophagy adaptor/substrate p62/SQSTM1.
Figure 3A. (Top) BrdU-sensitized U-2-OS (Flp-In T-Rex) cells stably expressing GFP-NLS-CDKL5 were treated with indicated transcription inhibitors before subjecting to spot micro-irradiation (405 nm). (Bottom) Quantitation of spot intensities. Data represent the mean ± SEM of two independent experiments; > 50 micro-irradiated cells per point. Scale bar is 10 μm BrdU-sensitized U-2-OS (Flp-In T-Rex) cells stably expressing GFP-NLS-CDKL5, mCherry-XRCC1 or mCherry-FAN1 were pre-incubated with indicated transcription inhibitors prior to line micro-irradiation (355 nm) and time lapse imaging. One of three independent experiments is shown. Scale bar is 10 μm BrdU-sensitized U-2-OS (Flp-In T-Rex) cells stably expressing GFP-NLS-CDKL5, mCherry-XRCC1 or mCherry-FAN1 were pre-incubated with indicated transcription inhibitors prior to line micro-irradiation (355 nm) and time lapse imaging. One of three independent experiments is shown. Scale bar is 10 μm BrdU-sensitized U-2-OS (Flp-In T-Rex) cells stably expressing GFP-NLS-CDKL5, mCherry-XRCC1 or mCherry-FAN1 were pre-incubated with indicated transcription inhibitors prior to line micro-irradiation (355 nm) and time lapse imaging. One of three independent experiments is shown. Scale bar is 10 μm E. Same as B-D. except that BrdU-sensitized cells stably expressing GFP-NLS-CDKL5 were also pre-incubated with olaparib as control. Cells were subjected to line micro-irradiation, fixed and then subjected to indirect immunofluorescence using antibodies against GFP, PAR and γH2AX. Scale bar is 10 μm F. Stable cell lines were permeabilized and incubated with RNase A or PBS before irradiation and imaging. Scale bar is 10 μm
Reduced cardiac dysfunction in mice with cardiac LXRα activation in response to pathological pressure overload.Figure 3A Echocardiographic assessment of percent fractional shortening in mice subjected to 5 weeks of TAC; n = 19 Wt sham, n = 22 LXRα-Tg sham, n = 24 Wt TAC, n = 26 LXRα-TgTAC. **P < 0.00001 versus Wt sham, *P = 0.0005 versus LXRα-Tg sham, #P = 0.009. B-D Hemodynamic measurements obtained in situ in sham- and TAC-operated mice; n = 6-8/group. (B) Mean arterial pressure (MAP) showed no significant differences. (C) LV end-systolic pressure (LVESP). **P < 0.00001 versus Wt sham, *P = 0.00004 versus LXRα-Tg sham, *P = 0.003. (D) LV end-diastolic pressure (LVEDP). *P = 0.02 versus Wt sham. B-D Hemodynamic measurements obtained in situ in sham- and TAC-operated mice; n = 6-8/group. (B) Mean arterial pressure (MAP) showed no significant differences. (C) LV end-systolic pressure (LVESP). **P < 0.00001 versus Wt sham, *P = 0.00004 versus LXRα-Tg sham, *P = 0.003. (D) LV end-diastolic pressure (LVEDP). *P = 0.02 versus Wt sham.
Figure 6A. (1) A tethered LE/Lys initiates movement. (2) Upon movement, the tether remains intact and the ER is able to hitchhike along with the LE/Lys. (3) When the tethered pair encounter an ER tubule, junction formation may be initiated. (4) After formation the ER may remain tethered. B. ER hitchhiking is likely facilitated by a range of ER- LE/Lys tethers yet will require motor protein recruitment to drive hitchhiking.
Residues connecting the FICD dimer interface with the inhibitory α-helix stabilise FICD and repress AMPylation.Figure 4A Ribbon diagram of the FICD dimer interface with monomers in purple and blue ribbons (PDB: 6I7G). Residues involved in a H-bond network linking the dimer interface to the αinh (as well as Gly299 and Glu234) are shown as green sticks. Sub-3.50 Å hydrogen bonds made by Asn236, Leu238 and Lys256 are depicted as dotted cyan lines. B Size-exclusion chromatography elution profile of wild-type and mutant FICD proteins (each at 20 µM). Protein absorbance at 280 nm is plotted against elution time. The elution times of protein standards are indicated as a reference. C Radioactive in vitro AMPylation reactions containing the indicated FICD proteins, [α-32P]-ATP, and BiPT229A-V461F were analysed by SDS-PAGE. The radioactive BiP-AMP signals were detected by autoradiography and proteins were visualised by Coomassie staining of the gel. D Representative autoradiograph of thin layer chromatography (TLC) plates revealing AMP produced from reactions containing [α-32P]-ATP and the indicated FICD enzymes in the presence of the co-substrate BiP. The radioactive signals were quantified and the AMP signals were normalised to the total nucleotide signal in each sample. The graph shows mean AMP values ± SD from three independent experiments. E Melting temperatures (Tm) of the indicated FICD mutants (at 2 µM) were measured by differential scanning fluorimetry (DSF). Shown is the mean Tm ± SD of three independent experiments. The inset shows melt curves with their negative first derivatives from a representative experiment. F A plot of the melting temperature of the indicated FICD proteins in absence (Apo) or presence of nucleotides. Shown are the mean Tm values ± SD of three independent DSF experiments. Monomeric FICDL258D (mFICD) and FICDL258D-E234G (mFICDE/G) as well as dimeric wild-type FICD (dFICD) and FICDE234G (dFICDE/G) were tested. ADP and ATP concentrations in mM are given in parentheses.
Figure 10A Expression of intracellular HIV-1 mRNA was determined from pools of CD4+ T cells from aviremic HIV-infected patients (n= 3, Appendix Table S4) following 24 h treatment as indicated. Results have been shown as fold induction relative to untreated controls. B Quantitative viral outgrowth assays (qVOA) were used to measure reactivation of HIV-1 provirus from latency in pools of cells from patients. Cells were treated with the indicated concentrations of PH02 and/ or PEP005 for 24 h, prior to analysis of virus production. As indicated in the Appendix Table S5, five different CD4+ T cells pools (n=5) were used for this analysis.
a) Wild-type (Mfn2+/+) and Mfn2 knockout (Mfn2–/–) MEFs stably expressing mCherry-Parkin were incubated in the absence or presence of CCCP (30 μM) for 4 h, fixed, permeabilized and subjected to immunofluorescence analysis with antibodies to FKBP38 and to cytochrome c.f8(a) Wild-type (Mfn2+/+) and Mfn2 knockout (Mfn2-/-) MEFs stably expressing mCherry-Parkin were incubated in the absence or presence of CCCP (30 μM) for 4 h, fixed, permeabilized and subjected to immunofluorescence analysis with antibodies to FKBP38 and to cytochrome c. The fluorescence of mCherry-Parkin was also monitored directly. (b) MEFs as in a but expressing EGFP-Parkin were treated with CCCP (30 μM) for the indicated times and then subjected to immunoblot analysis. The asterisk indicates a nonspecific band. (c) Wild-type MEFs stably expressing EGFP-Parkin and FLAG-VAP-A were incubated in the absence or presence of CCCP (30 μM) or nocodazole (10 μM) for 6 h as indicated, fixed, permeabilized and subjected to immunofluorescence analysis with antibodies to cytochrome c, to FLAG and to FKBP38. The fluorescence of EGFP-Parkin was also monitored directly. (d) Higher-magnification views of the boxed areas in (c,e). Wild-type MEFs stably expressing FLAG-Parkin and EGFP-FKBP38 were incubated in the absence or presence of CCCP (30 μM) for 10 h and then subjected to immuno-electron microscopic analysis with antibodies to GFP. Black arrowheads indicate the mitochondria. White arrowheads indicate small vesicle. (f) Wild-type (Fkbp38+/+) and Fkbp38 knockout (Fkbp38−/−) MEFs stably expressing FLAG-Parkin and either EGFP or EGFP-FKBP38 were incubated in the absence or presence of CCCP (30 μM) for 24 h and then stained with allophycocyanin (APC)-labelled annexin V and propidium iodide (PI) for flow cytometric analysis of the percentage of apoptotic cells (annexin V+ and PI+ cells). Data are means±s.d., from three independent experiments. (g) Model for FKBP38 and Bcl-2 translocation from the mitochondria. Damaged mitochondria accumulate PINK1 and recruit Parkin. FKBP38 and Bcl-2 then translocate from the damaged mitochondria to the ER in order to escape degradation during mitophagy. Scale bars (a,c,d), 10 μm; (e), 500 nm.
YAP stimulates expression of EGF-like ligands and EGFR in cervical cancer cell to promote cervical cancer cell proliferation and migration.Figure 5RT-PCR results showing that YAP stimulates the mRNA expressions of EGFR, AREG, and TGF-α in ME180 cervical cancer cell. Data were analyzed for significance using one-way ANOVA in GraphPad Prism 5 with Tukey's post hoc tests. Each bar represents mean ± SEM (n = 4). Bars with different letters are significantly different from each other (YAP mRNA: MXIV vs. YAP, P = 0.0003; MXIV vs. YAPS127A, P = 0.0001; EGFR mRNA: MXIV vs. YAP, P = 0.0066; MXIV vs. YAPS127A, P = 0.0003; TGF-α mRNA: MXIV vs. YAP, P = 0.0235; MXIV vs. YAPS127A, P = 0.0040; AREG mRNA: MXIV vs. YAP, P = 0.0002; MXIV vs. YAPS127A, P < 0.0001). Proliferation of ME180 cells incubated in medium containing 1% FBS in the absence (control) or presence of 10 ng/ml TGF-α. Data were analyzed with unpaired t-test in GraphPad Prism 5 with Welch's correction. Each point represents the mean ± SEM of four independent repeats. ***P < 0.0001 versus control on day 5. TGF-α treatment (10 ng/ml, 24 h) promotes ME180 cell cycle progression. G1, S, and G2 indicate cells in G1 phase, DNA synthesis phase, and the G2/M phase, respectively, of cell cycle. Apo: apoptotic cells. Representative images showing the morphology of ME180 cells with or without TGF-α (10 ng/ml) treatment for 24 h (scale bar: 50 μm) or 108 h (scale bar: 25 μm). Please note the elongation of ME180 cells after TGF-α treatment for 24 h (TGF-α, 24 h) and the formation of multiple layers in ME180 cells after TGF-α treatment for 108 h (TGF-α, 108 h). Effect of TGF-α on the migration of ME180 cells. TGF-α treatment (100 ng/ml, 10 h) drastically stimulated the migration of ME180 cells.
Characterization of PiCoVacc.Fig. 1(A) SARS-CoV-2 maximum likelihood phylogenetic tree. The SARS-CoV-2 isolates used in this study are depicted with black lines and labeled. The continents where the virus strains were from are colored differently. (B) Growth kinetics of PiCoVacc (CN2) P5 stock in Vero cells. (C) Flowchart of PiCoVacc preparation. (D) Protein composition and purity evaluation of PiCoVacc by NuPAGE 4-12% Bis-Tris Gel. (E) Representative electron micrograph of PiCoVacc.