EMBO/sd-character-level-ner huggingface dataset:
Figure 2A. Representative anti-YAP immunofluorescence images of sparsely cultured RPE cells transfected with the indicated siRNAs. Green, YAP. Blue, DAPI. Scale bar, 10 μm. B. The nuclear YAP intensities of the cells shown in (A) were quantified using the Image J software. At least 70 cells from five or six random fields were analyzed. Quantified values were normalized by adjusting the average of "siControl Dense cells" to 1. We performed two independent experiments and obtained similar results. Data from one representative experiment are presented. The red bar indicates the average value. S, Sparse. D, Dense (***p < 0.001; paired Student's t-test). C. Sparsely cultured RPE or MCF10A cells were transfected with the indicated siRNAs, fractionated into cytosolic and nuclear extracts, and analyzed by Western blotting for the indicated proteins. D. Representative anti-YAP immunofluorescence images of densely cultured RPE cells transfected with the indicated plasmids. Green, GFP or V5. Red, YAP. Blue, DAPI. Scale bar, 10 μm. E. The nuclear YAP intensities of transfected cells in (D) were quantified using the Image J software. At least 70 cells from five to six random fields were analyzed. Quantified values were normalized by adjusting the average of "EGFP transfected cells" to 1. Similar results were obtained from two independent experiments. Data from one representative experiment are presented. The red bar indicates the average value. (***p < 0.001; paired Student's t-test). F. Densely cultured stable RPE clones integrated with empty vector (Vec), USP9X-V5 WT or CS mutant were fractionated into cytosolic and nuclear extracts, and analyzed by Western blotting for the indicated proteins. G. Densely cultured RPE cells transduced with CRISPR-SAM targeting USP9X were fractionated into cytosolic and nuclear extracts, and analyzed by Western blotting for the indicated proteins.
Figure 1A. Characteristics of IGR39 /IGR37 and WM115/WM266.4 melanoma cell lines (Upper Panel). Pigmentation of melanoma cells pellets (lower panel). B. Growth curve of primitive IGR39 and metastatic IGR37 cell line (N=3 biological replicates). Data are mean ±SD. C. Phase-contrast images of IGR39 and IGR37 cells. Scale bar is 100µm. D. Analysis of IGR39 and IGR37 speed of migration by time-lapse microscopy.
BRAVO and WOX5 are required for the QC identity and stem cells maintenance.Figure 1Confocal images of mPS-PI stained 6-day-old seedlings of Col-0 (A), bravo-2 (B), wox5-1 (C) and bravo-2 wox5-1 (D) mutants. Left black arrows indicate QC cells and right white arrows indicate CSC. Scale bar: 50 µm. Quantification of the QC divisions in 6-day-old roots expressed in percentage (n>50, 3 replicates). D: QC divided; ND: QC non divided. Quantification of CSC layers in 6-day-old roots expressed in percentage (n>50, 3 replicates). Lateral root density (number of lateral roots per mm of root length) of 7-day-olf WT, bravo-2, wox5-1 and bravo-2 wox5-1 mutants (n>40, 2 replicates). Different letters indicate statistically significant differences (p- value < 0.05 Student´s t-test). In the boxplot, box width represents the interquartile range (IQR=Q3-Q1), with the horizontal line denoting the median, while whiskers extend from Q1-1.5IQR to Q3+1.5IQR. White dots are the outliers.
Bcl-xL retrotranslocates Bak.Figure 2A Confocal images of HCT116 Bax/Bak DKO cells transfected with GFP-Bak in the absence (left) or the presence (right) of Bcl-xL overexpression. Scale bar 15 µm. n ≥ 10. B FLIP (Fluorescence Loss in Photobleaching) of mitochondrial GFP-Bak in the absence (●, straight line) or the presence (○, broken line) of overexpressed Bcl-xL. Fluorescence of the neighboring cell (in Fig. 2C) is shown as control (, dotted line). Data represent averages ± SEM from 16 (-Bcl-xL) and 40 (+Bcl-xL) ROI measurements. C FLIP of GFP-Bak in the absence (top) or the presence (bottom) of overexpressed Bcl-xL diminishes GFP-Bak fluorescence in the cytosol of targeted cells (circled) completely after 90 s and GFP fluorescence is detected only on the mitochondria (arrows). During FLIP measurements mitochondrial GFP-Bak fluorescence is monitored, while the cytosol is bleached repeatedly. Time points in seconds are displayed above the images.
Figure 6(A) Immunoblot α-showing neurexin isoform distribution in cortex, hippocampus , midbrain and cerebellum dissected from 8-week-old mice. (B) Quantification of lower molecular weight neurexin (LMW; white arrowhead) in relation to total levels of α-neurexins [upper band (line) + LMW]. Bar graphs show mean ± SEM of 3 independent replicates***, p<0.001 by 2-way ANOVA and Holm-Sidak tests, comparing to levels in cerebellum. (C) α-Neurexin immunoprecipitated from forebrain (Fb) and cerebellum (Cb) using an anti-pan-neurexin antibody was heparinase-treated ('Heps') and analyzed by immunoblotting with antibodies against neurexin and the 3G10 monoclonal (to detect the HS stub). Stars indicate non-neurexin bands (see results). (D) Quantification of low molecular weight neurexin (LMW; white arrowhead) in relation to total levels of α-neurexins [upper band (line) + LMW]. Bar graphs show mean ±SEMof 3 biological replicates. ***, p<0.001 by 2-way ANOVA and Holm-Sidak tests comparing forebrain and cerebellum. (E) Quantification of the 3G10 signal normalized to the signal of the corresponding neurexin band. Bar graphs show mean ±SEM of 3 biological replicates. ***, p<0.001 by 2-way ANOVA and Holm-Sidak tests comparing forebrain and cerebellum.
Figure 4Representative blots showing the expression of YAP and RB1 in hOSE-MXIV, hOSE-YAP and hOSE-YAPS127A cells with or without pRB knockdown. Cells were collected at the 7th passage and protein levels were analyzed using Western blotting. β-Actin was used as a protein loading control. Growth curves of hOSE-MXIV, hOSE-YAP and hOSE-YAPS127A cells after RB1 knockdown. Representative images showing SA-β-gal staining in the hOSE-MXIV, hOSE-YAP and hOSE-YAPS127A cells with or without RB1 or TP53 knockdown. Cells were collected at the 7th passage.
Phenotypic analysis of loss-of-function (LOF) and gain-of-function (GOF) lines of the TFs under control conditions.Figure 4A At 22 days after stratification (DAS), leaf series of the LOF lines were made and the rosette area was calculated as the sum of the area of all individual leaves (n = 10 plants). The rosette area is presented relative to the corresponding wild type. B Average area of the individual leaves of rap2.6L and myb51, two knock-out lines with a smaller average rosette area. C Rosette area of two or three independent GOF lines per TF (calculated as the projected area or the sum of the leaves, n = 10 plants) germinated and grown on DEX-containing medium. Measurements were performed at 22 DAS relative to the control line. "Independent line 1" is the line with the highest overexpression level. D A representative picture of the rosette of GOF lines with significant growth phenotypes in both independent lines at 22 DAS. Scale bar is 1 cm. E, F Pavement cell number and area of the third leaf at 22 DAS of LOF (E) that showed a significant rosette area phenotype. Data information: In A and C, data are presented as mean ± SEM, n = 3 independent experiments, *=P<0.05 (Tukey's test). In B, data are presented as mean ± SEM, n = 3 independent experiments, *=P<0.05 (mixed model, partial F-tests). In E and F, data are presented as mean ± SEM, n = 3 independent experiments, . = P<0.1, * = P<0.05, ** = P<0.01, *** = P<0.001 (Tukey's test). E, F Pavement cell number and area of the third leaf at 22 DAS of GOF lines (F) that showed a significant rosette area phenotype. Data information: In A and C, data are presented as mean ± SEM, n = 3 independent experiments, *=P<0.05 (Tukey's test). In B, data are presented as mean ± SEM, n = 3 independent experiments, *=P<0.05 (mixed model, partial F-tests). In E and F, data are presented as mean ± SEM, n = 3 independent experiments, . = P<0.1, * = P<0.05, ** = P<0.01, *** = P<0.001 (Tukey's test).
Hh activity is modulated by Lsd-2A-B'') Representative images showing that Lsd2YFP is localised to the surface of LDs (yellow arrows).Figure 3A-B'') Representative images showing that Lsd2YFP is localised to the surface of LDs (yellow arrows). C-D'') Lsd2YFP co-localises with Hh antibody staining in the cortex glia (yellow arrows) but not surface glial cells (white arrow). E-G) Lsd-2 knockdown in cortex glial cells (NP2222-GAL4) where hh is overexpressed effectively reduces LD number in CB (outlined in yellow dashed lines). H-K) Representative images showing that NB EdU index is rescued upon Lsd-2 knockdown in cortex glial cells (NP2222-GAL4) where hh is overexpressed, quantified in (K) (n=11, 14; 16, 14; 6, 14 brain lobes). The NP2222-GAL4>w1118 vs hhOE columns depict the same data as Fig 2K. EdU+ NBs are circled with yellow, dashed lines.
B56δ retains PPM1G in cytoplasm.Figure 2(A) MCF7 and MDA-MB-231 cells transfected with MYC-B56δ were fixed and imaged using a confocal microscope after staining with antibodies against MYC tag and PPM1G to determine PPM1G localization. Representative images from three independent experiments are shown, Scale bars 5 µm. (B) Quantification of cytoplasmic to nuclear (Cyto/Nuc) ratio of PPM1G intensity per each cell was plotted (n=35 for MCF-7 cells; n=25 for MDA-MB-231 cells). Error bars indicate SD. ***p < 0.001 (one-way ANOVA, post hoc test: Bonferroni's multiple comparison test). (C) Western blot showing expression levels of SFB tagged B56δ along with endogenous B56δ using in dicated antibodies. (D) Cells expressing either vector or SFB-B56δ were fractionated into nuclear and cytoplasmic fractions and levels of indicated proteins were determined using western blotting. Tubulin and Histone H3 were used to mark cytoplasmic and nuclear fractions respectively. (E) Cells treated with control or B56δ shRNA were fractionated into cytoplasmic and nuclear fractions and levels of indicated proteins were determined using immunoblotting. Tubulin and Histone H3 were used to mark cytoplasmic and nuclear fractions respectively. (F) Localization of PPM1G in cells expressing either empty vector (control), full length B56δ (FL) or various indicated mutants was determined by confocal imaging after staining with Flag and Myc antibodies. Representative images from two independent experiments are shown, Scale bars 10 µm. (G) Cytoplasmic to nuclear (Cyto/Nuc) ratio of PPM1G intensity per each cell expressing either vector, B56δ full length (FL) or mutant versions was quantified and plotted (n=10). Error bars represents SD. ***p < 0.001 (one-way ANOVA, post hoc test: Bonferroni's multiple comparison test).
FYCO1 decorates cytosolic vesicles and cytosolic punctuated structures containing the protein markers of autophagosomes/LEs/lysosomes.Figure 3.(A) Endogenous FYCO1 localizes to the cytosolic punctuated structures. HeLa cells cultured in complete growth medium (left) or in HBSS for 3 h (right) were fixed and stained with anti-FYCO1 antibody (green) and Draq5 (blue). (B) Overexpressed mCherry-FYCO1 localizes to the cytosolic punctuated structures and cytosolic vesicles in stably transfected HeLa cells. (C) FYCO1 partially colocalizes with LC3B, LAMP1, ATG5, ATG16L, p62, and HuntingtinQ64 and does not colocalize with EEA1. HeLa cells or mouse embryonic fibroblasts (for ATG16L staining) were transfected with the indicated vectors or stained with the indicated antibodies and imaged 24 h after transfection. For p62 staining, HeLa cells were incubated with 0.2 µM BafA1 for 12 h before fixation. (D) FYCO1 colocalizes with LC3B on the rim but not inside of the vesical structures. HeLa cells cotransfected with GFP-FYCO1, mCherry-LC3B, and myc-p62 were imaged by confocal microscopy 48 h after transfection. (E and F) FYCO1-decorated structures are heterogenous in their luminal content. (E) HeLa cells transfected with CFP-FYCO1 and mCherry-YFP-p62 were imaged 48 h after transfection. (F) HeLa cells transfected with GFP-FYCO1 were labeled with LysoTracker red for 60 min and Alexa Fluor 647-dextran (10,000 D) for 4 h in normal growth medium to stain late endocytic compartments (left). Relative number of FYCO1 structures ± SD colocalized with LysoTracker red or Alexa Fluor 647-dextran (10,000 D; right).
Fig.Figure 2(A) Tyrosine phosphorylation of EGFR in HBMEC pre-treated with 20 μM S1P2 antagonist (JTE-013) or vehicle control and infected with GBS strain K79 infection for 60 min. EGFR phosphorylation in control and S1P2 knockout HBMEC infected with K79. (B) Relative invasion frequency of strain K79 in HBMEC with or without SphK1 and SphK2 inhibitors. All the inhibitors were used at 10 μM. From left to right, p value of DJB-V-39 is 0.0047, p value of SKI is 0.0062, p value of FTY720 is 0.0022, and p value of HB56 is 0.0045. (C) S1P2 protein expression in S1P2 knockout HBMEC using CRISPR/Cas9 (left panel). Relative invasion frequency of 8 meningitis isolates of GBS in S1P2 knockout HBMEC. From left to right, p value of K79 is 0.0018, p value of K160 is 0.0091, p value of K161 is 0.0054, p value of K181 is 0.00040, p value of K226 is 0.00032, p value of K237 is 0.040, p value of K238 is 0.041 and p value of P539 is 0.0025. (D) GBS strain K79 traversal of HBMEC monolayer was significantly decreased in S1P2 knockout HBMEC compare to control. (E) Bacterial counts recovered from the blood and brain of wild type (n = 5) and S1P2-/- mice (n = 5) infected with strain K79 for 1h.
Figure 6Immunoblotting of BM67 cell extract for ELP3 after stable shRNA-mediated ERα knockdown in BM67 cells. Graphs represent mean ± standard deviation of n=3. qPCR analysis and immunoblotting of BM67 cell extract for indicated proteins with its quantitation following deletion of Esr1 gene by CRISPR-Cas9 technology. Graphs represent mean ± standard deviation of n=5. qPCR analysis and immunoblotting of BM67 cell extract for ELP3 and DEK with its quantitation following deletion of Elp3 gene by CRISPR-Cas9 technology. Graphs represent mean ± standard deviation of n=4. Immunoblotting and qPCR analysis comparing cell extracts obtained from BM67 cells transduced with pLentiCRISPRV2 empty vector, Elp3-null BM67 cells and Elp3-null BM67 cells overexpressing ELP3 protein containing silent mutations in the sgRNA binding site. Graphs represent mean ± standard deviation of n=3-4.
Figure 1(A) Principal component analysis of WT CD4+ naïve T cell (gray), WT Treg cells (blue), and Tet2/3 DKO Treg cells (red). The percent of variation explained by principle components 1 (PC1) and 2 (PC2) are shown in parenthesis. (B) Scatter plots showing the comparison of mean gene expression between WT Treg cells vs CD4+ naïve T cells (upper panel) and WT Treg cells vs Tet2/3 DKO Treg cells (lower panel). Differentially expressed genes (FDR ≤ 0.05 and fold-change ≥ 2) are shown in color (blue: WT Treg cells; black: CD4+ naïve T cells; red: Tet2/3 DKO Treg cells) and selected genes are labeled in the plots. The scale is log2(FPKM+1). (C-E) Gene set enrichment analysis of gene expression changes between WT Treg cells vs CD4+ naïve T (upper panels) and WT Treg cells vs Tet2/3 DKO Treg cells (lower panels) for the hallmark gene sets IL2/STAT5 signaling (C), E2F Target genes (D) and Treg signature gene set that are upregulated in Treg cells compared to conventional T cells (GSE7460) (E). The green line represents the enrichment score with enrichment in WT Treg cells shown to the left and enrichment in CD4+ naïve T (upper panels) or Tet2/3 DKO Treg cells (lower panels) shown to the right. Overlap of genes in the given gene set is shown below and denoted by black ticks. (F) Examples of expression levels of Treg signature genes Foxp3, Il10ra, Nrp1, Ikzf4 and Ccr6; and examples of expression levels of genes upregulated in Tet2/3 DKO Treg cells Eomes, Ccl4, Chek1 and Fn1. The Y-axis shows FPKM (Fragment Per Kilobase per Million reads), the X-axis shows the different cell types. Error bars show mean ± S.D. from two (CD4+ naïve T cells) or four (WT and Tet2/3 DKO Treg) biological replicates.
Figure 3Internalization of EV20/MMAF (10 nM) at indicated times in BT474 and BTRH cells. Scale bar: 20 µm. HER3 staining: red; DAPI: blue. Internalization of biotin-S-S-EV20/MMAF in BT474 and BTRH cells. These cell lines were pulsed with 10 nM of biotin-S-S-EV20/MMAF for 1 hour at 4°C and chased at 37°C at the indicated times. Subsequently, surface accessible biotin was cleaved, cells were lysed, and extracts precipitated with streptavidin-sepharose. EV20/MMAF was analyzed by Western blotting using anti-human-HRP. Quantitative plot of the Western studies shown in (B). Data are presented as the mean + SD of two independent experiments. Co-localization (yellow) between EV20/MMAF and LAMP1 at the indicated incubation times with EV20/MMAF in BT474 and BTRH cells. Scale bar: 5 µm. HER3 staining: red; LAMP1: green, DAPI: blue. Western blot analyses of the effect of EV20/MMAF (10 nM) on the levels of HER2 and HER3 in BT474 and BTRH cells at the indicated incubation times with the ADC. The same Western was probed for HER2 or HER3 expression using two differently labelled species-specific secondary fluorescent antibodies. Quantitative representation of the Western studies shown in (E).
Figure 1(A, B) In situ cryo-electron tomogram displaying the proximal region of a mature mother centriole (A) and procentriole (B). Mature centriole, MC; procentriole, PC; mitochondria, mito; vacuole, vac; white dashed line, lamella edge. Scale bars, 100 nm. (C) Side view z-projection of cartwheels containing the central hub and several CIDs from a mature centriole. Central hub, CH; cartwheel inner densities, CID. Scale bar, 20 nm. (D) Cross section of the cartwheel-containing regions from a mature centriole. Microtubule triplet, MTT; spokes, SP. Scale bar, 200 nm. (E) Side view z-projection of cartwheels containing the central hub and several CIDs from a procentriole. Scale bar, 20 nm. (F) Cross section of the cartwheel-containing regions from a procentriole. Scale bar, 200 nm. (G) Nine-fold symmetrized cross sections of the cartwheel-containing region from a mature centriole (left side) and a procentriole (right side). Dashed white circle, central hub. Scale bars, 100 nm. (H) Longitudinal periodicity measurements of the central hub and CIDs. Central hub, blue; CID, red. Mean values are displayed above the data range. Blue data points are measured distance between individual units of the central hub, red data points are measured distances between individual units of the cartwheel inner density. Mature centriole, central hub, n = 30, mean = 4.1 +/- 0.67 (S.D); mature centriole, cartwheel inner density, n = 18, mean = 8.7 +/- 1.6 (S.D); procentriole, central hub, n = 10, mean = 4.0 +/- 0.66 (S.D); procentriole, cartwheel inner density, n = 5, mean = 8.6 +/- 1.2 (S.D). (I, J) Nine-fold symmetrized central hub z-projections, starting at the proximal end of the cartwheel and continuing distally along the cartwheel by 5.4 nm steps in a mature centriole (I) and a procentriole (J). Red arrow, CID; blue arrow; central hub. Scale bar: 20 nm.
Immuno-EM of SipB-transfected COS-2 cells.Figure 4. Immuno-EM of SipB-transfected COS-2 cells. COS-2 cells were transfected with a plasmid expressing SipB-YFP, and 24 h after transfection, cells were fixed and prepared for immuno-EM using an antibody specific for GFP and a gold-labeled anti-rabbit antibody. SipB was seen localized to structures made up of closely apposed membranes that were often in close proximity to mitochondria (A). Mitochondria were often seen apparently fusing to the SipB-induced membranous structure (B, see arrows in inset on top; C, inset to the right). Mitochondria were often seen contained with the SipB-induced membrane structures (D). Bars, 250 nm.
Figure 3Double immunofluorescence of the proteasome subunit PSMC4 and poly-GA inclusions in spinal cord of GA149-CFP transgenic mouse Scale bar denotes 20 µm. Double immunofluorescence of the proteasome subunit PSMC4 and poly-GA inclusions in cortex of C9orf72 patients compared to controls. Scale bar denotes 20 µm. Co-culture model of HeLa cells transfected with iRFP or GA175-iRFP in the donor compartment and an UbG76V-GFP proteostasis reporter in donor and receiver compartment (48 h). (C) Separate analysis of both compartments by immunoblot and (D) immunoblot quantification. For quantitative analysis of immunoblots UbG76V-GFP was normalized to calnexin. n=3 biological replicates. Scatter plot with mean ± SD. One-way ANOVA with Tukey's multiple comparisons test. Red dashed line indicates the control's expression level. * denotes p<0.05, *** denotes p<0.001. (E) GFP mRNA expression levels were measured by qPCR. RNA levels were normalized to GAPDH, β-actin and β2-microglobulin mRNA. Bar-graphs of mean ± SD. n=3 biological replicates. Unpaired two-tailed t-test with Welch's correction. (F,G) Flow cytometry analysis of a UbG76V-GFP reporter cell line incubated 48h with conditioned media from RFP or GA175-RFP transfected cells upon immunodepletion of poly-GA or control depletion using unspecific IgG. n=3 biological replicates. Scatter plot with bar-graphs of mean ± SD. One-way ANOVA with Tukey's multiple comparisons test. * denotes p<0.05. (G) Comparisons of the corresponding histograms for compensated RFP and UbG76V-GFP fluorescence from one representative experiment that shows specific transmission of GA175-RFP associated with accumulation of UbG76V-GFP in cells incubated with GA175-RFP conditioned media.
Figure 3Immunoblot of conditioned media collected from HEK293T cells transiently transfected with ERdj3WT or ERdj3H53Q and incubated with an affinity resin consisting of native (N) or guanidinium hydrochloride (GdnHCl)-denatured (D) RNAse A covalently coupled to sepharose resin. Resin was incubated with media for 2 h at ambient temperature, followed by washing and elution in Laemmli's reducing buffer.  Plot showing the time-dependent increase in ThioT fluorescence (ex:440 nm, em:485 nm) of A1-40 incubated (10 µM, 37 OC, pH 7.2) with regular agitation in the presence or absence (black) of recombinant ERdj3 (RERdj3). The RERdj3 concentrations and molar ratios of RERdj3 to A1-40 are as follows: Violet: 1.5 nM, 1:6561; light blue: 4.6 nM, 1:2187; dark blue: 14 nM, 1:729; dark green: 41 nM, 1:243; light green: 120 nM, 1:81; yellow: 370 nM, 1:27; orange: 1.1 M, 1:9; red: 3.3 M, 1:3, or maroon: 10 M, 1:1. All traces represent the average of three replicates.  Plot showing the time-dependent increase in ThioT fluorescence of A1-40 incubated (10 µM, 37 OC, pH 7.2) with regular agitation in the presence or absence of BSA (14 µg/mL) or RERdj3 (14 µg/mL; 370 nM, RERdj3: A=1:27). All traces represent the average of three replicates. Immunoblot of A1-40 incubated (10 µM, 37 OC, pH 7.2) with regular agitation for 90 h in the presence or absence of RERdj3 (14 µg/mL; 370 nM, RERdj3: A1-40 =1:27) or BSA (14 µg/mL). The reaction solution in the well was collected (Nonadhered) and separated into soluble and insoluble fractions by centrifugation. These samples, as well as the washed wells of the plate (Adhered), were denatured in 8 M GdnHCl with sonication and analyzed by SDS-PAGE/immunoblotting.