EMBO/sd-character-level-ner huggingface dataset:
GemC1 directly activates the upstream regulatory sequences of MCIDAS and FOXJ1.Figure 3MCIDAS (A) regulatory elements (see materials and methods) cloned upstream of the luciferase gene were co-transfected into HEK293T cells with vectors expressing GemC1, McIdas and Geminin, as indicated or an empty vector (-) as a control. All luciferase experiments (A, B, D) were normalized for transfection efficiency with an expression vector for Renilla luciferase. Fold induction is the ratio between the normalized luciferase activity induced by the expression constructs and that induced by the empty expression vector. Data are the mean values of at least three independent experiments and error bars indicate ± SEM. **p<0.01, ***p<0.001. P-values were calculated by the non-parametrical two-tailed Mann-Whitney test. Abbreviations: RLF: Relative Luciferase Fold induction. FOXJ1 (B, D) regulatory elements (see materials and methods) cloned upstream of the luciferase gene were co-transfected into HEK293T cells with vectors expressing GemC1, McIdas and Geminin, as indicated or an empty vector (-) as a control. All luciferase experiments (A, B, D) were normalized for transfection efficiency with an expression vector for Renilla luciferase. Fold induction is the ratio between the normalized luciferase activity induced by the expression constructs and that induced by the empty expression vector. Data are the mean values of at least three independent experiments and error bars indicate ± SEM. **p<0.01, ***p<0.001. P-values were calculated by the non-parametrical two-tailed Mann-Whitney test. Abbreviations: RLF: Relative Luciferase Fold induction. McIdasmRNA levels after McIdas RNAi were assessed by qPCR (C). FOXJ1 (B, D) regulatory elements (see materials and methods) cloned upstream of the luciferase gene were co-transfected into HEK293T cells with vectors expressing GemC1, McIdas and Geminin, as indicated or an empty vector (-) as a control. In D, McIdas siRNAs or a control siRNA were also co-transfected. All luciferase experiments (A, B, D) were normalized for transfection efficiency with an expression vector for Renilla luciferase. Fold induction is the ratio between the normalized luciferase activity induced by the expression constructs and that induced by the empty expression vector. Data are the mean values of at least three independent experiments and error bars indicate ± SEM. **p<0.01, ***p<0.001. P-values were calculated by the non-parametrical two-tailed Mann-Whitney test. Abbreviations: RLF: Relative Luciferase Fold induction.
Figure 8A-C Micrographs of sections of E15 cerebral cortex 2 days after IUE with plasmids and magnifications indicated. V= ventricle; VZ: ventricular zone. Scale bars: 20 µm and 10 µm for magnifications. Yellow dotted shapes show examples of heterochromatin spots in nuclei of electroporated cells from the VZ and the non-VZ. D-E, Violin plots depicting the number (D) and size (E) of condensed chromatin spots from cells electroporated as in A-C. Note that Trnp1 overexpression is sufficient to significantly increase the size of condensed chromatin spots, while the ∆1-16Trnp1 acts as a dominant negative reducing the number and size of heterochromatin spots. N=10 cells per region from 5 embryos for GFP and Trnp1 and 8-15 cells from 4 embryos for ∆1-16Trnp1 of at least 4 litters. Each dot represents a cell. F, Summary of the splicing changes observed in GFP+ cells isolated from E14 cerebral cortex 24 h after IUE with plasmids expressing the shRNA against Trnp1 and its control as determined by RNA-seq analysis. The number of events per category are shown in the bar. SE, skipped exon; RI, intron retention; MXE, mutually exclusive exon; A5SS, alternative donor site; A3SS, alternative acceptor site. G-H Translation assay using OPP-click reaction in P19 cells transfected for 24 hours with GFP, Trnp1-IRES-GFP or ∆1-16Trnp1-IRES-GFP plasmids and analyzed by immunofluorescence (G) or FACs sorting (H) . Graph in (G) is representative from three different biological replicates. Each dot represents a cell. Control=102; Trnp1=126; ∆1-16Trnp1=97. Graph in (H) shows analysis of 2,000 transfected cells from 1 representative experiment with 2 biological replicates for control and 3 biological replicates for Trnp1 and ∆1-16Trnp1 conditons. The same FACs sorting experiment was repeated two more times. The dotted vertical line shows the mean of the OPP click signal for each condition. I, Bar and dot plots depicting rRNA transcription for the 18S, 28S, 45S and 47S measured by qPCR in P19 cells transfected for 24h with plasmids expressing Trnp1-IRES-GFP or ∆1-16Trnp1-IRES-GFP compared to control cells expressing GFP . Each dot represents a biological replicate. N=4 for each condition.
Characterization of CATSPER2-deficient human sperm.Figure 1Representative Ca2+ signals in sperm from a patient with deafness-infertility syndrome lacking functional CatSper channels (CATSPER2-/-) (red) and a healthy donor (black), evoked by progesterone, PGE1, NH4Cl, or ionomycin. NH4Cl increases the intracellular pH. Bar graph: Amplitudes (n = 4; mean ± SD) of Ca2+ signals in CATSPER2-/- sperm Representative monovalent CatSper currents in CATSPER2-/- sperm (blue, green, orange, purple, brown) and in sperm from a healthy donor (black) and corresponding current-voltage relationship (right). The membrane voltage was stepped from -100 mV to +100 mV in increments of 10 mV from a holding potential of -80 mV. Outward and inward current amplitudes (mean ± SD) at +100 mV and -100 mV, respectively, in CATSPER2-/- sperm (color code: panel B) and sperm from healthy donors (black). Representative immunocytochemical staining of control sperm from healthy donors and CATSPER2-/- sperm form DIS patients using antibodies directed against CatSper 3 (D) or CatSper 4 (E); DNA was labelled with DAPI (blue). Scale bars represent 10 µm. 3D-STORM image in xy-projection of sperm from a healthy donor labelled with the anti-CatSper 3 antibody (left). Axial projection of the boxed region (right). Scale bars represent 5 µm in xy-projections and 200 nm in axial projections. 3D-STORM images in xy-projection of CATSPER2-/- sperm (left) labelled with the anti-CatSper 3 (upper panel) and anti-CatSper 4 (lower panel) antibody. Axial projection of the boxed regions (right). Scale bars represent 5 µm in xy-projections and 200 nm in axial projections.
Figure 5Schematics of HR and NHEJ reporter assays. Obtained results showing enhanced HR efficiency upon knockdown of endogenous cGAS in U2OS cells. Immunoblot inserts in depict knockdown efficiency of cGAS and Histone H1.2. Obtained results showing enhanced HR efficiency upon knockdown of endogenous cGAS in U2OS cells. Immunoblot inserts in depict knockdown efficiency of cGAS and Histone H1.2. Results showing the effect of hcGAS, hcGAS△cGAMP, hcGAS△DNA or hcGAS△Oligo on HR in HEK293 cells. Corresponding immunoblot inserts depict cGAS expression. Results showing the effect of hcGAS, hcGAS△cGAMP, hcGAS△DNA or hcGAS△Oligo on NHEJ in HEK293 cells. Corresponding immunoblot inserts depict cGAS expression.
Svb acts downstream of Wnt and EGFR mitogenic signaling pathways in the adult midgutA esgtsF/O midguts expressing GFP alone (control), or expressing RasV12, and RasV12 + svb-RNAi (top panels).Figure 4esgtsF/O midguts expressing GFP alone (control), or expressing RasV12, and RasV12 + svb-RNAi (top panels). Bottom panels show esgts midguts expressing GFP alone (control), or expressing EGFR-DN, and EGFR-DN + OvoB. Samples were stained for GFP (green). The graph shows quantification of the number of GFP-positive cells in esgts midguts expressing GFP alone (ctrl), or expressing EGFR-DN, EGFR-DN + OvoB, and EGFR-DN + svb-RNAi. The Y axis is plotted as log(10). esgts midguts expressing GFP alone (control), or expressing ArmS10, ArmS10 + svb-RNAi, ArmS10 + OvoB ,TCF-DN, and TCF-DN + OvoB. Samples were stained for GFP (green). The graph shows quantification of the number of GFP-positive cells in esgts midguts expressing GFP alone (ctrl), or expressing TCF-DN, TCF-DN + OvoB, and TCF-DN + svb-RNAi. The Y axis is plotted as log(10). The drawing at left schematizes the svb locus, with position of the E3N enhancer. Close-up shows E3N sequence that correspond to binding sites for Pnt (red) and TCF (green). Right subpanels are pictures of posterior midguts showing expression of wild-type E3N-lacZ (E3Nwt), or E3N-Pnt-mt, and E3N-TCF-mt, as seen from X-Gal staining (cyan blue).
Figure 1(A-C) AFM and TEM analysis of unloaded (u-NPs) and cholesterol loaded NPs (NPs-Chol). AFM "height" images (left column), 3D reconstruction (middle column) and TEM micrograph (right column) of u-NPs (A), NPs-Chol1 (B) and NPs-Chol3 (C). (D) Release profile in water of cholesterol (continuous line, -) and NBD-Chol (dotted line, - - -) from NPs-Chol1 and NPs-NBD-Chol1, respectively. (E) In vitro release of NBD-Chol from NPs at different time-intervals in NS cells. Data in the graph represent mean (µg) ± SEM of total NBD-Chol (embedded into and released from NPs; red columns) and NBD-Chol released after NPs degradation (purple columns) present in the homogenates of NS cells treated with NPs-NBD-Chol1
Figure 4Schematic diagram showing Kdr gene deletion by sCreER. Schematic figure showing experimental design. Immunostaining for VEGFR2 on Npr3-sCreER;Kdrflox/flox heart section from mice without tamoxifen treatment (No Tam). Immunostaining for VEGFR2 on mouse heart sections after tamoxifen treatment (Tam). A, atrium; V, ventricle; TM, trabecular myocardium. Immunostaining for tdTomato, VEGFR2 and CDH5 on heart sections from mice of four different genotypes. Quantification data showed the percentage of VEGFR2+ endocardial cells (ECs) in tdTomato+ (tdT+) cells in the TM regions. Cartoon images (bottom) indicate tdTomato+ cells (red) and VEGFR2+ cells (green) in the TM or compact myocardium (CM). Data are mean ± s.e.m.; n = 5. Scale bars, 100 µm. Each image is a representative of 5 individual biological samples.
Lmna null mice display defective primary cilia in the skeletal muscle, kidney, uterus and ovary.Figure 2A The skeletal (tabialis anterior) muscles from control (Lmna+/+ and Lmna+/-) and Lmna-/- mice were stained for acetylated tubulin (green) and nucleus (blue). The representative images with low and high magnification are shown. The insets are enlarged images for cilia. B The kidneys from control and Lmna-/- mice were stained for acetylated tubulin (green) and AQ1 (a marker for the proximal tubules, red). The insets show the cilia of proximal renal tubule cells. C The uterus from control and Lmna-/- mice was stained for acetylated tubulin (green) and keratin 8 (red). The insets show the cilia at the stroma of the uterus. D The ovary from control and Lmna-/- mice were stained for acetylated tubulin (green) and keratin 8 (red). The insets show the cilia of the granulosa cells in the follicles of the ovary. E, F The cilia length (E, n=150-180) and cilia positive cells (F, n ≥ 1197) in each organ from 4-week-old Lmna-/- mice (n=3) and their littermate controls (n=3) were measured.
Degradation of SQSTM1/p62 and long-lived proteins is not impaired in CTNS-deficient cells.Figure 3Analysis of the expression of endogenous SQSTM1 in wild-type or Ctns−/− mouse fibroblasts under fed (serum) or starved (15 h serum starvation) conditions. In these assays, 20 μg of total protein lysates were resolved by SDS-PAGE and analyzed by Western blot (WB; A). Equal loading was established by silver staining (B). Quantification (mean ± SEM) of five different areas of each silver-stained lane are shown in (C). Degradation of long-lived proteins using metabolic labeling was performed as described in Supplementary Materials and Methods. The data represent the mean ± SEM of nine independent samples analyzed in three independent experiments. *P = 0.009 for WT and *P = 0.0013 for Ctns−/−, respectively. NS, not significant (unpaired t-test).
Figure 7Immunostaining of STING (anti-STING, red) in THP-1 cells stimulated with FITC-ecGAMP (5 μg/ml, green) for 2 h, nucleus in blue (DAPI). Data are representative of 3 independent experiments. Scale bar, 10 μm. Cellular localization of ecGAMP in WT and CGAS KO THP-1 cells stimulated with FITC-ecGAMP (5 μg/ml, green) for 2 h, nucleus in blue (DAPI). Data are presentative of 5 independent experiments. Scale bar, 10 μm. Frequency of perinuclear accumulation of FITC-ecGAMP in WT and CGAS KO THP-1 cells stimulated with FITC-ecGAMP (5 μg/ml) for 2 h. Data are means+SD averaged from 5 independent experiments and approximately 100 cells were imaged and counted in each experiment. Each symbol represents the percentage of THP-1 cells with perinuclear cGAMP aggregates in every independent experiment. Mann-Whitney U test was used for statistical analysis. **p<0.01. Western blot detection of the presence of GST-cGAS and His-STING in the immunoprecipitates of cGAMP agarose. Purified GST-cGAS and His-STING were incubated separately or together with cGAMP agarose. The input and immunoprecipitates were immunoblotted. Western blot detection of the presence of His-STING in the immunoprecipitates of Glutathione Sepharose. Purified GST-cGAS and His-STING were incubated together in IP lysis buffer overnight at 4°C. Western blot detection of the presence of GST-cGAS and His-STING in the immunoprecipitates of Glutathione Sepharose. Purified GST-cGAS and His-STING were incubated together in the absence or presence of increasing 2′3′-cGAMP. Western blot detection of the presence of cGAS and STING in the immunoprecipitates of streptavidin beads. THP-1 cells were stimulated with biotin-ecGAMP (5 μg/ml) or biotin-icGAMP (1 μg/ml) for indicated time and cell lysates were harvested for IP with streptavidin beads. Data (A-B, D-G) are representative of 3 independent experiments.
Figure 5(A-C) Control and GRASP55 KO (#2) cells were transfected for 16 hours with the indicated HA-tagged enzymes and processed for cryo-immunolabeling with anti-HA antibody (10-nm gold particles) and anti-GM130 antibody (15-nm gold particles). Representative images of the distribution of HA-tagged enzymes are shown. Red circles indicate GM130 labelling. Arrow heads represent the peri-Golgi vesicles. Scale bar 150 nm. Quantification of the distribution of enzymes in vesicles represented as normalized linear density (D) COPI vesicles were reconstituted using the two-stage incubation system (detailed in the Methods section). After the second-stage incubation, samples were centrifuged to obtain the pellet fraction that contains Golgi membranes and the supernatant fraction that contains reconstituted COPI vesicles. Both fractions were immunoblotted for LCS, GCS and transferrin receptor (Tfr) to show their relative distributions on Golgi membranes and in COPI vesicles. (E) The COPI vesicle reconstitution system was performed as described above (in D), and then the fraction of LCS and GCS in COPI vesicles versus their total distribution (on Golgi membranes and in COPI vesicles) was calculated. (F) HeLa cells co-transfected with HA-tagged GCS and GRASP55-GFP were incubated for 16 hours and were processed for cryoimmunolabeling with anti-HA antibody (10-nm gold particles) and anti-GFP antibody (15-nm gold particles). Representative images of the distribution of GCS and GRASP55-GFP are shown. Red arrowhead indicates the clathrin-coated vesicle that marks the TGN area and black arrowheads indicate the presence of GCS-HA in TGN. Scale bar 150 nm. Distribution of GCS across the Golgi stack and TGN was quantified and represented as fraction of gold particles. (G) Model represents GRASP55-mediated compartmentalization of GCS and LCS. A cyclical and balanced activity of GRASP55 and GOLPH3 compartmentalizes LCS/GCS to the trans-Golgi. The anterograde transport of enzymes (forward direction arrow; cis to trans direction) counterbalances their retrograde transport (reverse direction arrow; trans to cis direction) resulting in the compartmentalization of these enzymes.
TIGAR expression modulates autophagy in response to nutrient starvation or metabolic stress.Figure 2TIGAR expression modulates autophagy in response to nutrient starvation or metabolic stress. (A) (Left panel) confocal microscopy images of the fluorescence in U2OS cells stably over-expressing Flag-tagged-TIGAR (clone TIGAR#7) or control cells (clone Cont#1) and infected with an adenovirus expressing GFP-LC3 for 16 h. Cells were then left untreated, exposed to nutrient starvation for 6 h or to metabolic stress for 24 h. (Right panel) Quantitation of the percentage of GFP-LC3-positive cells displaying GFP puncta from three independent experiments. The mean values with standard deviation are presented. TIGAR expression modulates autophagy in response to nutrient starvation or metabolic stress.(B) (Left panel) Confocal microscopy images of the fluorescence in U2OS cells stably expressing GFP-LC3 and transfected with scrambled or TIGAR siRNAs. After 48 h transfection, cells were then left untreated, exposed to nutrient starvation for 5 h or to metabolic stress for 18 h. (Right panel) Quantitation of the percentage of GFP-LC3-positive cells displaying GFP puncta from three independent experiments. The mean values with standard deviation are presented. TIGAR expression modulates autophagy in response to nutrient starvation or metabolic stress. (C) (Left panel) Western blot showing the expression levels of endogenous LC3-I, LC3-II and TIGAR in U2OS cells transfected with scrambled or TIGAR siRNAs, and 48 h later exposed to nutrient starvation for 0, 2.5 and 6 h. (Middle panel) Western blot showing the expression levels of endogenous LC3-I, LC3-II and TIGAR in U2OS stably over-expressing Flag-tagged-TIGAR (clones TIGAR#5 and TIGAR#7) or control cells (clones Cont#1 and Cont#3) and left untreated. (Right panel) Western blot showing the expression levels of p62, COX-IV and TIGAR in U2OS cells transfected with scrambled or TIGAR siRNAs and left untreated. Actin expression was examined as a loading control. TIGAR expression modulates autophagy in response to nutrient starvation or metabolic stress.(D) Western blot showing the expression levels of endogenous TIGAR in U2OS cells after exposure to nutrient starvation or metabolic stress for 0, 1, 3, 5 and 8 h; * represents significant difference from control conditions (P<0.05); # represents a lack of significant difference from control conditions (P>0.05).
Beclin-1 haploinsufficiency extends lifespan in AR113Q males.Figure 6A. Left panel, Kaplan-Meyer survival curve of AR113Q males (red line, n = 12) and AR113Q, Beclin-1 +/− males (blue line, n = 15). *p<0.05 by log-rank analysis. Right panel, mean survival +/− SEM. *p<0.05 by Student's t-test. B, C. Body weight (panel B) and grip strength (panel C) at different ages for wild type (wt, green line, n = 7), Beclin-1 +/− (yellow line n = 9), AR113Q (red line, n = 12), and AR113Q, Beclin-1 +/− (blue line, n = 15) male mice. B, C. Body weight (panel B) and grip strength (panel C) at different ages for wild type (wt, green line, n = 7), Beclin-1 +/− (yellow line n = 9), AR113Q (red line, n = 12), and AR113Q, Beclin-1 +/− (blue line, n = 15) male mice. D. Age of disease onset as measured by a decrease of 5% or more in forelimb grip strength (not significant by log rank analysis).
Figure 5(A) Representative images of E14.5 cortices stained with antibodies against Centrin (green), γ-Tubulin (red) and DAPI (blue). White and yellow arrows indicate cells with bipolar and monopolar spindles, respectively. Insets showing zoomed in view of 2 representative cells. Dashed lines showing the orientation of the cleavage plane relative to the ventricular surface. Scale bar = 10 μm. (B) Percentage of bipolar, monopolar and multipolar cells at the ventricular surface of E14.5 cortices. WT n = 201 cells, N = 3 embryos; Cep63T/T;Usp28-/- n = 205 cells, N = 3 embryos; Sas4cKO;Usp28cKO n = 308 cells, N = 3 embryos; #, chi-square test with *, post-hoc analysis, comparisons are made to WT. WT data are from Figure 1F and shown alongside for comparison. (C) Ratio of mitotic (PH3+) to cycling (Ki67+) cells in E14.5 cortices. WT N = 5, Cep63T/T;Usp28-/- N = 3, Sas4cKO;Usp28cKO N = 3; two-tailed Mann-Whitney t-test. 3 WT data points are from Figure 1H and shown alongside for comparison. (D) Representative images from live-cell movies of Nuclear-mCherry+ WT, Sas4cKO and Sas4cKO;Usp28cKO NPCs. White arrows showing the nuclei of daughter cells following cytokinesis. (E) Plot showing quantification of mitotic duration of WT, Sas4cKO and Sas4cKO;Usp28cKO NPCs. Triangles represent individual cells; triangles of the same color represent cells derived from the same embryo. Circles represent the average mitotic duration of cells from each embryo. WT n = 202 cells, N = 4 embryos; Sas4cKO n = 138 cells, N = 3 embryos; Sas4cKO;Usp28cKO n = 191 cells, N = 3 embryos; two-tailed Welch's t-test. WT and Sas4cKO data are from Figure 1K and shown alongside for comparison. (F) Graph showing the mitotic duration of NPCs and the fate of their progeny in primary cultures generated from Sas4cKO;Usp28cKO E14.5 embryos. Each bar represents one cell; its height represents the amount of time the mother cell spent in mitosis; bar color represents the fate of the progeny. Dashed line is set at 30 min. Sas4cKO;Usp28cKO n = 271 cells, N = 3 embryos. (G) Percentage of proliferating, arrested and apoptotic progeny within each group of the indicated mitotic duration, calculated from data shown in F; #, chi-square test with *, post-hoc analysis, comparisons are made to the 0-30min group. Graph showing the mitotic duration of NPCs and the fate of their progeny in primary cultures generated from Sas4cKO E14.5 embryos. Each bar represents one cell; its height represents the amount of time the mother cell spent in mitosis; bar color represents the fate of the progeny. Dashed line is set at 30 min. (H and I) Data from Figures 2C and 2F shown alongside for comparison with F and G.
Epg5 deficiency causes a defect in autophagosome maturation and impairs endocytic trafficking.Figure 5.(A) Number of LC3 puncta in Epg5+/− (control) and Epg5−/− MEFs. (B) Western blot showing levels of LC3-I and LC3-II in control and Epg5−/− MEFs upon indicated treatment (Starv., starvation; Rapa., rapamycin; Bafilo., bafilomycin A1). (C) Proteinase K (ProK) protection assay in different MEFs. PNS, postnuclear supernatant; P, pellet fractions. (D) Colocalization ratio of LC3 and LAMP-1 in control and Epg5−/− MEFs. 50 cells were examined per time point in A and D. (E) Colocalization of LC3 puncta and lysosomes in control and Epg5−/− MEFs 4 h after starvation. (F) Under nutrient repletion conditions, almost no autophagic elements are detected in Epg5+/− MEFs. RER, rough ER; M, mitochondrion; N, nucleus. The arrowhead indicates a vacuole with a late residual body-like appearance (aAV-III). (G) Under nutrient repletion conditions, Epg5−/− MEFs accumulate a large number of autophagic vacuoles. Red arrow, a likely autophagosome; red arrowheads, complex vacuoles of the aAV-I type; white arrowheads, aAV-II vacuoles (H and I) High magnification of autophagic elements in unstarved Epg5−/− MEFs. (H) The red arrow indicates a complex autophagic vacuole containing multilayered membrane structures (white arrowheads) and a lipid-like region (L). (I) The red arrow indicates an autophagosome containing cytoplasm and ribosomes. The red arrowhead points to an autophagosome containing a mitochondrion and cytoplasmic material with membranous structures. The white arrow shows an aAV-III vacuole. (J) Compared with control cells, Rab5-labeled early endosomes are significantly enlarged in Epg5 siRNA-treated cells. (K and L) EGFR degradation in control and Epg5 knockdown cells. (M and N) Distribution of Alexa Fluor 488-conjugated transferrin at different time points in control siRNA- and Epg5 siRNA-treated cells. Higher magnification views are shown in the insets. Error bars indicate SEMs.