EMBO/sd-character-level-ner huggingface dataset:
BAG3 facilitates aggresome formation.Figure 1(A) Upper panel: immunoblot analysis of COS7 cells treated for 12 h with MG132 (25 μM) and vinblastine (25 μM). Lower panel: real‐time PCR analysis showing gene expression changes in COS7 cells following MG132 exposure (25 μM, 12 h). (B,C) Immunofluorescence staining of indicated proteins in COS7 cells treated as in (A). Scale bar, 20 μm. (D) COS7 cells were transfected for 24 h with nonsense or bag3 siRNA and treated with MG132 (25 μM, 12 h). Aggresomes were visualized by ubiquitinimmunostaining ( supplementary Fig S1D online). Diagram shows the percentage of aggresome‐positive cells. (E) Aggresome quantification as in supplementary Fig S1D online from cells analysed in (H). Images are shown in supplementary Fig S1G online. Diagram shows the percentage of aggresome‐positive cells at 4, 24 and 48 h after MG132 wash out. (F) Cells were treated as in (H); however, 4 h after MG132 wash out, BafA1 (5 μM) was added for 20 h to inhibit aggresome clearance. Diagrams show the percentage of aggresome‐positive cells and the mean size of aggresomes ( supplementary Fig S1H online). Images are shown in supplementary Fig S1G online. (G) Dot‐blot (upper panel, quantification in supplementary Fig S1B online) and western blot (lower panel) analyses of 0.5% NP40‐soluble and ‐insoluble proteins from cells shown in Fig 1D; supplementary Fig S1D online. (H) COS7 cells transfected with BAG3 or empty vector were treated with either MG132 (25 μM, 18 h) or DMSO as a control. After MG132 wash out, cells were allowed to recover for 4, 24 and 48 h and 0.5% NP40‐soluble and ‐insoluble proteins were analysed by immunoblotting. (I) COS7 cells were transfected for 24 h with GST or GST fused to DIC (GST‐DIC) and treated with MG132 (25 μM, 12 h). Indicated proteins in whole‐cell lysates and in precipitates obtained by GST pull‐down with glutathione beads were analysed by immunoblotting. BafA1, bafilomycin A1; ctrl, control; DIC, dynein intermediate chain; DMSO, dimethyl sulphoxide; GST, glutathione‐S‐transferase; Hsp70, heat‐shock protein 70; nons, nonsense; siRNA, short‐interfering RNA; vinblast, vinblastine.
depletion impairs neuronal differentiation (a) SATB2 staining revealed a reduction in SATB2 and GFP colocalization when TCF20 was depleted.Figure 3(a) SATB2 staining revealed a reduction in SATB2 and GFP colocalization when TCF20 was depleted. White arrows represent SATB2, GFP double-positive cells. (b) Statistical analysis of the SATB2 GFP double-positive cell ratio in GFP positive cells. n=3 brains. Data information: Error bars represent the means ± S.E.M. Two tailed unpaired T-tests were used to analyze the data, n.s. (no significant difference), p<0.05(*), p<0.01(**), p<0.001(***). Scale bar: 25 μm (c) Staining with CTIP2 and TBR1 antibodies shows TCF20 knockdown decreases the colocalization of CTIP2 and TBR1 with GFP in E13.5-E16.5 brains. White arrows represent CTIP2, GFP double-positive cells. White arrow heads represent TBR1, GFP double-positive cells. (d, e) Statistical analysis of CTIP2, GFP and TBR1, GFP double-positive cell ratio in GFP positive cells. n=3 brains. Data information: Error bars represent the means ± S.E.M. Two tailed unpaired T-tests were used to analyze the data, n.s. (no significant difference), p<0.05(*), p<0.01(**), p<0.001(***). Scale bar: (d) 50 μm (f) A diagram of the BrdU labeling time point in electroporation experiments. (g) Immunostaining of BrdU. White arrows represent BrdU GFP double-positive cells. (h) Percentage of BrdU and GFP double-positive cells in GFP-positive cells n=3 brains for each group Data information: Error bars represent the means ± S.E.M. Two tailed unpaired T-tests were used to analyze the data, n.s. (no significant difference), p<0.05(*), p<0.01(**), p<0.001(***). Scale bar: f) 25 μm
Figure 2(A) CD144-enriched EVs were isolated from serum of IPAH patients (n = 37) and HCs (n = 34). The levels of miR-483-3p/-5p were measured by qPCR. Data information: Values are expressed as median ± interquartile range. Statistical test: Mann Whitney U test. PAECs were transfected with miR-483-3p/-5p mimic or scramble RNA for 36 hr before RNA isolation and then analyzed by RNA-seq. Data are results from two biological repeats. (B) PAH-related GO enrichment delineated by DAVID for the top 300 up-regulated or down-regulated genes with the cutoff of p<0.05. PAECs were transfected with miR-483-3p/-5p mimic or scramble RNA for 36 hr before RNA isolation and then analyzed by RNA-seq. Data are results from two biological repeats. (C) Heat map comparison of log2 fold changes of the indicated genes. Data information: Values are expressed as median ± interquartile range. Statistical test: Mann Whitney U test.
Figure 2A,B) Real-time qPCR analysis of ANG-2 levels in retina and RPE/choroid complexes of JR5558 and C57Bl/6J (C57) mice. A) Relative expression levels of ANG-2 were significantly increased in the RPE/choroid complexes of JR5558mice in comparison to C57, at 50 and 62 days old (P = 0.022 at D50 and P = 0.042 and at D62). B) By contrast, retina levels of ANG-2 were not significantly different between C57 and JR5558mice, indicating neovascularization is driven locally by ANG-2. SEM is shown as error bar with n = 4 animals per group. *Denotes statistical significance of JR5558mice compared to wild type C57bl6 using unpaired T-test for each time point analyzed separately. C) Schematic presentation of experimental design. Mice received IP injections of CrossMAb anti-VEGF-A/ANG-2 (species crossreactive, B20.4.1 and LC10), anti ANG-2 (LC10), an anti-VEGF-AIgG1 (B20.4.1) or IgG control at postnatal D14 and D19 followed by fluorescence angiography at D26 (early intervention), results demonstrated in D-K. Alternatively, mice received antibody at postnatal D47 and D55 followed by fluorescence angiography at D60 (late intervention), results demonstrated in L and M. D-I) Representative examples of fluorescence angiograms of IgG control (D), anti-VEGF-A (E), anti-ANG-2 (F) (all the 5 mg/kg) and three doses of anti-VEGF-A/ANG-2 (G,H,I), (at 3, 5, and 10 mg/kg).J,K) Bar graph of numbers of spontaneously occurring lesion number (J) and area by fluorescence angiography (K) after two weekly doses of antibody (antibodies at 5 mg/kg IP and anti-VEGF-A/ANG-2 at 10 [high], 5 [mid], and 3 [low] mg/kg IP) followed by analysis a week later in the early intervention model.L,M) Bar graph of numbers of spontaneously occurring lesion numbers (L) and area by fluorescence angiography (M) after two weekly doses of antibody (3 mg/kg IP) followed by analysis a week later in the late intervention model.
Traction force maps and distributions of signaling components in oscillatory cells.Figure 3A: Sequence of snapshots of the LimE-GFP distribution and stress T for an oscillatory cell together with snapshots of the GFP-myo distribution for a separate oscillatory cell. Corresponding speeds are 7.0µm/min and 4.6µm/min. The sequences show an entire cycle of oscillation (spreading and contraction). White arrows represent the direction of motion before the cell spreads and contracts during which no net motion exists. B: Top: Basal area (black line) and limE fluorescent intensity (green line) as a function of time, for the same cell as in A. Inset: Temporal CF of the area and the limE fluorescent intensity (blue) and of the area change rate and the limE fluorescent intensity (magenta). Bottom: Kymograph of the LimE-GFP intensity along the cell outline. C: Top: Basal area (black line) and myosin fluorescent intensity (green line) as a function of time for a separate cell. Inset: Temporal CF of the area and the myosin fluorescent intensity (blue) and the area change rate and the myosin fluorescent intensity (magenta). Bottom: Kymograph of the GFP-myo intensity along the cell outline. D: Top: Basal area (black line) and total force F (blue line) as a function of time, for the same cell as in A. Inset: Temporal cross-correlation function (CF) of the area and F (blue) and of the area change rate and F (magenta). Bottom: Kymograph of T along the cell outline. E: Median shift in the CF of the area and F (33.8 (22.5/42.2) s; N=45 biological replicates), the area and the LimE-GFP intensity (-11.2 (-19.6/ -10.3) s; N=21 biological replicates), and the area and the GFP-myo intensity (52.5 (41.3/62.8) s; N=11 biological replicates). F: Median shift in the CF of the area change rate and F (-3.7 (-11.2/3.8) s; N=42 biological replicates), the area change rate and the LimE-GFP intensity (16.9 (7.5/30.0) s; N=20 biological replicates), and the area change rate and the GFP-myo intensity (3.8 (0/7.5) s; N=11 biological replicates). Data bellow the dashed line indicates cells for which no significant correlation was found. G: Time-averaged total force F as a function of the cell area A.
Figure 4A-F. Bclaf1 WT, KO-1 and -2 HeLa cells (A and D), as well as HeLa (B and E) and primary MEFs (C and F) which were transfected with siCtrl or siBclaf1-1 and -2 were treated with TNF (10 ng/ml) for 6 hours followed by western blotting analysis (A-C), or for 4 hours followed by total RNA extraction and RT-PCR analysis (D-F). Data information: Data are shown as mean ± SD. n=3 biological replicates. *p<0.05; ***p<0.001; ****p<0.0001. One-way ANOVA test. G. CFLAR full length (FL) promoter ranging from -1179 to +281 and three promoter regions P1 (-1179 to -788), P2 (-788 to -288) and P3 (-288 to +281) were fused with a luciferase reporter and then co-transfected with an increase dose of Bclaf1 in HeLa-Bclaf1-KO cells. At 24 hours after transfection, the cells were collected and luciferase activity was measured. The experiments were performed in triplicates with a Renilla reporter in the transfection mixture for normalization and the activity in the control was arbitrarily set to 1. Data information: Data are shown as mean ± SD. n=3 biological replicates. *p<0.05; ***p<0.001; ****p<0.0001. One-way ANOVA test. H, I. CHIP analysis of the binding of Bclaf1 to the promoter of CFLAR was performed in HeLa-Flag-Bclaf1 cells treated without (H) or with TNF (I). The regions amplified by RT-PCR were delineated. Data information: Data are shown as mean ± SD. n=3 biological replicates. *p<0.05; ***p<0.001; ****p<0.0001. One-way ANOVA test.
H3R antagonist binds with H3R and subsequently releases its binding with CLIC4.f8H3R antagonist binds with H3R and subsequently releases its binding with CLIC4. This process inhibits PI3K/Akt/GSK-3β/mTOR signalling, that activates autophagy.
a, AMBRA1 downregulation in 2FTGH cells using specific short interfering RNA (siRNA) oligoribonucleotides (1 and 2).figf3a, AMBRA1 downregulation in 2FTGH cells using specific short interfering RNA (siRNA) oligoribonucleotides (1 and 2). AMBRA1 mRNA and protein levels were analysed by quantitative polymerase chain reaction (PCR, left) and western blotting (right), respectively. An unrelated oligoribonucleotide was used as the control. Tubulin was used as the protein-loading control. RL, relative levels. The arrowhead indicates the AMBRA1-specific band. b, Rapamycin-induced autophagy requires AMBRA1. After AMBRA1 downregulation, 2FTGH cells were treated with rapamycin; after 48 h, the occurrence of autophagy was analysed by appearance of GFP-LC3 punctate staining or by LC3-I to LC3-II conversion (western blot in inset). Representative results are accompanied by a graph reporting data from three experiments. Scale bar, 20 µm. c, Nutrient-starvation-induced autophagy requires AMBRA1. After AMBRA1 downregulation, 2FTGH cells were starved for 4 h and analysed for appearance of GFP-LC3 punctate staining. d, AMBRA1 overexpression increases basal and rapamycin-induced autophagy. 2FTGH cells were transduced with full-length AMBRA1 (FL), AMBRA1 fragments (F1-3), BECN1 and β-gal (negative control) encoding retroviruses. They were then stimulated with rapamycin or left untreated, and analysed by appearance of GFP-LC3 punctate staining. e, BECN1-induced autophagy requires AMBRA1. After AMBRA1 downregulation, GFP-LC3-expressing 2FTGH cells were transduced with a BECN1-encoding retrovirus. Occurrence of autophagy was analysed 48 h later by appearance of GFP-LC3 punctate staining. f, AMBRA1 downregulation reduces the amount of Vps34 associated to BECN1 during autophagy. Forty-eight hours after AMBRA1 downregulation with oligo 2, 2FTGH cells were either starved or cultured in standard medium for an additional 4 h. Protein extracts were immunoprecipitated (IP) using an anti-BECN1 antibody. Purified complexes and corresponding total extracts were analysed by western blotting with anti-Vps34 (left panels) and anti-BECN1 (right panels) antibodies. The graph indicates the signal intensity of BECN1-associated Vps34 as determined by densitometry.
a) COS-7 cells expressing wild-type (Gln23; Q23) or mutant (Gln74; Q74) huntingtin (green) were labeled for S6 phosphorylated at Ser235 and Ser236 (red).figf3(a) COS-7 cells expressing wild-type (Gln23; Q23) or mutant (Gln74; Q74) huntingtin (green) were labeled for S6 phosphorylated at Ser235 and Ser236 (red). Percentages of cells expressing wild-type or mutant huntingtin that did (+agg) or did not (−agg) contain aggregates were scored for negative staining for phosphorylated S6 (p-S6). (b) Brain sections from control (WT) and mice expressing mutant huntingtin (HD) were stained for phosphorylated S6 (red) and with antibody to huntingtin (EM48; green). (c,d) Impaired mTOR-dependent TOP translation in cells expressing mutant huntingtin. (c) PC12 cells expressing wild-type (Gln23; Q23) or mutant (Gln74; Q74) huntingtin were transfected with luciferase constructs containing 5′ untranslated sequence with (+) or without (−) a TOP sequence along with β-gal. Each bar represents the difference between the luciferase activities in the presence versus the absence of rapamycin. Data shown are from one representative experiment (of three) done in sextuplicate (error bars represent s.e.; raw data are given in Supplementary Fig. 4 online). (d) COS-7 cells were cotransfected with wild-type or mutant huntingtin, a nontypical TOP or control luciferase vector and β-gal. The experiment was done and analyzed as in c (raw data are given in Supplementary Fig. 4 online) (e-g) Relevance of mTOR sequestration to other polyglutamine expansion diseases (e) COS-7 cells expressing Xpress-tagged ataxin-1 with 92 polyglutamine repeats (Q92) for 48 h were doubly labeled with antibodies to Xpress (green) and to mTOR (red). (f) mTOR immunohistochemistry in brains of individuals with dentatorubral-pallidoluysian atrophy (DRPLA) and spinocerebellar ataxia types 2, 3 and 7 (SCA2, SCA3 and SCA7). (g) Staining for phosphorylated S6 (red) in COS-7 cells transiently transfected with mutant (ataxin Q92; green) or wild-type (ataxin Q2) ataxin-1 constructs. Results were significant and similar to those in a. Arrows in a, b, e and g show cells containing aggregates.
Data-driven modeling reveals signaling correlates of BRAF-mutant melanoma cellular response to RAF/MEK inhibition.Figure 1Overview of the systematic measurements using single-cell phenotypic and multiplex biochemical assays to profile signaling biochemistry and cell state in 10 BRAFV600E/D melanoma cell lines exposed to four RAF inhibitors and one MEK inhibitor at multiple doses and times. Multiplex single-cell immunofluorescence assays were used in the follow-up experiments on selected cell lines for treatments with vemurafenib, JNK-IN-8, and their combination. Dose-response curves representing 72-h viability measurements for six selected BRAFV600E melanoma cell lines after treatment with vemurafenib. Ranges of estimated IC50 and Emax for the selected lines are shown. Data are represented as mean ± SD. Pairwise distribution and correlation of estimated IC50 and Emax values for responses (72-h viability) of 8 cell lines to four RAF inhibitors and one MEK inhibitor. Each dot represents one drug/cell line combination. Sensitive cell lines are arbitrarily defined based on their IC50s (the most widely used metric for evaluation of drug response; log10[IC50] < −6.5). Cell line-specific model calibration for two selected cell lines C32 and WM115. Left: R2, Q2, and MSPE for C32 and WM115 models built with increasing numbers of PLSR components. Middle: Relative viability as measured experimentally (z-score-scaled) or as predicted by the three-component PLSR models using tenfold cross-validation. R2 reports model fit, and Q2 reports model prediction accuracy. Right: PLSR score plots for the first two components of C32 and WM115 models. Shown PLSR models were developed using RPPA data from Supplementary Dataset S3. PLSR loading plots for the first two components of C32 and WM115 models.
Time‐course analysis of Rab8A, Rab8B and Rab13 Ser111 phosphorylation.Figure 8Time‐course comparison of PINK1‐mediated Rab8A, Rab8B and Rab13 Ser111 phosphorylation vs. Parkin Ser65 phosphorylation. Flp‐In T‐Rex HEK293 cells expressing WT PINK1‐FLAG were transfected with either WT or Ser111Ala‐(S111A) mutant HA‐Rab8A, HA‐Rab8B or HA‐Rab13, induced with doxycycline and stimulated with CCCP for the indicated time. In parallel, Flp‐In T‐Rex HEK293 cells expressing WT PINK1‐FLAG were transfected with either WT or Ser65 Ala (S65A)‐mutant Parkin. Whole‐cell lysates (0.25 mg) were immunoprecipitated with anti‐HA agarose and immunoblotted with indicated phospho‐Ser111 antibodies. Part of the immunoprecipitates was used to immunoblot for HA antibody as loading controls. For the lower panel, whole‐cell lysates (30 μg) were immunoblotted with indicated antibodies. Time‐course comparison of endogenous PINK1‐mediated Rab8A, Rab8B and Rab13 Ser111 phosphorylation vs. Parkin Ser65 phosphorylation. HeLa cells were transfected with either WT or S111A‐mutant HA‐Rab8A, HA‐Rab8B or HA‐Rab13 for at least 24 h before CCCP treatment for the indicated time. Whole‐cell lysates (1 mg) were immunoprecipitated with anti‐HA agarose and immunoblotted with indicated phospho‐Ser111 antibodies. In parallel, HeLa cells were transfected with either WT or S65A‐mutant Parkin and whole‐cell lysates (30 μg) were immunoblotted with indicated antibodies.
Figure 3Representative traces of cell-attached recordings performed in WT (blue), KO (red) and KO injected with either with AAV9-hSynI-mWwox (KO+A-mW shown in purple) or AAV9-hSynI-hWWOX (KO+A-hW shown in green) pups at P18-21 days. Traces represent spontaneous neocortical activity (action potentials shown). The panels represent 12 s recording with an inset presenting a zoom-in of an 0.5 s interval.   The graph shows the recordings (P18-21) averages over 30 neurons from WT (n=3), 30 neurons from KO+A-mWwox (n=3), 30 neurons from KO+A-hWWOX (n=3), and 45 neurons from KO (n=3).   Representative traces of cell-attached recordings performed in a WT (blue) and a KO+A-mWwox (purple) adult mice (6 months old). The graph shows the averages over 60 neurons from WT adult mice (n=3) and 60 neurons from KO+A-mWwox adult mice (n=3). There was no significant change observed between the average firing rate of the WT and the KO+A-mWwox adult mice. Immunofluorescence staining with anti-GFAP (shown in green) and anti-Iba1 (shown in magenta) antibodies showing the expression of astrocytes and microglia at P19 from WT, KO and KO+A-hWWOX. The square white box shows the magnified area from CA3 regions of hippocampus from all the genotypes. F, G. Graphs represents the quantification of GFAP (F) and Iba1 (G) positive cells from the images shown in E. The number of GFAP or Iba1 cells were counted from 3 identical sagittal sections from CA3 region per group (n=3 per each genotype). H. Immunocytochemical images showing astrocytes (anti-GFAP) and microglia (anti-Iba1) in CA3 region of hippocampus from WT (n=3) and rescued (KO+AAV-hWWOX) (n=3) mice at 9 months. Magnified area is shown with square box. I, J. Graphs represents the quantification of GFAP (I) and Iba1 (J) positive cells from the images shown in H. The number of GFAP or Iba1 cells were counted from 3 identical sagittal sections (paraffin embedded) from CA3 region per group (n=3 per each genotype).
Pharmacological inhibition of CaMKII decreases TNT's formation and function.Figure 2Representative confocal images of CAD cells cultured in control conditions or treated with 0.2% DMSO, 200 µM H2O2, 200 ng/mL Wnt7a, 10 μM KN-93, 200 ng/mL Wnt7a plus 10 μM KN-93, 1 μM TAT or 200 ng/mL Wnt7a plus 1 μM TAT for 4 h. Cells were fixed and stained with WGA (green) and DAPI (blue). Percentage of TNT-connected CAD cells with each of the indicated treatments. Representative confocal images of co-cultured donor and acceptor CAD cells. Cells were co-cultured in control conditions or treated with 0.2% DMSO, 200 µM H2O2, 200 ng/mL Wnt7a, 10 μM KN-93, 200 ng/mL Wnt7a plus 10 μM KN-93, 1 μM TAT or 200 ng/mL Wnt7a plus 1 μM TAT for 4 h. Yellow arrows point to DiD-labeled vesicles inside acceptor cells. Percentage of acceptor cells containing DiD-labeled vesicles in the co-culture systems with the indicated treatments. Representative confocal images of CAD cells stained for vinculin (red) and HCS CellMask™ Blue after being treated with 0.2% DMSO, 200 µM H2O2, 200 ng/mL Wnt7a, 10 μM KN-93, 200 ng/mL Wnt7a plus 10 μM KN-93, 1 μM TAT or 200 ng/mL Wnt7a plus 1 μM TAT for 4 h. The ring circumscribed between the green lines mark the area where the vinculin puncta were counted for each cell. This area is defined by the board of the HCS CellMask™ Blue staining and the vinculin staining around it. Average number of vinculin puncta per cell with the indicated treatments.
IVM decreases AR levels and activity in both in vitro and in vivo studies.Figure 6A. In LNCaP xenografts, AR and SDHB protein levels (left panel) and SDH activity (right panel) were reduced acutely after castration (3 days) but returned to baseline after castration resistance (3 months). SDH assay was performed on tumour tissues from 5 mice per group. Data shown as means ± SD B. IVM reduced AR activity in combination with ENZA in LNCaP cells. Data shown as means ± SD C. ENZA reduced cell proliferation more potently when combined with IVM in LNCaP cells. Data shown as means ± SEM In vitro data represent three independent experiments. Statistical analysis was performed for the last data point *, p<0.05, **, p<0.01 and ***, p<0.001 compared between groups, one-way ANOVA followed by Tukey's test. D. IVM reversed the cell proliferation benefit mediated by SDH repression in LNCaP cells. Data shown as means ± SEM In vitro data represent three independent experiments. Statistical analysis was performed for the last data point *, p<0.05, **, p<0.01 and ***, p<0.001 compared between groups, one-way ANOVA followed by Tukey's test. E. LNCaP xenografts treated with IVM (n=10) (10mg/kg) demonstrated lower serum PSA values (upper panel) and growth of tumours after castration (lower panel) compared to vehicle (n=10). PSA was measured every week. Data shown as means ± SEM F. IVM treatment reduced p-Hsp27 levels and therefore AR protein level, with subsequent p-CAMKK2 axis activation in castrated LNCaP xenografts. Bar graph in the right panel shows quantification of data from western blot normalized to loading control. Data shown as means ± SD