EMBO/sd-character-level-ner huggingface dataset:
Npas4 regulates dendritic spine density in D1R-MSNs under basal conditions and after repeated cocaine exposure.Figure 5a, Time course of AAV injections, habituation and treatments to assess the impact of Npas4 knock-down on MSN neuronal morphology under basal conditions and after repeated cocaine injections. b, Representative images of AAV-shscr-GFP- and AAV-shNpas4-GFP-infected cells together with the sparse labelling of D1R-MSNs achieved through a co-injection of AAV-Flex-tdTomato and AAV-PPTA-Cre (to label D1 MSNs) or AAV-ENK-Cre (to label D2 MSNs) in a 1/10,000 ratio. Scale bar = 100 µm. c, Representative confocal images of staining for Npas4 expression (red) in the NAc of mice infected with AAV shscr-GFP or AAV-shNpas4-GFP 1h after cocaine treatment. Scale bar = 20 µm. Note the presence of Npas4-positive neurons infected with the AAV shscr-GFP (arrowheads) and not in cells infected with the AAV-shNpas4. d, Quantifications of GFP-positive and Npas4-positive cells 1h post cocaine treatment. Data were normalized so that the number of GFP- and Npas4-positive cells was set to 100%. 20 fields of view were analysed from n = 3 mice in the case of AAV-shscr-GFP and 30 fields of view from n = 3 mice for AAV-shNpas4-GFP. Mean + SEM; unpaired Student's t-test; p* < 0.05, n = 3 mice per group. e, Confocal images of tdTomato-labelled D1-MSNs expressing shscr-GFP or shNpas4-GFP (left panels; scale bar: 20 µm). 3D volume-renderings of MSN dendrites from the NAc shell (right panels; scale bar: 2 µm). f, Quantification of spine density in D1-MSNs, defined as the number of spines normalized to 10µm of dendrite for saline control and after cocaine injection. Mean + SEM; two-way ANOVA, Bonferroni post-hoc test, p* < 0.05; p** < 0.01; p*** < 0.001. n=40 neurons from 4 mice in each experimental group. g, Confocal images of tdTomato-labelled D2-MSNs expressing shscr-GFP or shNpas4-GFP (left panels; scale bar: 20 µm). 3D volume-renderings of MSN dendrites from the NAc shell (right panels; scale bar: 2 µm). h, Quantification of spine density in D2-MSNs, defined as the number of spines normalized to 10µm of dendrite for saline control and after cocaine injection. Mean + SEM; two-way ANOVA, Bonferroni post-hoc test, all non-significant. n=40 neurons from 4 mice in each experimental group.
Molecular mechanisms of SOM230 action through sst1 for inhibition of Akt-4E-BP1 signalling pathways in CAF.Figure 4A-D Immunoblotting using an anti-P-Akt, anti-P-S6, anti-4EB-P1 or anti-GAPDH (loading control) antibody of protein extracts from CAFs pre-treated (+) or not overnight with PTX (100 ng/ml) (A), gallein (10 μM) (B), NSC87877 (10 μM) (C), Src inhibitor (5 μM) (D), treated (+) or not with SOM230 (10−7 M) for 30 min (representative of n = 3). E, F Immunoblotting using anti-P-Akt, anti-P-S6, anti-4EB-P1 or anti-GAPDH (loading control) antibody of protein extracts from CAF siCTR- or siSHP2- (E) or siPTPε- (F) transfected CAF treated or not with SOM230 for 30 min (representative of n = 3). G, H Immunoblotting using an anti-P-Akt, anti-P-S6, anti-4EB-P1 or anti-GAPDH (loading control) antibody of protein extracts from CAFs treated with the indicated molecules (PDGF receptor inhibitor, 5-10 μM; Jak1/2 inhibitor ruxolitinib, 5 or 10 μM; EGFR inhibitor, 150 or 300 nM and recombinant PDGF, 5 μg/ml) (representative of n = 3).
Figure 2A Recombinant His-p53 was incubated with or without purified Flag-NAT10 as described in Experimental Procedures. The reaction products were resolved by SDS-PAGE and immunoblotted using anti-acetyl-lysine antibodies. Flag-PCAF was used as a positive control. B In vitro acetylation was performed and the acetylated p53 was detected as in A. C In vitro acetylation assay was performed as described in A. The reaction products were resolved by SDS-PAGE and acetylated p53 was purified from the SDS-PAGE and subjected to mass spectrometry analysis. D His-p53 or His-p53-K120R fusion protein was incubated with purified NAT10 or NAT10 GE mutant as described in Experimental Procedures. Reaction mixtures were subjected to western blot using the site specific monoclonal anti-Ac-p53-K120 antibody. E H1299 cells were transfected with the indicated vectors. Total proteins and the anti-Flag antibody M2-specific immunoprecipitates were analyzed by western blot using the indicated antibodies.
Temporal metabolomics reveals the mammalian metabolome is rapidly reshaped by iron depletion.Figure 1a. Schematic of temporal metabolomics workflow in human ARPE-19 cells. b. Multivariate modelling of the metabolome reveals distinct transitions over time (states I-IV) in response to iron chelation using deferiprone (DFP). OPLS-DA was computed and generated from combined LC-MS and GC-MS datasets from three independent biological experiments each with four technical replicates per time point in each experiment.
Figure 6A. siRNA transfected HEK293 cells were serum deprived and stained for acetylated-tubulin, TBC1D31 and DRAQ5. B. Quantitative analysis of the experiments shown in A. A mean value ± SD of four independent experiments is shown. Student's t test *p< 0,05. C C. HEK293 cells expressing flag-OFD1 or flag-S735A were serum deprived and subjected to staining analysis for acetylated α-tubulin, flag and DRAQ5. Images were collected by super-resolution microscopy (upper panels). Where indicated, flag-OFD1 or flag-S735A was co-transfected with cilia-APEX-GFP vector (middle panels). NIH3T3 expressing flag-OFD1 or flag-S735A were stained with anti-ARL13B and anti-flag antibodies. D, E. Statistical analysis of the experiments shown in D. A mean value ± SD of three independent experiments is shown. For each experimental group, a minimum of 40 cilia were analyzed. Student's t test p***<0,001. F F. HEK293 cells transiently transfected with flag-OFD1 or flag-S735A were serum deprived, treated with FSK (6 hours) and stained for flag, acetylated-tubulin and DRAQ5. G. Statistical analysis of the experiments shown in F. A mean value ± SD of three independent experiments is shown. Cells analysed for each experiment: 300 for NT, 80 for flag-OFD1 and 80 for flag-S735A. Student's t test, p*<0,05, p**<0,01. H H. Quantitative RT-PCR analysis showing levels of Gli2 mRNA in NIH3T3 fibroblasts transfected with vectors for flag-OFD1 and flag-S735A. An empty vector (CMV) was used as control. Serum deprived cells were stimulated with the SHH ligand purmorphamine (1μM) for 24 hours. Where indicated, cells were treated with FSK for 6 hours before harvesting. The data represent a mean value ± SD of three independent experiments. Student's t test, p**<0,01.
Figure 5A. Cell lysates from HeLa cells KD or OE for either GOLPH3 or LCS were processed for SDS-PAGE and Western blotting with antibodies aimed at evaluating the activation status of the mTOR pathway. Data are representative of at least 3 independent experiments. B. Clonogenic assay of HeLa cells in adherence. 250 HeLa cells KD for either GOLPH3 or LCS were allowed to form colonies for 10 days. Afterward, colonies were fixed and stained as detailed in Materials and Methods. The graph on right reports the number of colonies per well in each indicated condition (data are means ± SEM of 3 independent experiments; *p <0.05, **p <0.01 [Student's t-test]). C. Soft agar colony formation assay of HeLa cells transfected with different concentration of either GOLPH3 (left upper graph) or LCS (left bottom graph) encoding plasmids. Soft agar colony formation assay of HeLa cells KD for either GOLPH3 or LCS (right upper graph). Soft agar colony formation assay of HeLa cells KD for LCS and GOLPH3 OE alone or in combination with LCS KD (right bottom graph). Colonies were stained and quantified as described in Materials and Methods; (individual data points are shown in the graphs; data are means ± SD of at least 3 independent experiments; *p <0.05, ***p <0.001, n.s. = not significant [Student's t-test]). D. Protein lysates from parental (left), GCS-KO (middle), and LCS-KO (right) HeLa cells, CTRL or GOLPH3-OE were processed for SDS-PAGE and WB with the indicated antibodies. Data are representative of at least 3 independent experiments. E. Soft agar colony formation assay of parental and LCS-KO HeLa cells expressing LCS-HA or GOLPH3. Colony number was quantified; (individual data points are shown in the graphs; data are means ± SD of at least 3 independent experiments; ***p <0.001 [Student's t-test]). F. Quantification of soft agar colony formation assay of LNCaP and DU145 prostatic cancer cell lines treated with increasing concentrations of siRNA targeting GOLPH3 or LCS (data are means ± SEM of at least 3 independent experiments; ***p <0.001 [Student's t-test]).
Upregulation of IL-1β expression in infiltrating microglia potentiates photoreceptor apoptosis.Figure 8Microglia infiltrating the rd10 retina upregulate IL-1β expression during degeneration. Iba1+ microglia (green) were immunonegative for IL-1β (red) at P18 but become prominently IL-1β immunopositive at P22 (arrows). Scale bars, 25 μm. Depletion of infiltrating microglia in the rd10 retina decreases IL-1β levels. Cytokine levels in rd10/CreDTA mouse retinas were assayed following tamoxifen-induced microglial depletion (from P21 to P37-50, green bars; n = 9 animals) and compared with their untreated littermate controls (white bars, n = 8 animals; values normalized to control animals in the same litter). IL-1β protein levels were significantly lowered following microglial depletion, but IL-6, CCL2, or TNFα were not significantly changed. Inhibition of microglial phagocytosis results in decreased microglial IL-1β expression. Eyes of rd10 animals were treated with intravitreal injections of cRGD at P20, and analyzed at P23 by immunohistochemistry for Iba1 (green) and IL-1β (red), and compared to uninjected eyes and with eyes injected with inactive analog, cRAD (n = 3 eyes in each category). Immunopositivity was high in infiltrating microglia (indicated by arrows) for untreated and cRAD-injected control eyes and significantly reduced in microglia in cRGD-injected eyes (upper panel), as demonstrated by the quantification of IL-1β expression (area of immunopositivity as a fraction of the ONL) (lower left panel). ONL thickness correlated negatively with the extent of IL-1β immunopositivity (lower right panel). Scale bar, 25 μm. IL-1R signaling during rod degeneration was inhibited by intravitreal injections of recombinant IL-1 receptor antagonist, anakinra (from P20-22 to P26-27) in one eye of each rd10 animal, while the contralateral eye was injected with PBS as a control. Upper panels show representative retinal sections from control PBS-injected and IL-1RA-injected eyes from the same animal. ONL atrophy in control eyes was more advanced compared to IL-1RA-injected eyes, with greater mean ONL thickness and lower mean density of TUNEL+ ONL nuclei in IL-1RA-injected eyes (lower panels) (n = 15 animals; measurements in IL-1RA-injected eyes normalized to contralateral control eyes, paired t-test). Scale bar, 25 μm.
Figure 9Huh-7 cells were first starved with serum free medium and then incubated with oleic acids for 12 hours. Condition 1 (control), the cells were not treated with BFA at any time point (left column). Condition 2, the cells were incubated with both 5 μg/ml of BFA and oleic acid incubation for 6 h and then in oleic acid alone for 10 h (middle column). Condition 3, the cells were treated with 5 μg/ml of BFA starting at 12 hours after oleic acid incubation (right column). Bar = 10μm.
From integrated proteomic subtypes to drug sensitivity prediction.Figure 3 Figure 3 - From integrated proteomic subtypes to drug sensitivity prediction (A) Heat map of standardized, log2-transformed and median-centred giBAQ protein quantification (z-scores) across the combined CRC65/CPTAC dataset. Cell lines/patients are displayed as columns and proteins are shown as rows. Black bars to the left of the heat map indicate the presence of the respective protein in the CMS classifier. Annotation bars on top of the heat map visualise the membership of the different cell lines/patients in five annotation categories. (B) Elastic net regression was used to model drug sensitivity as a function of protein profile with the CCLE, CTRP, GDSC and Cetuximab datasets as input. The resulting models were used to predict the drug sensitivity of cell lines/patients (see also Appendix). In the heat map of standardized predicted AUC values (z-scores), cell lines/patients are displayed as columns with the same annotations and ordering as in (A), while drugs or combinations thereof are shown as rows. Coloured bars to the left of the heat map indicate drugs, which are more effective in a respective subtype (see main text and Appendix for details). See also Figures EV2-4.
Evolutionary conservation patterns in APA and gene expression.Figure 72D clustering of Pearson correlation coefficients between all pairwise sample combinations of C57BL/6J (BL6) or SPRET/EiJ (SPR) allelic PAS usages from 5,264 genes (see Methods). 2D clustering of Pearson correlation coefficients between all pairwise combinations of BL6 or SPR allelic gene expression levels from 5,264 genes. All clustering was performed hierarchically using Pearson correlations between samples. Scatterplot of maximum (X-axis) and minimum (Y-axis) allelic PAS usage divergence (BL6 - SPR) across tissues for each gene. The total number of APA divergent genes is indicated in the legend. The number of APA divergent genes with opposite sign between maximum and minimum is in parentheses. Scatterplot of maximum (X-axis) and minimum (Y-axis) allelic gene expression divergence log2(BL6 / SPR) for each gene. The total number of gene expression divergent genes is indicated in the legend. The number of gene expression divergent genes with opposite sign between maximum and minimum is in parentheses.
amiRSUL levels are reduced in hst, unlike its loading into AGO1 or its silencing activity(A) Northern analysis of indicated miRNAs, in biological duplicates, in leaves with the indicated genotypes.Figure 3Northern analysis of indicated miRNAs, in biological duplicates, in leaves with the indicated genotypes. U6 was probed as endogenous control. Average relative U6-normalized band-intensity quantifications are indicated. Left: silencing phenotype of homozygous (+/+) versus hemizygous (+/-) pSUC2::amiRSUL plants. Right: RT-qPCR analysis of SUL mRNA levels in leaves of (+/+) or (+/-) pSUC2::amiRSUL versus WT Arabidopsis. Error-bars: SD. t-test p-values are indicated. n=3. amiRSUL northern analysis, in biological triplicates, in leaves of (B). miR159 and U6 were probed as endogenous controls. Band-intensity quantifications: as in (A). amiRSUL northern analysis in input and AGO1-immunoprecipitated (AGO1-IP) fractions isolated from leaves of pSUC2::amiRSUL plants in the specified genotypes. No Ab: No antibody added to the HST input extract (IP negative control). miR165/166 and U6 were probed as endogenous controls. AGO1 western analysis and Coomassie blue (Coom.) staining of the western blot membrane are provided as controls. amiRSUL band-intensity quantifications, U6-normalized for input samples, are indicated. amiRSUL northern analysis in pSUC2::amiRSUL (amiR) scions (S) and non-transgenic WT and hst-1 rootstocks (R) in the indicated grafting combinations. U6: as in (A). RT-qPCR analysis of SUL mRNA levels in WT and hst-1 rootstocks grafted onto WT or amiR scion. Error-bars: SD. Tukey's multiple comparisons test p-values are indicated. n=4.
Figure 3qRT-PCR analysis of INH1 mRNA levels. Data are mean ± SD from three biological replicates. Western blot analysis of Inh1 protein levels. Whole cell lysates were extracted and equal amount of proteins was loaded for western blot. Deletion of INH1 benefits the proliferation of ρ0 cells at 37 ℃. Serial dilutions (tenfold dilution) of the indicated strains were analyzed on SCD plates at 30℃ and 37 ℃ for 2 days. Western blot analysis of copper-inducible overexpression of Inh1. A cassette expressing Inh1 under the control of CUP1 promoter was inserted into the HO locus. Inh1 overexpression was induced by 100 μM CuSO4 for 12 hours. Overexpression of Inh1 decreases ∆ψm in ρ0 cells. Cells were cultured in SCD or SCD plus 100 μM CuSO4 for 12 hours to mid-log phase. Cells were then stained with 125 nM TMRM for FACS analysis. Overexpression of Inh1 represses the proliferation of ρ0 cells. Serial dilutions (tenfold dilution) of the indicated strains were analyzed on SCD or SCD plus 100 μM CuSO4 plates at 30℃ for 2 days. Overexpression of Inh1 reduces the protein levels of mitochondrial ISC biosynthesis proteins (Nfs1 and Yah1) and nuclear ISC-containing protein Pol3. Cells were cultured in SCD or SCD plus 100 μM CuSO4 for 12 hours to mid-log phase. Whole cell lysates were extracted and equal amount of proteins was loaded for western blot. Nfs1, Yah1 were endogenously tagged with FLAG, and Pol3 was endogenously tagged with HA. Cartoon illustration of the translational repression of Hap4 and the subsequent transcriptional downregulation of Inh1 to maintain ∆ψm in ρ0 cells.
Figure 3A. Transmitted light micrographs of MDGI-silenced (shMDGI1 and shMDGI2) and control (Scr) patient-derived BT12 and BT13 spheroids 11 days after gene silencing. Representative images of at least 3 independent experiments are shown. Scale bar: 200 μm. B. Methylcellulose assay demonstrating the ability for self-renewal of control (Scr) and MDGI-silenced (shMDGI1 and shMDGI2) patient-derived BT12 and BT13 cells after three weeks of culture. Representative images of at least 3 independent experiments are shown. Scale bar: 200 μm. C. Quantification of the colony size in methylcellulose of MDGI-silenced (sh1 and sh2) cells relative to the controls (Scr) that is set as 100 (n = 3). Data are represented as mean ± SD. ***P < 0.001, two-tailed, nonparametric Mann-Whitney's U-test. D. MTT-proliferation assay of control (Scr) and MDGI-silenced (shMDGI1) BT12 and BT13 cells. The values of each cell line were normalized to the values at day 1 marked with a dashed line (n = 3). Data are represented as mean ± SD. ***P < 0.001, two-tailed, nonparametric Mann-Whitney's U-test. E, F Representative whole coronal section micrographs of murine brain injected intracranially with control (BT12 Scr, n = 5) and MDGI-silenced (BT12 sh1, n = 5) BT12 cells. After four weeks animals were sacrificed, the brains were excised and prepared for immunohistochemistry. Control (Scr) cells grew very similarly to the parental BT12 cells forming a tumour mass with invasive tumour cells (single cell invasion) that eventually co-opted existing blood vessels (angiotropism) and formed secondary tumours (upper panels). No tumour growth was observed when MDGI-silenced cells were implanted (lower panels). Glioma cells were visualised by using anti-human vimentin (hVim red) and blood vessels using anti-podocalyxin antibodies (green). Nuclei were visualised by using DAPI (white). Scale bar: 1 mm (E) and 50 µm (F).
PPM1G-B56δ complex controls cell migration by dephosphorylating α-Catenin (A, B) MDA-MB-231 cells were transfected with vector control, PPM1G wild type, catalytically inactive PPM1G D496A mutant or α-Catenin and transwell migration assay was performed.Figure 5(A, B) MDA-MB-231 cells were transfected with vector control, PPM1G wild type, catalytically inactive PPM1G D496A mutant or α-Catenin and transwell migration assay was performed. Phase contrast microscopic images (A) were taken after staining the migrated cells with crystal violet and (B) Quantification data for migrated cells per field (derived from average of 3 different fields) from three independent experiments is shown. Error bars indicate standard deviation (n=3), ***p<0.001 (one-way ANOVA, post hoc test: Bonferroni's multiple comparison test). (C, D) MCF-7 cells were transfected with SFB-vector, SFB-B56δ or indicated mutants and transwell migration assay was performed. Phase contrast microscopic images (C) were taken after staining the migrated cells with crystal violet and (D) Quantification data for migrated cells per field (derived from average of 3 different fields) from three independent experiments is shown. Error bars indicate standard deviation (n=3), ***p<0.001 (one-way ANOVA, post hoc test: Bonferroni's multiple comparison test). (E, F) SFB-B56δ was expressed in control shRNA and PPM1G shRNA transduced MCF7 cells. Representative images from transwell migration assay (E) are shown and (F) quantified data for migrated cells per field from three independent experiments was plotted. Error bars indicate standard deviation (n=3), ***p<0.001 (one-way ANOVA, post hoc test: Bonferroni's multiple comparison test). (G, H) SFB-PPM1G was expressed in control shRNA and B56δ shRNA transduced MCF7 cells. Representative images from transwell migration assay (G) are shown and (H) quantified data for migrated cells per field from three independent experiments was plotted. Error bars indicate standard deviation (n=3), ***p<0.001 (one-way ANOVA, post hoc test: Bonferroni's multiple comparison test). (I, J) Control shRNA and B56δ shRNA transduced MCF7 cells were transfected with MYC-PPM1G and MYC-PPM1G ΔNLS. Representative images from transwell migration assay (I) performed with these cells are shown and (J) quantified data for migrated cells from three independent experiments per field was plotted. Error bars indicate standard deviation (n=3), ***p<0.001 (one-way ANOVA, post hoc test: Bonferroni's multiple comparison test). (K, L) SFB-tagged α-catenin S641A and S641D mutants were expressed in PPM1G shRNA and B56δ shRNA transduced MCF7 cells as shown. Representative images from transwell migration assay (K) performed with these cells are shown and (L) quantified data for migrated cells per field from three independent experiments was plotted. Error bars indicate standard deviation (n=3), ***p<0.001 (one-way ANOVA, post hoc test: Bonferroni's multiple comparison test).