EMBO/sd-character-level-ner huggingface dataset:
Increased TGIF1 phosphorylation in human PDAC A Specificity of anti-phospho-TGIF1 (pTGIF1) antibody.Figure 6Specificity of anti-phospho-TGIF1 (pTGIF1) antibody. Cell extracts from Tgif1-/- MEFs cells reconstituted with empty vector (EV), wild-type (TGIF1) or non-phosphorylated form (TGIF1.2TA) of Flag-TGIF1 were immunoblotted with anti-phospho-TGIF1 or β-Actin as a loading control. Expression of pTGIF1 and Twist1 in human tissue microarrays of human PDAC samples was analyzed by immunohistochemistry. Representative pictures at different stages (40x) are shown. The percentages of samples with high versus low expression of pTGIF1 and Twist1 in normal tissues versus PDAC tissues are shown. Scale bars, 100 μM. MIAPaCa-2 cells were transfected with TGIF1 or TGIF1.2TD expression vectors, selected with G418 and resistance colonies were pooled (n=6). Expression of Twist1 was examined by qRT-PCR (D). MIAPaCa-2 cells were transfected with TGIF1 or TGIF1.2TD expression vectors, selected with G418 and resistance colonies were pooled (n=6). Binding of TGIF1 to the Twist1 promoter was examined by ChIP and agarose gel. BxPC3 cells were transfected with TGTLuc together with TGIF1 or TGIF1.2TA in the presence or absence of KrasG12D. Luciferase activity was measured 48 hr following transfection and normalized on the basis of co-transfected Renilla luciferase (n=6). BxPC3 cells were transfected with CDH1Luc or p16Luc reporter together with TGIF1 or TGIF1.2TA in the presence or absence of KrasG12D and luciferase activity was measured 48 hr following transfection as described in (E) (n=6).
Grk2 acts upstream of Gli processing.Figure 3A Prox1a and Eng expression in the myotome of 30hpf wild type and MZgrk2 embryos injected with mRNAs encoding Shh, dnPKA or mSmoA1-GFP. Each panel shows Prox1a in green, Eng in red, and the merged images with DAPI staining in blue. The co-labelling for Prox1a and Eng (orange) is indicative of MP differentiation (n=30 for each sample) Scale bar, 50μm. B Western blot showing levels of Gli2a-FL and Gli2a-R forms in 20hpf wild type and MZgrk2 embryos injected with mRNA encoding Shh, dnPKA or mSmoA1-GFP, respectively. γ-tubulin was used as loading control. Three biological replicates of this analysis were performed.
Figure 2A. Traces showing average changes (mean±s.e.m.) in intracellular Ca2+ (Fura2 ratio) over time in response to perfusion of different [Ca2+]o as indicated in the upper bar in MEF Stim1/Stim2-/- cells transfected with Stim1- (black trace, n=111), Stim1A (red trace, n=116), the combination Stim1 with Stim1A (blue trace, n=86) or with vector only (grey trace, n=79). B. Quantification of changes in ratio of resting, influx rate (Δratio/time), Δpeak and Δplateau measured in A. *** p<0.001, Kruskal-Wallis Anova. C. Traces showing average changes (mean±s.e.m.) in intracellular Ca2+ (Fura 2 ratio) in HEK293 cells co-transfected with Orai1 and either Stim1- (black trace, n=119) or Stim1A IRES-mCherry (red trace, n=118). D. Quantification of changes in ratio of resting, influx rate (Δratio/time), Δpeak and Δplateau measured in C. ** p<0.01 *** p<0.001 Mann-Whitney test. E. Data information: Data were obtained from three biological replicates (e.g. transfections) with each three measured dishes (technical replicates) with multiple cells each (yielding a total number n) and is shown as mean±s.e.m. E. Traces showing average changes (mean±s.e.m.) in intracellular Ca2+ (Fura 2 ratio) in primary astrocytes after transfection with non-silencing (siCTL, black trace, n=216) or exonA-specific siRNA (siS1A, red trace, n=263). Data information: Data were obtained from three biological replicates (e.g. transfections) with each three measured dishes (technical replicates) with multiple cells each (yielding a total number n) and is shown as mean±s.e.m. F. Efficiency of siRNA as determined by qRTPCR. Quantification of parameters shown in (E).*p<0.05, ** p<0.01, Mann-Whitney test. Data information: Data were obtained from three biological replicates (e.g. transfections) with each three measured dishes (technical replicates) with multiple cells each (yielding a total number n) and is shown as mean±s.e.m. H. Quantification of parameters shown in (E).*p<0.05, ** p<0.01, Mann-Whitney test. Data information: Data were obtained from three biological replicates (e.g. transfections) with each three measured dishes (technical replicates) with multiple cells each (yielding a total number n) and is shown as mean±s.e.m. I. Traces showing average changes (mean±s.e.m.) in intracellular Ca2+ (Fura 2 ratio) in primary astrocytes from C57BL/6 wt (CTRL,black trace, n=49) and splice deficient C57BL/6 10ACon mice (10ACon, red trace, n=82). J. Quantification of parameters determined in [I]. *** p<0.001 Mann-Whitney test. Data information: D were obtained from three biological replicates (e.g. transfections) with each three measured dishes (technical replicates) with multiple cells each (yielding a total number n) and is shown as mean±s.e.m.
Figure 7A, ColE9 R-domain binds BtuB with nM affinity, positioning the T-domain and IUTD above a neighbouring OmpF trimer. B, The ColE9 IUTD translocates through subunit 2 of OmpF to deposit the TBE in the periplasm and capture TolB at the expense of the OM lipoprotein Pal. The TBE allosterically promotes displacement of TolB's N-terminus which constitutes the TolA binding site. We propose this complex equates to the full translocon cryo-EM structure. C, ColE9 OBS1 binds subunit 1 of OmpF from the periplasm. Threading through two of OmpFs three subunits triggers dissociation of the ColE9 R-domain-BtuB complex and reorients the T-domain above subunit 2 below which TolB is positioned in the periplasm. The docking of OBS1 also results in TolB moving closer to the opening of subunit 2. This complex, which we propose equates to the partial translocon cryo-EM structure, is now primed for contact with TolA in the inner membrane. TolA extension through the periplasm is coupled to the PMF via its stator proteins, TolQ and TolR. D, Retraction of TolA (the molecular mechanism of which remains to be established) provides the driving force for pulling ColE9 bound to TolB into the periplasm through subunit 2 of OmpF, accompanied by unfolding of its constituent domains. It is at this point the immunity protein Im9 (not shown) would be displaced at the cell surface. E, The TolA-TolB complex is thought to retract through the cell wall, which would bring ColE9 close to the cytoplasmic membrane. The toxin likely refolds prior to transport across the cytoplasmic membrane, which involves the AAA+ATPase/protease FtsH (not shown) Walker et al., 2007().
Figure 5A. Confocal microscopy of tau (red pseudocolor) and Hsp70 (green pseudocolor) colocalization (merged image) levels in wild type primary neurons overexpressing GFP-AAV9 or FLAG-DnaJC5-AAV9. Boxed area are 5X digital zoom on a 60X lens shown below. Scale bars are 20 µm and 5 µm as indicated. Only neurons overexpressing virus were imaged.B. Quantification of tau-Hsp70 co-localization (mean Pearson's coefficient ± SEM, n=18, *p <0.05, one-way analysis of variance with Tukey's multiple comparison post-hoc analysis). C. Western blot of immunoprecipitatedtau from HEK293T reveals tau exists in an intracellular complex with DnaJC5 and Hsc70. DnaJC5 was detected with FLAG antibody. D. Western blot of HEK293T intracellular lysates show the DnaJC5/Hsc70/tau complex is disrupted by the addition of increasing amounts of YM-01 (0, 10, 30, 100 µM) in a dose-dependent fashion. DnaJC5 was detected by FLAG antibody. E. The DnaJC5/Hsc70 complex is disrupted when Hsc70 activity is inhibited by a dominant-negative point mutation, E175S. Western blot of immunoprecipitated DnaJC5 from HEK293T cells overexpressing WT or DN E175S Hsc70. DnaJC5 is immunopreicipitated by DnaJC5 antibody (Synaptic Systems). DnaJC5 was detected on Western blot by FLAG antibody. F. Dot blot of tau levels in media of M17 neuroblastoma cells treated with YM-01. Quantification of extracellular tau levels are mean ± SEM, n=6, *p<0.05 one-way analysis of variance with Tukey's multiple comparison post-hoc analysis. Intracellular levels shown by Western blot. DnaJC5 was detected on Western blot by FLAG antibody. G. Dot blot of tau levels in media of primary neurons from transgenic overexpressing tau P301L mice treated with YM-01 (10µM). FLAG-DnaJC5 was transduced into neurons using AAV9. Quantification of extracellular tau levels are mean ± SEM, n=6, ***p<0.001 one-way analysis of variance with Tukey's multiple comparison post-hoc analysis. DnaJC5 was detected on Western blot by FLAG antibody. H. Schematic illustrating the Hsc70/DnaJC5/tau complex can be disrupted by both chemical (YM-01) inhibition.
TP53INP1-deficient mice are highly susceptible to HFD-induced obesity owing to their chronic oxidative stress.Figure 1TP53INP1-KO (−/−) and WT (+/+) male mice were subjected to a high-fat diet (HFD, 60% fat) or a control diet (CTRL) for 16 weeks. Mice drank tap water or tap water supplemented with NAC (10 mg/ml or 1%).Curves show mice body weight recorded every week. CTRL: P (−/− versus +/+; t = 8w) = 0.047; P (−/− versus +/+; t = 9w) = 0.023. HFD: P (−/− versus +/+; t = 7w) = 0.039; P (−/− versus +/+; t = 8w) = 0.029; P (−/− versus +/+; t = 9w) = 0.021; P (−/− versus +/+; t = 10w) = 0.014; P (−/− versus +/+; t = 11w) = 0.0046; P (−/− versus +/+; t = 12w) = 0.0028; P (−/− versus +/+; t = 13w) = 0.0025; P (−/− versus +/+; t = 14w) = 0.00051; P (−/− versus +/+; t = 15w) = 0.00027; P (−/− versus +/+; t = 16w) = 0.00013. At the end of protocol, mice were sacrificed; liver and epididymal and renal fat masses were taken and weighed. Histograms show organ weight. Liver: P (−/− versus +/+; HFD) = 0.014; P (HFD versus CTRL; +/+) = 0.00063; P (HFD versus CTRL; −/−) = 0.0010; P (HFD versus CTRL; +/+ NAC) = 0.034; P (HFD versus CTRL; −/− NAC) = 0.027; P (NAC versus no NAC; −/− HFD) = 0.014. Epididymal fat mass: P (−/− versus +/+; HFD) = 0.011; P (HFD versus CTRL; +/+) = 0.028; P (HFD versus CTRL; −/−) = 0.000017; P (HFD versus CTRL; +/+ NAC) = 0.019; P (HFD versus CTRL; −/− NAC) = 0.0054; P (NAC versus no NAC; +/+ CTRL) = 0.037; P (NAC versus no NAC; −/− HFD) = 0.0025. Renal fat mass: P (−/− versus +/+; HFD) = 0.0041; P (HFD versus CTRL; +/+) = 0.028; P (HFD versus CTRL; −/−) = 0.000013; P (HFD versus CTRL; +/+ NAC) = 0.019; P (HFD versus CTRL; −/− NAC) = 0.0078; P (NAC versus no NAC; −/− HFD) = 0.0047.
Genetic and pharmacological inhibition of Apelin impairs mammary tumor growth and tumor angiogenesis in a paracrine manner.Figure 1(A) Kaplan Meier plot for survival in NeuT;Apln+/+ (n=11) and NeuT;Apln-/- (n=10) mice with mammary cancer after tumor onset. *P=0.0185; Log rank test. (B) Tumor volumes, followed over time, of control mammary tumor E0771 cells (shRenilla) and Apelin-depleted (shApln) E0771 cells orthotopically-injected into both syngeneic C57BL/6J Apln+/+ and Apln-/- mice (5x105 cells/mouse), respectively. Tumor volumes were determined using calipers and are shown as mean tumor volumes ± S.E.M. Data shown is pooled from 2 independent experiments. Apln+/+;shRenilla (n=18), Apln+/+;shApln (n=17), Apln-/-;shRenilla (n=14), Apln-/-;shApln (n=15); ** P<0.01, *** P<0.001; two-way ANOVA. (C) Mean percentages (± S.E.M.) of CD31+ area in E0771 shRenilla (n=3) or shApln (n=3) mammary tumors, assessed on day 23 post-orthotopic injection into C57BL/6J Apln+/+ or Apln-/- mice, respectively. **P<0.01; t test. (D) Mean percentages (± S.E.M.) of extravasated Dextran in E0771 shRenilla (n=9) or shApln (n=12) mammary tumors, assessed on day 19 post-orthotopic injection into C57BL/6J Apln+/+ or Apln-/- mice, respectively. **P<0.01; t test. Right panel shows representative immunofluorescence of Dextran (red), CD31+ vessels (green), and DAPI (blue). The white arrows indicate regions of Dextran extravasation. Scale bars = 100 μm (E) Mean counts (± S.E.M.) of pimonidazole positive foci, assessed on day 26 post-orthotopic injection of E0771 shRenilla (n=6) or shApln (n=4) into C57BL/6J wild-type mice (5x105 cells/mouse). *P<0.05; t test. Right panels show representative immunohistochemical pimonidazole staining at two different magnifications; Scale bars = 200 μm (upper panels) and 50 μm (lower panels). (F) Mean percentage (± S.E.M.) of tumor-infiltrating immune cells normalized to CD45+. E0771 shRenilla (n=8) or shApln (n=6) were orthotopically injected into C57BL/6J Apln+/+ or Apln-/- mice (5x105 cells/mouse), respectively and tumors were harvested day 25 post-injection. *P<0.05, **P<0.01; t test. All immune cell populations were gated for viable CD45+ cells and then further defined as: CD8 T cells (Thy1.2+, CD8+), CD4 T cells, (Thy1.2+, CD4+), Inflammatory Monocytes (Ly6C+, Cd11b+, Ly6G-), PMN-MDSCs (Ly6G+, Cd11b+), natural killer cells (Thy1.2-, Ly6G-, NK1.1+), natural killer T cells (Thy1.2+, CD4-, CD8-, NK1.1+) and peripheral dendritic cells (Ly6G- ,Ly6C+, Cd11b- , PDAC1+, B220+).
Figure 2(A) Representative flow cytometry gating and fluorescence microscopy images of sorted GFPhigh stem cells and GFPlow progenitor cells from Lgr5-EGFP-IRES-CreERT2 mice. (B) Representative brightfield images of organoids generated from GFPhigh stem cells isolated from tamoxifen treated AhR WT mice (Lgr5-GFPCreERT2 X CDX2P-CreERT2 [GC]) and KO mice (AhRf/f X Lgr5-GFPCreERT2 X CDX2P-CreERT2 [HGC]), treated with DMSO (control) or TCDD for 5 d. Data are from ≥4 mice, scale bar 200 µm. (C, D) The organoid forming efficiency (C) and diameter (D) from sorted GFPhigh stem cells in AhR WT or KO mice treated for 5 d with DMSO or TCDD (10 nM). n=4-5 mice per group in (C). Each dot represents an individual organoid and grey line indicates mean in (D). Bars with different letters are significantly different (p<0.05). Organoid growth from GFPlow progenitor cells. (E) Representative brightfield images of organoids. (F) Organoid forming efficiency and (G) diameter. n=4-8 mice per group in (F). Each dot represents an individual organoid and grey line indicates mean in (G). Scale bar 200 μm. Bars with different letters are significantly different (p<0.05). (H, I) (H) Lgr5 expression in organoids and (I) sorted stem cells in the absence or presence of TCDD treatment (n≥3 independent samples per group). (J) Representative brightfield images for secondary organoid formation pretreated with DMSO or 10 nM TCDD for 2d. Scale 200 µm.
Mutant p53 and YAP share a common transcriptional program.Figure 1A. Over-representation analysis was performed with Gene-Signatures highlighting activation of specific pathways using Gene Set Enrichment Analysis (GSEA) on microarray data obtained from control and mutant-p53 silenced MDA-MB-231 mammary cells. A negative normalized enrichment score (NES; highlighted in blue) indicates signatures that are more active in control cells, i.e., that have been repressed upon the silencing of mutant p53 proteins; a positive NES (highlighted in red) indicates signatures that have been activated by mutant p53 silencing (i.e., signature normally not active in MDA-MB-231). The false discovery rate (FDR) is the estimated probability that a gene set with a given NES represents a false positive; we considered signatures to be significantly enriched at FDR<0.05. Gene expression data has been obtained from n=3 biological replicates for each condition. See Appendix Table S2 for a GSEA analysis including also Biocarta gene sets. B. Upper panel: Venn diagram showing the overlap of functional analyses. Starting from the TP53mutant genes list or YAP/TAZ genes list, the biological process (BP) and molecular functions (MF) were obtained using the ClusterProfiler tool. An overlap of the functional annotations was performed to obtain the list of the common ones. Lower panel: common functional annotations. For the common functional annotations, we represent the enriched score (that is the minus log of the p-value) for both gene lists. In particular colour horizontal bars represent the functional themes that are overrepresented in TP53mutant genes (blue bars) or YAP/TAZ genes list (red bars). C-E. Western blot analysis for CCNA, CCNB, Cdk1 and GAPDH (loading control) protein expression in lysates from control (si-GFP) and p53-depleted (si-p53; si-p53_1) (C) MDA-MB-231 and CAL27 cell lines. Representative images are shown. C-E. Western blot analysis for CCNA, CCNB, Cdk1 and GAPDH (loading control) protein expression in lysates from control (si-GFP) and YAP-depleted (si-YAP; si-YAP_1) (D) MDA-MB-231 and CAL27 cell lines. Representative images are shown. C-E. Western blot analysis for CCNA, CCNB, Cdk1 and GAPDH (loading control) protein expression in lysates from control (si-GFP) and TAZ-depleted (si-TAZ) (E) MDA-MB-231 and CAL27 cell lines. Representative images are shown.
Figure 4A Doughnut chart for RNA-seq analysis showing the differentially regulated alternative splicing events (ASEs) and their corresponding genes in ESRP1 knockdown cells compared to control cells; SE: skipped exon, RI: retained intron, MXE: mutually exclusive exons, A5SS: alternative 5' splice site, and A3SS: alternative 3' splice site). The "percent spliced in" (PSI or ψ) value was estimated. Differences in inclusion levels (∆ψ) between samples and their significance were calculated (P<0.05 and IΔψI>0.1). Circle 1: all ASEs; Circle 2: all genes corresponding to all ASEs; Circle 3: significant ASEs; Circle 4: significant genes corresponding to significantly altered ASEs; Circle 5: significant ASEs with ESRP1 motifs; Circle 6: significant genes corresponding to significantly altered ASEs with ESRP1 motifs. ESRP-binding motifs were obtained from Dittmar et al.31[] and analyzed based on the following assumption: the presence of the motif in the downstream region of an exon results in inclusion of the exon, while upstream or intraexonic location leads to exclusion of the exon. B Radar plot showing that the EMT gene signature was not altered at the gene level in the ESRP1 knockdown model (LCC2 set). C Radar plot showing that the EMT gene signature was altered at the ASEs level (SE) in the ESRP1 knockdown model (LCC2 set). D Radar plot showing that the EMT gene signature was not altered at the gene level in the ESRP1 knockdown model (LCC9 set). E Radar plots showing that the EMT gene signature was altered at the ASEs level (SE) in the ESRP1 knockdown model (LCC9 set).
Figure 8A. A flow chart depicting the in vivo experimental design. B. Alterations of tumor volume in different groups with or without pep-AP OE (n = 6, biological replicates, mean±SEM). Data information: * for P < 0.05; ** for P < 0.01; NS for no significance. Control referred to the corresponding plasmid vector. C. (a) Pictures of the tumors in all groups in B. (b) Further validation of the effect of pep-AP in vivo. D. Weight of the tumors in C(a) (n = 6, mean±SEM, Dunnett-t test). Data information: * for P < 0.05; ** for P < 0.01; NS for no significance. Control referred to the corresponding plasmid vector. E. RT-qPCR quantification of lnc-AP within the tumors in C(a) (n = 6, mean±SEM, Dunnett-t test). Data information: * for P < 0.05; ** for P < 0.01; NS for no significance. Control referred to the corresponding plasmid vector. F WB analysis of pep-AP, TALDO1, PARP, cleaved-PARP and cleaved-caspase-3 within the tumors in C(a). G. Relative NADPH/NADP+ ratio within the tumors in C(a) (n = 6, mean±SEM, Dunnett-t test). H. GSH contents within the tumors in C(a) (n = 6, mean±SEM, Dunnett-t test). Data information: * for P < 0.05; ** for P < 0.01; NS for no significance. Control referred to the corresponding plasmid vector. I. IF detection of pep-AP, TALDO1 and H2AFX, TUNEL of DNA fragmentation and IHC of Ki67 within the tumors in C(a). J. A proposed mechanistic model in this study.
Figure 3Semi-thin cross sections of femoral quadriceps nerves stained with alkaline methylene blue. Note the smaller size of Ccp1-/- and Ccp1-/-Ttll7-/- nerves (A). Semi-thin cross sections of femoral quadriceps nerves stained with alkaline methylene blue. These sections were quantified for (B) number of myelinated axons, (C) nerve area, (D) nerve perimeter (Fig. EV2) of femoral quadriceps nerves from wild-type, Ccp1-/-, Ttll7-/-, Ccp1-/-Ttll7-/-, Ttll1-/- and Ccp1-/-Ttll1-/- mice. Scatter dot plots in which each point represents one nerve of an individual mouse are shown. The black lines indicate means ±SEM. Significance was tested with one-way ANOVA and Holm-Šídák's multiple comparisons test. Note that all three parameters are decreased for Ccp1-/- and Ccp1-/-Ttll7‑/- mice, while Ccp1-/-Ttll1-/- mice showed no difference to the Ttll1-/- control. E,F) Representative electron microscopy images of cross sections of femoral quadriceps nerves. (E) Nerves from Ttll1-/- and Ccp1-/-Ttll1-/- mice show no abnormalities of myelinated axons (green asterisks), indicating that loss of TTLL1 prevents axonal degeneration caused by the absence of CCP1. (F) Ccp1-/- and Ccp1-/-Ttll7-/- mice both show the presence of foamy macrophages (magenta arrowheads), which are signs for myelin phagocytosis during axon degeneration. This indicates that loss of TTLL7 cannot prevent the CCP1-induced degeneration of femoral quadriceps nerves.
Figure 2A. HeLa cells, untreated or treated with SA, were fixed and visualized by fluorescence microscopy using anti-Sec24C Ab, anti-eIF3 Ab, and DAPI (blue). Data information: Scale bars, 10 µm. B. Quantification of Sec24C redistribution to SGs over time after SA treatment (the ratio between Sec24C (mean fluorescence intensity) in SG puncta and cytosolic Sec24C). Mean ± s.e.m. n = 50 cells per experiment, N = 3. C. Quantification of residual Sec24C in ERES after SA treatment. The data are expressed as percentage of steady state values (CTRL). Mean ± s.e.m. n = 100 cells per experiment, N = 3. D. Representative images of TRAPPC2 localization in Sec23AB-KD and Sec24ABCD-KD cells treated with SA. Cells were fixed and visualized by fluorescence microscopy using anti-TRAPPC2 Ab, anti-G3BP Ab (to label SGs) and DAPI (blue). Data information: Scale bars, 10 µm. E. Quantification of TRAPPC2 redistribution to SGs after KD of the indicated Sec23 and Sec24 combinations, calculated as the ratio between TRAPPC2 (mean intensity) in SG puncta and cytosolic TRAPPC2 and expressed as % of CTRL. Mean ± s.e.m., n = 40-60 cells per experiment, N = 3. ns: not significant, One-way ANOVA with Dunnett's multiple comparison test. F. Representative images of Sec24C localization at SGs (stained for G3BP) in TRAPPC3-KD and TRAPPC2-KD cells treated with SA. Depletion of the entire TRAPP complex (via TRAPPC3 depletion) or of only TRAPPC2 reduces Sec24C recruitment. Graph, quantification of Sec24C at SGs, calculated as in (E). Mean ± s.e.m., n = 40-60 cells per experiment, N = 3. ****p< 0.0001, One-way ANOVA with Dunnett's multiple comparison test. Data information: Scale bars, 10 µm. G. Representative images of Sec24C localization at SGs (stained for G3BP) in TRAPPC2-KO cells treated with SA. Data information: Scale bars, 10 µm.