EMBO/sd-character-level-ner huggingface dataset:
Klf2 and Stat3 are required for EpiSC resetting, but not for naïve state maintenance.Figure 6(A) 0.782 cABN predictions of essential or dispensable factors for EpiSC resetting, compared with the experiment results shown in panel (B). (B) siRNA knockdown effects measured by Oct4-GFP+ colony formation at Day 6 of resetting. n=4 independent experiments (C) Left, resetting capacity of Klf2 and Klf4 KO EpiSCs measured by Oct4-GFP+ colony formation at Day 6 of resetting. Data points represent two independent experiments. Right, representative fluorescent and bright field images of wild type and Klf2 KO EpiSCs at Day 6 of resetting in 2i+LIF (D) Expression of naïve pluripotency, transition and somatic lineage markers in wild type and Klf2 KO EpiSCs during a resetting time course in 2i+LIF. Expression is normalised to wild type EpiSCs in A/F, and β-actin was used as internal control. The values shown correspond to the average expression of three technical replicates from one representative experiment out of two (E) Rescue of Klf2 KO EpiSC resetting by forced expression of individual network components measured by Oct4-GFP+ colony formation at Day 6 of resetting in 2i+LIF. Data points represent two independent experiments (F) Comparison between the effect of single factor knockdowns on ESC maintenance (n=4 independent experiments) and EpiSC resetting using experimental results (G) EpiSC resetting in 2i+LIF measured by Oct4-GFP+ colony formation after Stat3 siRNA in wild type EpiSCs (left), or Klf4 KO EpiSCs transfected with Tfcp2l1 and Gbx2 siRNAs. n=3 independent experiments in wild type cells (box plots indicate min, median, max), n=2 for Klf4-/- cells (box plots indicate min, mean, max) (H) EpiSC resetting in 2i+LIF measured by Oct4-GFP+ colony formation of Stat3 knockdown EpiSCs transiently transfected with Tfcp2l1, Gbx2 and Klf4. n=4 independent experiments: Student"s t-test, p-value indicated on plot. Box plots indicate min, median, max. See also Appendix Fi (I) The 0.717 cABN, used to illustrate the kinetics of EpiSC resetting as determined experimentally. Left: Genes coloured according to the order of activation during resetting in 2i+LIF. Right: Genes coloured according to their potency in enhancing the efficiency of resetting. TFs with a green border are the common factors required for ESC self-renewal and EpiSC resetting
Fig.Figure 1(A) Schematic of protein degradation using AID and SMASh systems. The SMASh tag consists of the self-cleaving viral HCV NS3 protease fused in cis to a constitutive destabilising peptide. The tag detaches itself from the target protein in the off condition. Upon inhibition by the protease inhibitor, asunaprevir (Asv), the tag remains on the target protein and exposes it to the cellular degradation machinery, while the old protein is depleted depending on its specific half-life. The AID-tag relies on the expression of the plant F-box protein TIR1 that combines with mammalian Skp1 and Cul1 to form a functional SCF E3 ligase that is activated to ubiquitylate the AID degron tag upon stimulation by the hormone Auxin (IAA). In the case of the double tag combination newly synthesised proteins are depleted by the two independent degradation systems. Old proteins are destabilised by the mAID-tag (mini-AID). (B) Knock-in strategy for inducible OsTIR1 expression at the human Rosa26 locus, and endogenous cyclin A2 and B1 protein tagging. We integrated mAID-SMASh-T2A-neomycin in frame at the C-terminus of the coding sequence of cyclins A2 and B1. The chosen length for the homologous regions for gene targeting was approximately 1kb. (C) To verify gene targeting primers were designed in the genomic locus and in the cassette (indicated in (B)) and used to amplify genomic DNA from different cell lines: wild-type RPE-1, RPE-1 expressing OsTIR1, CycA2dd, CycB1dd, and primers amplifying a region of the Kif23 gene were used as a positive control (D; E) (D) Representative immunoblots and (E) quantitation showing timecourse of induced degradation of double degron tagged cyclin A and cyclin B (A2dd, B1dd). * indicates crossreacting band. (n=3 experiments, s.d. indicated by error bars). Doxycycline/IAA/Asv (DIA was added to asynchronous cells for indicated time before extract preparation and immunoblotting. (F) Comparison of protein degradation efficiency using different degrons. Cells were incubated for indicated time periods with Asv or DOX/IAA or both (DIA, see methods) then collected for western blot analysis. Doxycycline was added 2 hours before addition of IAA or Asv/IAA. To determine protein degradation, either cyclin A2 or cyclin B1 were probed as indicated. Anti-myc antibody was used to check OsTIR1-myc expression. Asterisks indicate non-specific protein crossreacting with the cyclin B1 antibody. (G) Timecourse of DIA induced cyclin A and B degradation, cross checking levels of each mitotic cyclin. Treatments were performed as for DIA treated Cell extracts were checked for both cyclin A2 and B1 levels following DIA addition (H) Comparison of DIA and siRNA depletion. Cells were treated with DIA for four hours or pre-transfected with CycB1 SMARTpool siRNAs 48 hours earlier. Cell extracts were analysed by immunoblotting for cyclin B1 (L.E, long exposure, S.E. short exposure) using mouse monoclonal antibody (I) Assaying cyclin B1 depletion in mitotic cells using a rabbit mono-clonal antibody. Extracts from asychronous or mitotic cells were collected and analysed by immunoblotting using the indicated antibodies.
Figure 5Immunoblots of BTV-NS3-transfected 293T cells treated with proteasome inhibitors MG or LC. Membranes were probed for STAT1, STAT2, BTV-NS3 (FLAG) and GAPDH. (B-D) Immunoblots of BTV-NS3-transfected 293T cells treated with increasing concentration of lysosome acidification inhibitors (B) CQ or (C) NH4Cl or with increasing concentration of the (D) autophagosome formation inhibitor 3-MA, and probed (IB) for STAT1, STAT2, BTV-NS3 (FLAG) and GAPDH expression. 293T cells were co-transfected with NS3 expression plasmid (or empty plasmid as control) and siRNA for Atg7 Lysates were obtained from FACS-sorted transfected cells. Immunoblots of sorted cells were probed for STAT1; STAT2; Atg7 FLAG-tagged NS3; LC3 and GAPDH. 293T cells were co-transfected with NS3 expression plasmid (or empty plasmid as control) and siRNA for Beclin-1 (or siRNA control or no RNA as controls). Lysates were obtained from FACS-sorted transfected cells. Immunoblots of sorted cells were probed for STAT1; STAT2; Beclin-1 FLAG-tagged NS3; LC3 and GAPDH. Representative flow cytometry dot plots of Vero cells infected with rgBTV-8 or BTV-8 ΔNS3 and stained for BTV-VP7. Gating on VP7+ cells was used for sorting to obtain lysates from infected cells. Immunoblot of FACS-sorted Vero cells infected with rgBTV-8 or BTV-8 ΔNS3 and treated with IFN-U for 30min prior to fixation, staining and sorting. Membranes were probed for STAT1, P-STAT1, STAT2 and GAPDH. Immunofluorescence confocal images of Vero cells infected with rgBTV-8, BTV-8 ΔNS3 or mock-infected and treated with IFN-U for 30min prior to fixation and staining. Cells were stained for BTV-VP7 and (I) P-STAT1 or (J) STAT2. Nuclei were counterstained with DAPI. (Scale bar = 30 μm). (I) Arrowhead in merge image indicate partial inhibition of STAT1 phosphorylation in BTV-8 ΔNS3 infected cells. (J) Arrowhead in merge image shows STAT2 translocation to nucleus in BTV-8 ΔNS3-infected cells.
a) HL-1 cells transfected with control or p53-specific siRNAs no.f4(a) HL-1 cells transfected with control or p53-specific siRNAs no.1 and cultured in complete medium for 24 h in the presence or absence of 0.02 μM DOX. After treatment with 20 μM CCCP for 6 h, the fractionation experiment was performed. Endogenous Parkin translocation to mitochondria and ubiquitination were assessed by immunoblotting. Representative immunoblots are shown from three independent experiments. (b) These samples were subjected to immunoprecipitation with the anti-VDAC antibody to evaluate ubiquitination. Representative immunoblots are shown from three independent experiments. (c,d) Representative images of YFP-Parkin overexpressing p53−/− HCT116 cells re-transfected with WT p53 and various mutants of p53 and treated with 60 μM CCCP for 8h (c) and 36 h (d) before the immunostaining of mitochondria with anti-TOM20 (red) and endogenous poly-ubiquitin with a specific antibody FK-2 (white); original magnification, × 630; scale bar, 20 μm. (e-g) Parkin mitochondrial translocation (e) and ubiquitination (f) 8 h after CCCP treatment and mitochondrial clearance (g) 36 h after CCCP treatment were quantified. A minimum of 300-400 YFP-positive cells were scored in three independent experiments. The single-letter amino-acid code is used. NES indicates nuclear export signal; NLS, nuclear localization signal; ER, endoplasmic reticulum; Ub, ubiquitin. Data are shown as the means±s.d. *P0.05, compared with the corresponding control (two-tailed unpaired Student's t-test).
Figure 7(A) Representative AP traces and (B) averaged activity of triggered and spontaneous activity in hiPSC-CMs in the absence of NA. (C) Representative AP traces and (D) averaged activity of triggered and spontaneous activity in hiPSC-CMs in the presence of NA.
Figure 6A Cartoon of the N-terminal region (residues 1-180) of mouse MLKL (PDB accession 4BTF;(Murphy, Czabotar et al. 2013)) showing the four lysine residues identified from MS analysis as yellow sticks. B WT and 4KR mutant MLKL were inducibly expressed in Mlkl-/- MDFs by doxycycline for 6 hrs and cells were untreated (UT) or treated with TSI, followed by UBA-pull down. Representative of three independent experiments.
Figure 4Boxplots of 5´UTR (A), 3´UTR (B) lengths for mRNAs whose abundance decreased ('DOWN', dark green) or increased ('UP', light green) in PABP-depleted cells as compared to cells expressing PABP. Solid horizonal middle lines in box plots denote the median; lower and upper box limits correspond to first and third quartiles; whiskers extend from box limits to the most extreme values. Wilcoxon-Mann-Whitney tests (two-sided) were used to determine differences between upregulated mRNAs and downregulated mRNAs relative to each other and to the background (mRNAs whose abundance did not change in the absence of PABP relative to PABP-expressing cells). Boxplots of poly(A) tail lengths for mRNAs whose abundance decreased ('DOWN', dark green) or increased ('UP', light green) in PABP-depleted cells as compared to cells expressing PABP. Solid horizonal middle lines in box plots denote the median; lower and upper box limits correspond to first and third quartiles; whiskers extend from box limits to the most extreme values. Wilcoxon-Mann-Whitney tests (two-sided) were used to determine differences between upregulated mRNAs and downregulated mRNAs relative to each other and to the background (mRNAs whose abundance did not change in the absence of PABP relative to PABP-expressing cells). (D and E) Comparison of log2-fold change in transcript abundances within PABP-depleted cells relative to cells expressing PABP versus their reported P-body log2-fold enrichment status (Hubstenberger et al., 2017). The Pearson's correlation coefficient (R=0.73) and the corresponding p-value (p<2.2e-16) are reported with a corresponding trendline (black) and 95% confidence interval (gray). (D) Points in dark green indicate mRNAs that are downregulated in PABP-depleted cells and depleted from P-bodies. Points in light green indicate mRNAs that are upregulated in PABP-depleted cells and enriched in P-bodies. Transcript numbers in each quadrant are listed. (E) Nuclear-encoded mRNAs encoding cytoplasmic ribosomes (orange), mitochondrial ribosomes (blue) and proteins with mitochondrial functions (green) that are downregulated in PABP-depleted cells relative to PABP-expressing cells and are predominantly depleted from P-bodies. Proteins with mitochondrial functions were defined based on an inventory of human mitochondrial proteins (Calvo et al, 2016).
a) Inhibition of ROCK1 kinase activity prevents metabolic stress-mediated phosphorylation of Beclin1.f4(a) Inhibition of ROCK1 kinase activity prevents metabolic stress-mediated phosphorylation of Beclin1. HeLa cells left untreated or treated with a ROCK1 inhibitor, Y27632 (10 μM), were starved of glucose (4 h) as indicated, and endogenous Beclin1 was immunoprecipitated and blotted against phospho-Ser/Thr antibody. Whole cell lysates were run as inputs for Beclin1 and β-actin. (b) Phosphorylation of Beclin1 by ROCK1. Recombinant ROCK1 was used for in vitro kinase assay in the presence or absence of Y27632, using recombinant His-Beclin1 (left) or Flag-Beclin1 immunopurified from transfected HeLa cells (right). Proteins were resolved by SDS-PAGE; phosphorylated protein was visualized with autoradiography, and Beclin1 by Coomassie staining. (c) Identification of ROCK1-mediated phosphorylation domains in Beclin1. The indicated Flag-Beclin1 fragments were purified from HeLa cells and used as substrates for in vitro ROCK1 kinase assay. 32P-autoradiogram (centre) analysed phosphorylation and western blotting (WB; right) determined protein levels. Schematic representation of Beclin1 domain structure and deletion constructs are shown (left). (d) Identification of the phosphorylation site on Beclin1. Representation of point mutations in the different domains of Beclin1 are shown (left). In vitro kinase assay using Flag-Beclin1 WT and mutants (T38A and T119A), immunoprecipitated from transfected HeLa cells, as substrate and recombinant ROCK1 was performed. Phosphorylation was detected by 32P-autoradiogram, and Flag-Beclin1 levels were examined by WB. (e) Flag-Beclin1 Wt or T119A transfected HeLa cells were incubated in glucose-rich or nutrient-free (HBSS) media, in the presence or absence of ROCK1 inhibitor Y27632. Total cell extracts were used for IP using Flag agarose. Eluted protein was analysed by WB against phospho-T119 (Beclin) antibody and Beclin1. Input for Beclin1 was run on 7.5% gel and immunoblotted.
Upregulation of mTOR signalling in TAM-MG but not TAM-BMDM in GBM mouse models.Figure 1(A) Representative images of a PDGFB tumour: H&E on the right to identify the tumour, an adjacent section stained for Iba1 (red), p-S6 (green) and DAPI (blue) at the centre and the region selection for quantification on the left (non-tumour in light blue, tumour edge in orange and tumour core in dark blue). (B) Representative images of core, edge and adjacent non-tumour brain tissue for Iba1, p-S6 and DAPI staining in Pten-/-p53-/- (right) and GL261 (left) tumours. Percentage of Iba1+ cells co-expressing p-S6 in the three defined regions. Five high grade glioma models were analysed - GL261 (n=3), PDGFB (n=3), Ntv-a;PDGFB+shp53 (n=2), Pten-/-p53-/- (n=3), and Pten-/-p53-/-Idh1mut (n=3) (mean ±SEM; Two-Way ANOVA Tukey test). (C) Venn diagram identifying significantly deregulated genes in healthy versus GL261 TAM-MG and/or TAM-BMDM (GSE68376 dataset). (D) Top-most deregulated canonical pathways in GL261 TAM-MG, as identified by the IPA software. Threshold indicates p≤0.05. Z-score indicates the orientation of the deregulation. Ratio indicates the number of deregulated genes in the pathway. (E) mTOR-related deregulated canonical pathways in GL261 TAM-MG and TAM-BMDM, as identified by the IPA software. (F,G) Expression levels of (F) Rps6 and (G) Eif4ebp1 in healthy versus GL261 microglia and macrophage (n=3; mean ±SEM, likelihood ratio test in edgeR).
Figure 7Phospho-site clusters in the carboxyl-terminus of V2R and AT1aR are underlined and color-coded to reflect the PXPXXP and PXXPXXP patterns as proposed in a previous study (Zhou et al., 2017). Two different AT1aR mutants were generated by site-directed mutagenesis to mimic the spatial distribution of Ser/Thr as in V2R with respect to double-Thr cluster. The red dotted box highlights the resulting sequence upon either the delition of Leu337 or Leu337Thr mutation. HEK-293 cells expressing the indicated AT1aR constructs, in absence (CTL; control) or presence of βarr1 knock-down (βarr1-KD) were stimulated with indicated concentrations of angiotensin II (AngII) for 10 min. Subsequently, ERK1/2 phosphorylation was measured by Western blotting and signal intensities were quantified using densitometry and presented as bar graphs. Data were normalized with respect to the signal at 100nM agonist concentration in control cells (treated as 100%) and represent mean±SEM of five independent experiments. Data were analyzed using Two-way-ANOVA with Bonferroni multiple comparisons test (***p<0.001, p<0.05). HEK-293 cells expressing the indicated AT1aR constructs, in absence (CTL; control) or presence of βarr1 knock-down (βarr1-KD) were stimulated with indicated concentrations of angiotensin II (AngII) for 10 min. Subsequently, ERK1/2 phosphorylation was measured by Western blotting and signal intensities were quantified using densitometry and presented as bar graphs. Data were normalized with respect to the signal at 100nM agonist concentration in control cells (treated as 100%) and represent mean±SEM of five (six for AT1aRΔL337) independent experiments. Data were analyzed using Two-way-ANOVA with Bonferroni multiple comparisons test (***p<0.001, p<0.05).
Figure 6Dots indicate rate-limiting molecular events; arrows indicate connections between these events. Both antibiotics, NIT and TMP, induce DNA stress, reported here by the recA promoter. For NIT, oxidative stress (reported here by the ybjC promoter) is likely upstream in the same chain of molecular events as DNA stress. Other chains of molecular events (gray) that may run in parallel were not probed here. For TMP, DNA stress is preceded by fewer rate-limiting events compared to NIT, and it is not in the same chain as acid stress (reported here by the gadB promoter); both are generated by different upstream molecular events that were not probed here.
SDS AGE analysis of RHIM-based assemblies.Figure 6SDS AGE analysis of monomeric or assembled forms of YPet-RIPK3387‑518 and mCHERRY-M451-90WT or mCHERRY-M451-90AAAA. Monomeric forms of the proteins maintained in 8 M urea prior to electrophoresis, as indicated. Assembled samples incubated with 0 or 2% SDS at RT for 10 min before electrophoresis. Protein components as indicated above each lane. SDS AGE analysis of assemblies of mCHERRY-RIPK1497‑583 and YPet-RIPK3387‑518 with or without four times molar equivalents of His-Ub-M45. SDS AGE analysis of assemblies of YPet-RIPK3387‑518 and mCHERRY-ZBP1170-355 with or without four times molar equivalents of His-Ub-M45.
Figure 4(A) The effect of Beclin 1 Ser90/93 phosphorylation on the interaction between Beclin 1 and Bcl-2. GST-Beclin1 WT, AA mutant and DD mutant (bait) were incubated with or without recombinant CHK2 in the presence of ATP and then incubated with flag-Bcl-2 (preys). (B) Immunoprecipitation of Bcl-2 with Flag-Beclin 1 WT, AA mutant and DD mutant in HCT116 cells in normal medium or after H2O2 (500μM) treatment for 1h. (C-D) Autophagy levels were determined in Beclin 1-depleted H1299 cells with reconstituted expression of Beclin 1 WT, AA mutant or DD mutant in normal medium or after H2O2 (500μM) treatment for 3h in the presence (C) or absence (D) of CHK2. (E) Representative electron microscopic image of an autophagosome (arrow, left panel, AP) and an autolysosome (arrow, right panel, AL) in H1299 Beclin 1 WT cells treated with H2O2 (500μM) for 6 hr. Scale bars, 500 nm. Electron microscopic quantification of autophagosomes (AP) and autolysosomes (AL) in H1299 cells with the indicated plasmids cultured for 6 hr in normal or H2O2 (500μM) treatment. All quantitative data are presented as mean ± s.e.m. from 3 independent experiments; *P < 0.001 compared to Beclin 1 WT; #P < 0.001 compared to Beclin 1 WT treated with H2O2; &P < 0.001 compared to Beclin 1 WT treated with H2O2 in the absence of CHK2. (F) Western blot detection of p-CHK2 Thr68, CHK2, p-Beclin 1 Ser90, p-Beclin 1 Ser93, Beclin 1, p62 and LC3 in H1299 cells transfected with the indicated shRNA in normal medium or after H2O2 (500μM) treatment for 3h.
Figure 3(A) Quantification of body weight change in control (n=12) and cKO (n=13) mice after DSS treatment and DSS removal. (B) Fecal occult blood test in control and cKO mice at indicated timepoints. n=3 at each timepoint. (C) Histological images of colonic tissues from control (n=10) and cKO (n=10) mice at indicated timepoints after DSS treatment. The Panels with non DSS are identical to Panels in Fig EV1F. Scale bar: 100 μm. (D) Quantification of the clinical scores in control (n=12) and cKO (n=13) mice at indicated timepoints. (E) Gross images of colons and quantification of colon length from control (n=6) and cKO (n=6) mice two days after 5-day DSS treatment. (F) Double immunofluorescence for Ki67 and β-catenin in colons from control (n=4) and cKO (n=4) mice two days after 5-day DSS treatment. Quantification of percentage of Ki67+ epithelial cells. Scale bar: 100 m.
endocytic trafficking.Figure 2N2a cells treated with siBin1, siCD2AP or siControl.(A) Scheme illustrating APP endocytosis trafficking assayed in N2a cells transiently expressing APP-RFP using a pulse/chase assay with anti-N-terminal APP (22C11). A 10 min pulse to assay APP endocytosis (b) followed by a 60 min chase to assay the degradation of endocytosed APP (d). N2a cells treated with siBin1, siCD2AP or siControl.(B) Endocytosed APP detected after a 10 min pulse with 22C11 and APP-RFP (insets), analysed by epifluorescence microscopy. Scale bars, 10 µm.(C) The amount of endocytosed APP fluorescence at 10 min was quantified and normalized to APP-RFP fluorescence (n=3, NsiControl=56, NsiBin1=70, NsiCD2AP =42; mean ± SEM).(D) Non-degraded APP detected after 10 min pulse and 60 min chase with 22C11 and APP-RFP (insets), analysed by epifluorescence microscopy. Cells are outlined in white. Scale bars, 10 µm.(E) APP degradation was assessed by the decrease in the amount of endocytosed APP fluorescence at 60 min relative to time 0 (10 min pulse) in siControl cells normalized to APP-RFP fluorescence (n60min=3, NsiControl=68, NsiBin1=43, NsiCD2AP=64; ****PAPP60min<0.0001, t-test, mean ± SEM). (F) Scheme illustrating BACE1 endocytic trafficking assayed in N2a cells transiently expressing BACE1-GFP N-terminally tagged with FLAG using a pulse chase assay with anti-FLAG antibody (M1). A 5 min pulse to assay BACE1 endocytosis (g), a 10 min pulse and 20 min chase to assay BACE1 recycling to the plasma membrane (i) and the pool of endocytosed BACE1 that did not recycle (k). N2a cells treated with siBin1, siCD2AP or siControl.(G) Endocytosed BACE1 detected upon 5 min pulse with M1 and BACE1-GFP (insets), analysed by epifluorescence microscopy. Scale bars, 10 µm.(H) The amount of endocytosed BACE1 per cell was quantified as percentage of siControl normalized to BACE1-GFP (n=3, NsiControl=86, NsiBin1=72, NsiCD2AP=113; mean ± SEM).(I) Recycled BACE1 detected at the plasma membrane of non-permeabilized cells with M1, upon a 10 min pulse, acid stripping and 20 min chase, and BACE1-GFP (insets), analysed by epifluorescence microscopy. Scale bars, 10 µm.(J) The amount of recycled BACE1 was quantified as in (h) (n=3, NsiControl=94, NsiBin1=58, NsiCD2AP=109; ****P<0.0001 siBin1 vs. siControl, t-test, mean ± SEM).(K) Non-recycled BACE1 detected in acid-stripped permeabilized cells pulse-chased as in (i) with M1 and BACE1-GFP (insets), analysed by epifluorescence microscopy. Scale bars, 10 µm.(L) The amount of non-recycled BACE1 was quantified as in (h) (n=3, NsiControl=61, NsiBin1=51; ****P<0.0001 siBin1 vs. siControl, t-test; n=2, NsiCD2AP=37; mean ± SEM).