EMBO/sd-character-level-ner huggingface dataset:
PPP downregulates Akt signalling A) Western blot showing the levels of pAKT (Ser473), total Akt, pErk1/2 (T202/Y204) and total Erk1/2 after treatment with 1 µM PPP or 50 ng/ml IGF1 for 60 min.Figure 3Western blot showing the levels of pAKT (Ser473), total Akt, pErk1/2 (T202/Y204) and total Erk1/2 after treatment with 1 µM PPP or 50 ng/ml IGF1 for 60 min. Protein was normalised to GAPDH. Western blot quantification. PMN treated with PPP showed a significant decrease in Akt activation (* P=0.049, N=5 independent experiments, Student's T-test), but no significant difference was seen on Erk1/2 activation (P=0.07, Student's T-test, N=5 independent experiments). 50 ng/ml IGF1 treated neurons showed a significant increase in Akt activation (** P=0.008, Student's T-test, N=3 independent experiments). In contrast, no change in Erk1/2 activation (P=0.88, Student's T-test, N=3 independent experiments) was observed (data shown are mean ± SEM). Graph shows the average velocity of HcT-containing organelles treated with Capivasertib or Ipatasertib (10 nM) for 30 min compared to controls (DMSO: 483 endosomes, 34 axons, 41,897 movements; Capivasertib: 658 endosomes, 43 axons, 49,841 movements; Ipatasertib: 605 endosomes, 32 axons, 41,485 movements) (** P=2 x10-16, One-way ANOVA, Tukey's post-hoc test: DMSO-Capivasertib and DMSO-Ipatasertib *** P <0.0001; N=6 independent experiments, boxplot shows median, first and third quartiles. Upper/lower whiskers extend to 1.5 * the interquartile range). Percent of time HcT-containing organelles pausing per axon. Treatment with Ipatasertib and Capivasertib reduced the amount of time organelles spent pausing (* P=1.14 x10-7, One-way ANOVA, Tukey's post-hoc test: DMSO-Capivasertib P= 9.37x10-5, DMSO-Ipatasertib P= 2 x10-7; data shown are mean ± SEM, N=6 independent experiments).
Figure 1A Schematic diagram of sgRNA targeting mouse Mettl5 locus. sgRNA targeting site is highlighted in red. PAM sequence is underlined and highlighted in green. B Western blot analysis confirming two Mettl5 KO clones by specific METTL5 antibody. α-TUBULIN was used as a loading control. C LC-MS/MS analysis of m6A/A levels in purified 18S rRNA and 28S rRNA from the WT and Mettl5 KO (KO-6B) mESCs. D PAGE gel electrophoresis showing the PCR amplification of the elongated and ligated products of SELECT method for detecting m6A1832 site and A1825 site (for input control) in 18S rRNA of WT and Mettl5 KO (KO-6B) mESCs, respectively. E LC-MS/MS analysis of d3-m6A/A levels in 18S rRNA oligos after being incubated with recombinant WT or catalytically mutant mouse METTL5 proteins in vitro. No enzyme group was used as a negative control.
Figure 7Identification of LRPPRC as a novel ASS1 interacting protein. Cell extracts prepared from SW-1116 cells expressing Flag-ASS1, Flag-ASS1/LOC113230 300-800 or empty vector were incubated with M2 beads and the co-eluted proteins were resolved on SDS-PAGE gel and detected by silver staining assays. The specific band was collected and the potential proteins were identified by Mass spectrometry. RIP assays showed association of LOC113230 with LRPPRC in SW-1116 cells. Antibodies against GAPDH and control IgG served as controls. LOC113230 strengthens the interaction between LRPPRC and ASS1. HA-LRPPRC, Flag-ASS1, together with LOC113230 were co-expressed in SW-1116 cells, and co-IP assays were performed with α-Flag antibody. The co-eluted proteins were detected by western blot assays with Flag, HA antibodies, respectively. The endogenous interaction between LOC113230, ASS1 and LRPPRC was detected in SW-1116 cells by co-IP assays. The plasmids encoding HA-TRAF2, HA-TRAF3, Flag-TRAF6, Flag-ASS1 or LOC113230 were co-transfected into SW-1116 cells and western blot assays were performed using antibodies against ASS1, HA, Flag or GAPDH. in vitro binding assays showed the binding activity of TRAF2 and LRPPRC. GST-TRAF2 and His-LRPPRC proteins were prepared from E. coli BL21 cells. The interaction of endogenous TRAF2, ASS1 and LRPPRC was detected in SW-1116 cells by co-IP assays. GST-TRAF2, HA-LRPPRC, His-ASS1 and Biotin-LOC113230 were expressed in E. coli BL21 cells, and were used for in vitro binding assays. SW-1116-LOC113230 cells transiently transfected with siRNAs to LRPPRC or TRAF2 or scrambled control (SiRNA-NC) were treated with MG132 (5 µM) for 12 hrs. Cell lysates were immunoprecipitated with α-Flag antibody. The precipitate proteins and input proteins were analyzed by immunoblotting. NC: Negative control. Average urea production from each group was determined by a commercially available urea assay kit. The model describing the role of LOC113230 as a tumor suppressor by degrading ASS1 and inhibiting arginine synthesis in CRC cells.
Monomerising mutations de-repress FICD's AMPylation activity.Figure 2A Monomerising FICD mutations inhibit deAMPylation. Shown is a representative plot of data points and fit curves of the time-dependent deAMPylation of a fluorescent BiPV461F-AMPFAM by the indicated FICD proteins (at 7.5 µM) as detected by a change in fluorescence polarisation (FP). DeAMPylation rates calculated from independent experiments B-C Dimer interface mutants both AMPylate and deAMPylate BiP. Shown are representative autoradiographs of thin layer chromatography (TLC) plates revealing AMP produced from reactions containing [α-32P]-ATP and the indicated FICD enzymes in the presence or absence of the co-substrate BiP (arrow indicates direction of nucleotide migration). The radioactive signals were quantified and the AMP signals were normalised to the total nucleotide signal in each sample. Plotted below are mean values ± SD from at least three independent experiments. Unpaired t-tests were performed. D Cartoon depicting sequestration of AMPylated BiP by a covalently linked, disulphide-stapled, S-SFICDA252C-H363A-C421S dimer (trap). E Detection of the time-dependent accumulation of AMPylated BiPT229A-V461F in radioactive reactions, containing [α-32P]-ATP and the indicated FICD proteins, in the presence of excess trap. At the specified time-points samples were taken and analysed by SDS-PAGE. The autoradiograph (32P) illustrates the radioactive signals, which represent AMPylated BiP. Proteins were visualised by Coomassie staining. The radioactive signals were quantified and presented in the graph below. Mean values ± SD of three independent experiments are shown.
Figure 3A, HCT116 cells were treated with 100 nM Thapsigargin for the indicated times in the presence or absence of shRNA targeting USP22. Cells were harvested and whole-cell lysate was subjected to immunoblotting with the indicated antibodies. B, Cells were treated as in A, but over an extended series of time points. RNA was isolated and subjected to qRT-PCR against the indicated transcripts. Three independent experiments are represented as mean ± SEM. C, A Tetracycline (Tet)-inducible, shRNA-resistant USP22 transgene was stably introduced into HCT116 cells. Cells were treated as in A, with and without expression of the transgene via Tet treatment. Whole-cell lysates were subjected to immunoblotting with the indicated antibodies. D, RNA was isolated from conditions described in C subjected to qRT-PCR analysis against the CHOP mRNA. Three independent experiments are represented as mean ± SEM, with significance measured by Student's t-test. ***p<0.005.
R-III reduces CCl4-induced liver fibrosis.Figure 5Hydroxyproline content in the livers from control and CCl4-, CCl4/albumin-, and CCl4/R-III-treated mice (μg/g liver). *P = 0.037, two-sample t-test (n = 10) (CCl4 + R-III compared to CCl4-treated mice). Western blot analysis on liver extracts from the treated mice with antibody against type I collagen. Immunohistochemical analysis of α-SMA on representative liver sections from the treated mice. Scale bar, 200 μm.
Figure 3A). Recombinant human LIF inhibited growth of BAE cells in a dose-dependent manner. BAE cells were cultured in the presence of vehicle and indicated concentrations of recombinant human LIF (rhLIF). Cell proliferation was analyzed after 6 days, n=3. Data information: Bars and error bars represent mean±SD. All experiments were carried out in three independent studies. siNegative, negative control siRNA not targeting any known genes. Two-way ANOVA was used as statistical test. B). JAK inhibitor baricitinib blocked activation of STAT3 by LIF. BAE cells pre-incubated with DMSO and inhibitors for 1 hour were treated with vehicle and LIF (10 ng/ml) for 15 minutes. Whole-cell lysates were subjected to Western blotting with indicated antibodies. Ctrl, no pre-incubation with inhibitors; Ba, baricitinib (2 μM); Co, cobimetinib (150 nM); BE, BEZ235 (5 nM). C). The JAK inhibitor baricitinib reversed LIF-induced BAE growth inhibition. BAE cells pre-incubated with inhibitors for 1 hour were treated with vehicle and LIF (10 ng/ml, Sigma). Cell proliferation was analyzed after 6 days using alamar blue, n=3. Data information: Bars and error bars represent mean±SD. All experiments were carried out in three independent studies. siNegative, negative control siRNA not targeting any known genes. Two-way ANOVA was used as statistical test. D) Knockdown of STAT3 in BAE cells. BAE cells were transfected with siRNAs targeting STAT3. qRT-PCR was performed to examine STAT3 mRNA levels. STAT3 level in siNegative was set as 1. Data from three independent experiments were averaged and shown Data information: Bars and error bars represent mean±SD. All experiments were carried out in three independent studies. siNegative, negative control siRNA not targeting any known genes. Two-way ANOVA was used as statistical test. E). Knockdown of STAT3 in BAE cells. BAE cells were transfected with siRNAs targeting STAT3. In E, cells transfected with siRNAs were treated with LIF (10 ng/ml, Sigma) and vehicle for 15 minutes. Whole-cell lysates were subjected to Western blotting with indicated antibodies. F). LIF-induced BAE cell growth inhibition was abolished by knockdown of STAT3. BAE cells with STAT3 knockdown were cultured with LIF (10 ng/ml, Sigma) and vehicle. Cell proliferation was analyzed after 3 days. Fluorescence reading for each vehicle group was set as 1, n=3. Data information: Bars and error bars represent mean±SD. All experiments were carried out in three independent studies. siNegative, negative control siRNA not targeting any known genes. Two-way ANOVA was used as statistical test.
C medium removes epigenetic barriers during reprogramming (A-B) 5C and mES medium were used during reprogramming.Figure 2(A-B) 5C and mES medium were used during reprogramming. Representative alkaline phosphatase (AP) staining on day 15 was provided in (A). AP+Oct4GFP- and AP+Oct4GFP+ colonies were counted on day 15 (B). (C) Thirty-two epigenetic factors were selected because they had higher expression in 5C-Oct4GFP- than in mES-Oct4GFP-. Their expression in ESCs, iPSCs, and pre-iPSCs in two previous reported assay (GSE14012 and GSE10871) was also listed. (D) The expression of Bmi1, Ctcf, Ezh2, Kdm2b, or Wdr5 was modulated with overexpression or sh-RNA-mediated knockdown via a retrovirus system during reprogramming. AP+Oct4GFP- and AP+Oct4GFP+ colonies were counted on day 15. During reprogramming with mES medium, Bmi1, Ctcf, Ezh2, Kdm2b, and Wdr5 were overexpressed simultaneously with the four Yamanaka factors (OKMS+5F) or Oct4 (O+5F). All factors were delivered simultaneously via a retrovirus system on day 0 and 1. Reprogramming with only Yamanaka factors (OKMS) or Oct4 (O) served as control. The expression of pluripotency markers was determined with qPCR on day 6 during reprogramming (E). During reprogramming with mES medium, Bmi1, Ctcf, Ezh2, Kdm2b, and Wdr5 were overexpressed simultaneously with the four Yamanaka factors (OKMS+5F) or Oct4 (O+5F). All factors were delivered simultaneously via a retrovirus system on day 0 and 1. Reprogramming with only Yamanaka factors (OKMS) or Oct4 (O) served as control. The numbers of Oct4GFP+ colonies were determined on day 15 (F). During reprogramming with mES medium, Bmi1, Ctcf, Ezh2, Kdm2b, and Wdr5 were overexpressed simultaneously with the four Yamanaka factors (OKMS+5F) or Oct4 (O+5F). All factors were delivered simultaneously via a retrovirus system on day 0 and 1. Reprogramming with only Yamanaka factors (OKMS) or Oct4 (O) served as control. The histone methylation on core pluripotency loci was determined on day 6 with ChIP-qPCR (G).
a, b, Fetal liver chimaeric mice were given 5% DSS in drinking water for 7 days.figf4a, b, Fetal liver chimaeric mice were given 5% DSS in drinking water for 7 days. The survival (a) and weight loss (b) of each mouse genotype were plotted. The results shown are mean ± s.d. Statistical significance was determined by the Students t-test. c, Typical distal colon appearance 6  days after the initiation of DSS administration. The results shown are mean ± s.d. (n = 3, each group). d, Expression levels of IL-1b and IL-18 in serum (n55, each group). e, f, Atg16L1-deficient chimaeric mice given 5% DSS in drinking water were intraperitoneally injected with both anti-IL-1b and anti- IL-18 neutralizing antibodies (squares; n55) or isotype control IgG (circles;n55) at days 1, 3, 5 and 7. The survival (e) and weight loss (f) of each mouse genotype were plotted.
Figure 1A. Histograms of spindle angle of GFP-H2B U2OS cells transfected with either GAPDH or Spindly siRNA and filmed on rectangular micropatterns. Cells were filmed 48 hours post-transfection. B. Mitotic timing of control-depleted U2OS cells or CLIP-170-depleted cells. CLIP-170-depeleted cells are split in two categories; 'aligned' and 'misaligned', relating to the observed chromosome misalignment phenotype C. The histograms of spindle angles for the two different categories of CLIP-170-depleted U2OS cells (Left graph with alignment defect, right graph without alignment defect). D and E. Mitotic timing and spindle angle histograms of control (GAPDH)-depleted U2OS cells and CENP-E-depleted cells.
Bruton's tyrosine kinase mediates TLR9-dependent calcineurin activation.Figure 8A, B J774A.1 macrophages were pre-treated with the BTK inhibitor LFM-A13 (12.5 μM) and infected with swollen conidia. (A) After overnight stimulation, TNF-α was measured in the SN by ELISA. A, B J774A.1 macrophages were pre-treated with the BTK inhibitor LFM-A13 (12.5 μM) and infected with swollen conidia. (B) After 30 min of stimulation, NFATc2 translocation was quantified by confocal microscopy. Both TNF-α levels and NFATc2 translocation were significantly reduced by BTK blockade. C J774A.1 macrophages were treated with a control or BTK-targeting siRNA (25 nM), and NFAT translocation was quantified by confocal microscopy. BTK knock-down led to a significant reduction in NFATc2 translocation in response to AF. D, E J774A.1 macrophages were treated with a control, BTK (25 nM)- or TLR9 (75 nM)-targeting siRNA and additionally pre-treated with either ODN2088 (10 μM) or the BTK inhibitor LFM-A13 (12.5 μM). The cells were infected with swollen conidia (MOI = 5), and NFATc2 translocation was measured by confocal microscopy. Blocking both TLR9 and BTK signalling had no additive effect on NFAT translocation blockade.
a, Etoposide-treated Atg5-/- MEFs were assessed by EM (left) and by Lamp2 immunofluorescence (IF; middle).figf1a, Etoposide-treated Atg5 -/- MEFs were assessed by EM (left) and by Lamp2immunofluorescence (IF; middle). Right: reverse Lamp2 immunofluorescence image merged with the electron microscopy image. Arrows indicate Lamp2-positive autolysosomes. Magnified photos are provided in Supplementary Fig. 5 b, The size of each autophagic vacuole (AV) (n = 30 cells) in MEFs treated with etoposide. The total number of AVs in 30 cells was 483 in WT cells and 494 in Atg5-/- cells. c, Percentage autophagic area (n ≥ 20 cells) in MEFs treated with etoposide. d, e, Representative macroautophagy in etoposide-treated Atg5-/- MEFs. Conventional (d) and quick freezing and freeze-substitution (e) techniques were used. The autolysosomes (arrows) contain multilamellar bodies (arrowheads). f, The number of autophagosomes (white columns) and autolysosomes (black columns) in MEFs (n = 10 cells) treated with etoposide. g, Reduction of percentage autophagic area by 3-methyladenine (3-MA; n = 22 cells). . h, Time-course analysis of long-lived protein degradation (n = 4). i, Inhibition of long-lived protein degradation by bafilomycin A1, protease inhibitor (PI) cocktail and 3-MA (*P 0.05; n = 4). j, k, Similar experiments to those in c and i were performed by starvation. Red and blue lines in b, c, g and j indicate means and s.e.m., respectively. The dashed line in c, g and j indicates the autophagic cell border (6%). Black error bars indicate s.d.
Figure 4A. Schema of the experimental paradigm used for drug administration. Mice were injected daily with THC (5 mg/kg) or vehicle (Veh) during adolescence, from PND 30 to 45. SB258585 (SB, 2.5 mg/kg) or rapamycin (Rapa, 1.5 mg/kg) were administered concomitantly with THC or vehicle. Electrophysiological recordings were performed from PND 60. B. Left: representative traces of GABA mIPSCs recorded in layer V pyramidal neurons are illustrated. Right: the histograms represent means ± S.E.M. of GABA mIPSC frequency and amplitude measured during the last minute of recording. The mIPSC frequency is: 3.2 ± 0.4 Hz and 1.3 ± 0.2 Hz for Vehicle (n=9 from N= 4) and THC (n=7 from N= 4) conditions, respectively, p < 0.001 and 2.5 ± 0.2 Hz and 4.1 ± 0.4 Hz for THC+SB258585 (n=8 from N= 4) and THC+Rapa (n=8 from N= 4) conditions, respectively, p < 0.01 and p < 0.001 vs. THC-injected mice. C. Left: representative traces of AMPA mEPSCs recorded in layer V pyramidal neurons are illustrated. Right: the histograms represent means ± S.E.M. of AMPA mEPSC frequency and amplitude measured during the last minute of recording, n = neurons from 3 to 5 mice per group. The mEPSC frequency is: 4.7 ± 0.4 Hz for Vehicle (n=9 from N= 7) and 6.8 ± 0.4 Hz for THC (n=9 from N= 5) conditions, respectively, p < 0.01and 5.3 ± 0.5 Hz and 4.6 ± 0,3 Hz for THC+SB258585 (n=10 from N= 6) and THC+Rapa (n=9 from N= 5) conditions, respectively, p < 0.05 and p < 0.01 vs. THC-injected mice. In B and C, * p < 0.05; ** p < 0.01; *** p < 0.001, one-way ANOVA followed by Newman-Keuls test.
Figure 3A. Schematic representation of the domain structures of the two deletion mutants, Kap114p (Δ347-371) and Kap114p (Δ899-956). B. Pull-down assays of yTBP with Kap114p, Kap114p (Δ347-371) and Kap114p (Δ899-956). GST-fused yTBP (61-240) was incubated with recombinant Kap114p, Kap114p (Δ347-371) and Kap114p (Δ899-956). Unbound (S) and bound (B) samples were analyzed by SDS-PAGE and stained with Coomassie blue. C. Band intensities of Kap-βs from the SDS-PAGE gels in (B) were quantified and normalized by GST-yTBP intensity. Quantitative plots show percent Kap-βs binding on yTBP. D, E. ITC titration curves (upper) and binding isotherms (lower) of (D) Kap114p (Δ347-371) and (E) Kap114p (Δ899-956) with yTBP (61-240). Salt concentration and Kd values are indicated. F. Relative fluorescence GFP intensities in the nucleus and cytoplasm of wild type, KAP114 knockout strains, and KAP114 knockout strains rescued by KAP114 deletion mutants. G. His-tagged RanQ69L was incubated with recombinant Kap114p, Kap114p (Δ347-371) and Kap114p (Δ899-956). Unbound (S) and bound (B) samples were analyzed by SDS-PAGE and stained with Coomassie blue. H. Band intensities of Kap-βs from the SDS-PAGE gels in (G) were quantified and normalized by His-Ran intensity. Quantitative plots show percent Kap-βs binding on His-Ran. I. GST pull-down assays of yTBP (61-240) with Kap114p or Kap114p (Δ347-371) in the presence or absence of RanQ69L. GST-fused yTBP (61-240) was incubated with recombinant Kap114p and His-tagged RanQ69L at a molar ratio of 1:4:8 Kap114p:yTBP:RanQ69L. Unbound (S) and bound (B) samples were analyzed by SDS-PAGE and stained with Coomassie blue. J. Analysis of Kap114p dissociation from yTBP in the presence of RanQ69L. Band intensities of Kap114p and Kap114p (Δ347-371) from the SDS-PAGE gels were used to determine the average fraction of bound protein.
Figure 5HER-2-E0771 mammary tumour cells (2x105) were injected into the fourth inguinal mammary fat pads of female HER-2 TG mice. Six days after tumour injection HER-2 TG mice received total body irradiation (4 Gy) followed by the adoptive transfer of 6x106 FACS-purified Lck-Cre;Ptpn2fl/fl CD8+CD44hiCD62Lhi central memory HER-2 CAR-T cells. Mice were injected with IL-2 (50,000 IU/day) on days 0-4 after adoptive CAR-T cell transfer and monitored for survival. HER-2-E0771 tumours at day 10 post adoptive CAR-T cell transfer or from HER-2-E0771 tumours that had re-emerged after being cleared by Lck-Cre;Ptpn2fl/fl CD8+ HER-2 CAR-T cells were analyzed for HER-2 expression by immunohistochemistry. Scale bars: 200 μm (full size) and 70 μm (zoom). Normal mammary tissue or HER-2-E0771 tumours at day 10 post-adoptive CAR-T cell transfer, or from those had re-emerged after being cleared by Lck-Cre;Ptpn2fl/fl CD8+ HER-2 CAR-T cells were analysed for HER-2 expression by quantitative PCR. HER-2-E0771 versus HER-2 negative E0771 breast cancer cells (2x105) were injected into the contralateral fourth inguinal mammary fat pads of female HER-2 TG mice 50 days after adoptive CAR-T cell transfer and mice were monitored for tumour growth. At day 28 the number of CD45+CD3+CD8+ mCherry+ CAR-T cells in HER-2-E0771 versus E0771 tumours and dLNs were determined by flow cytometry. Ptpn2fl/fl versus Lck-Cre;Ptpn2fl/fl HER-2 CAR-T cells isolated from the spleens of HER-2 TG mice at f) 70 days Ptpn2fl/fl versus Lck-Cre;Ptpn2fl/fl HER-2 CAR-T cells isolated from the spleens of HER-2 TG mice at g) 10 days post adoptive transfer were stained for CD8, CD44 and CD62L and analysed by flow cytometry.