EMBO/sd-character-level-ner huggingface dataset:
Unless otherwise stated, all experiments were repeated three times and data shown are representative.figf6(a) HEK293 cells were transfected with the indicated plasmids. Cells were grown in the presence of amino acids and treated with NH4Cl to block autophagic turnover where indicated. Lysates were immunoblotted with the indicated antibodies. (b) HEK293 cells transfected with Beclin-1 ATG14L were grown in the presence or absence of amino acids. Lysates were immunoblotted with the indicated antibodies (left panel) and quantified by densitometry (right panel). Data represent mean+s.d. of three unique experiments. (c) Beclin-1-shRNA reconstituted lines (WT or mutant) and controls (scramble shRNA or Beclin-1 shRNA) were grown with or without amino acids and assessed for autophagy (left panel) and Beclin-1 levels (right panel). (d) Autophagosome (denoted by arrowheads) generation on amino-acid withdrawal was analysed by electron microscopy. Cell lines and conditions from c were used and representative images from the indicated amino-acid-starved lines are shown; scale bars, 0.4 μm. (d) Autophagosome (denoted by arrowheads) generation on amino-acid withdrawal was analysed by electron microscopy. Cell lines and conditions from c were used and representative images from the indicated amino-acid-starved lines are shown; scale bars, 0.4 μm. (e) Quantification of d. Fold induction was determined by arbitrarily making the nutrient-rich condition 1 (solid bars) for each line. Error bars represent the s.d. of the mean value over an average of 20 fields of view within a representative experiment. NS, not significant. (f) HA-Beclin-1 WT or S14D was transiently expressed in FIP200−/− MEFs grown under nutrient-rich conditions. Indirect immunofluorescence was performed using antibodies against endogenous LC3B and HA-Beclin-1. Scale bars, 20 μm. (g) Quantification of LC3B puncta from confocal in f. In the HA-Beclin-1- or HA-Beclin-1-S14D-transfected samples, only the HA-positive cells were counted for LC3B puncta. Error bars were processed as in e. Mean value shown; P values determined by Student's t-test using 10 unique fields of view from f. Uncropped images of blots are shown in Supplementary Fig. S4.
Figure 1A. Schematic diagram of high-throughput screening. B. Carfilzomib, Bortezomib and MLN9708 activate luciferase activity in M2 macrophages derived from Il-1β-luciferase transgenic mice. BMDMs from IL-1β-luciferase transgenic mice were treated by IL-4 (20 ng/mL) for 24 hours to differentiate into mature M2 macrophages, and then used for screening of FDA compounds (5 μM). The X axis stands for the code of the drug. C. Carfilzomib, Bortezomib and MLN9708 activate Il-1β-luciferase in M2 macrophages in a dose-dependent manner. BMDMs from Il-1β-luciferase transgenic mice were treated by IL-4 (20 ng/mL) for 24 hours, then treated by DMSO, Carfilzomib, Bortezomib or MLN9708 at indicated concentrations. Luciferase activity was monitored 12 hours later. Data from three experiments are presented as the mean ± SD. D. Carfilzomib, Bortezomib and MLN9708 activate Il-1β-luciferase in a dose-dependent manner as well as inhibit the transformation of M2 macrophages. BMDMs from Il-1β-luciferase transgenic mice were pretreated with DMSO, Carfilzomib, Bortezomib, or MLN970 at indicated concentrations for 1 hour, followed by treatment with IL-4 (20 ng/mL). Luciferase activity was monitored 12 hours later. Data from three experiments are presented as the mean ± SD. E. Carfilzomib, Bortezomib and MLN9708 activate Il-1β-luciferase in M0 macrophages in a dose-dependent manner. BMDMs from Il-1β-luciferase transgenic mice were stimulated by DMSO, Carfilzomib, Bortezomib, or MLN970 at indicated concentrations. Luciferase activity was monitored 12 hours later. Data from three experiments are presented as the mean ± SD. F, G. Carfilzomib, Bortezomib and MLN9708 promote the mRNA expression of Il-1β in M2 macrophages. BMDMs (F) or Raw264.7 cells (G) were pretreated by IL-4 (20 ng/mL) for 24 hours, then treated by DMSO, Carfilzomib (1 μM), Bortezomib (1 μM), or MLN9708 (2 μM). RNA was extracted from BMDMs or Raw264.7 cells 6 hours after stimulation and the expression of Il-1β were quantified through RT-qPCR. Data from three experiments are presented as the mean ± SD. T test was used for statistical analysis of differences between groups. ***p < 0.001 (student's t-test). H, I. Carfilzomib, Bortezomib and MLN9708 promote the secretion of inflammatory cytokine IL-1β in M2 macrophages. BMDMs (H) or Raw264.7 cells (I) were pretreated by IL-4 (20 ng/mL) for 24 hours, then stimulated by DMSO, Carfilzomib (500 nM), Bortezomib (500 nM) or MLN9708 (500 nM). Secretion of IL-1β were measured through ELISA 24 hours later. Data from three experiments are presented as the mean ± SD. T test was used for statistical analysis of differences between groups. **p < 0.01, ***p < 0.001 (student's t-test).
Figure 2A) Map of the region upstream of IL3 gene spanning the -34 kb pDHS and -37 kb inducible enhancer, together with the locations of the guide RNAs used to delete the -34 kb pDHS and the PCR primers used to detect the deletion. On the right is a PCR analysis confirming deletion of the -34 kb pDHS on both alleles in 2 out of 4 clones selected for the analyses shown below. B) Average IL3 (upper) and JUN (lower) mRNA expression in the -34 kb-/- clones A and B compared to the WT clones A and B stimulated for 2, 4 and 8 hours with PMA/I. mRNA levels were normalised first to GAPDH and then to the level of gene expression in untransfected Jurkat T cells. Values represent the average of two -34 kb-/- and two WT clones from at least two independent experiments. C) IL3 and JUN mRNA expression levels after two hours of stimulation with PMA/I normalised as in B. The standard error is shown from 5 independent experiments. D) Deletion of the -34 kb pDHS impairs induction of the iDHS at the -37 kb inducible enhancer. The -34 kb-/- clones A and B, the WT clones A and B, and untransfected Jurkat T cells were stimulated with PMA/I for 3 hours. A range of DNase I concentrations were used to determine the chromatin accessibility of the 37 kb iDHS in two independent clones, with values expressed relative to normal unstimulated Jurkat cells. Increased accessibility was detected by a reduction in signal detected by qPCR. The active TBP promoter and an inactive region on Chr18 are used as controls. Independent experiments for the -34 kb-/- and WT clones A and B compared to the untransfected Jurkat T cells are shown in the upper and lower panels respectively.
Figure 8Control shRNA or two different shRNAs against IL6RA were used to knock down IL6RA in the Tgfbr2-mutant cell line Tgfbr2mut1. Cell lysates were harvested and western blotting for IL6RA and tubulin. 3D culture: control or IL6RA knockdown Tgfbr2mut1 cells were seeded on poly-HEMA-coated 96-well plates and cultured for 4 days (5000 cancer cells for monoculture, 3000 cancer cells + 2000 NIH 3T3 for co-culture). 3D culture: control or IL6RA knockdown Tgfbr2mut1 cells were seeded on poly-HEMA-coated 96-well plates and cultured for 4 days (5000 cancer cells for monoculture, 3000 cancer cells + 2000 NIH 3T3 for co-culture). Control IgG, 2G8 and IL-6 neutralizing antibody (each 100 ng/ml, n = 5/group). Scale bars = 50 μm. P values by t test are shown (C). Subcutaneous tumors established from control and IL6RA knockdown Tgfbr2-mutant cell lines in C57Bl/6 mice received rat IgG Mac48 (control), 2G8 or anti-mouse IL-6 antibody MP5-20F3 (each 30 mg/kg 2x/week, n = 5/group). For NK cell depletion, prior to therapies, mice received 50 μg of control rabbit IgG or anti-Asialo-GM1 three days in a row. For maintenance, 25 μg of control rabbit IgG or anti-Asialo-GM1 were given 2X/week throughout the whole study. Therapy started at day 12 post tumor cell injection and mice were on therapy for 16 days. Tumor volume was measured twice per week. P values by t test are indicated. Subcutaneous tumors established from control and IL6RA knockdown Tgfbr2-mutant cell lines in C57Bl/6 mice received rat IgG Mac48 (control), 2G8 or anti-mouse IL-6 antibody MP5-20F3 (each 30 mg/kg 2x/week, n = 5/group). For NK cell depletion, prior to therapies, mice received 50 μg of control rabbit IgG or anti-Asialo-GM1 three days in a row. For maintenance, 25 μg of control rabbit IgG or anti-Asialo-GM1 were given 2X/week throughout the whole study. Therapy started at day 12 post tumor cell injection and mice were on therapy for 16 days. Tumor volume was measured twice per week. P values by t test are indicated. Subcutaneous tumors established from control and IL6RA knockdown Tgfbr2-mutant cell lines in C57Bl/6 mice received rat IgG Mac48 (control), 2G8 or anti-mouse IL-6 antibody MP5-20F3 (each 30 mg/kg 2x/week, n = 5/group). For NK cell depletion, prior to therapies, mice received 50 μg of control rabbit IgG or anti-Asialo-GM1 three days in a row. For maintenance, 25 μg of control rabbit IgG or anti-Asialo-GM1 were given 2X/week throughout the whole study. Therapy started at day 12 post tumor cell injection and mice were on therapy for 16 days. Tumor volume was measured twice per week. P values by t test are indicated. Immunohistochemistry with tumor samples from (D) for Ki67 (G and K), P-STAT3 (H and L), ZAP70, and CD3 (I-J and M-N) was performed. NK cells are highlighted as red (ZAP70+CD3-) and T cells are highlighted as yellow (ZAP70+CD3+) by ImageJ (N). Scale bars outside the magnification boxes = 50 μm, scale bars inside the magnification boxes = 10 μm. n = 5/group, P values vs. control by t test are shown.
Figure 3A. Immunoblot analysis of HCT116 cells transfected with indicated RADX siRNAs and RADX knockout cell lines (RADXΔ). B. Schematic for DNA fiber assays in (C-E). C. Cells in (A) were labeled with consecutive pulses of CldU and IdU as shown in (B). Replication fork speeds were calculated as length of labeled track divided by pulse time (bars, mean; n=400 fibers, pooled from two independent experiments, per condition). D. Bidirectional replication fork symmetry was calculated as percentage of shorter divided by longer tracks from (C). Concordance is 100%, representing equal rates of bidirectional elongation for both daughter forks (bars, mean; n=50 bidirectional forks, pooled from two independent experiments, per condition). E. Proportion of stalled forks (CldU-only tracks) among DNA fibers from (C) (bars, mean; 200 fibers analyzed per condition; n=2 independent experiments). F. Proportion of new origins (IdU-only tracks) among DNA fibers in (C) (bars, mean; 20 fields of view quantified per condition; n=2 independent experiments). G. Schematic for DNA fiber assays in (H) and (I). H. Proportion of stalled forks (CldU-only tracks) among DNA fibers from cells labeled as in (G) (bars, mean; 200 fibers analyzed per condition; n=2 independent experiments). I. Proportion of restarted forks (CldU- and IdU-positive tracks) among DNA fibers in (G) (bars, mean; 200 fibers analyzed per condition; n=2 independent experiments).
Figure 3Summary of METTL14 arginine methylated peptides identified by LC-MS/MS. METTL14 IDR arginine methylation occurs at multiple arginine residues within RGG/RG motifs. Mutation of five arginine sites identified from mass spectrometry reduces METTL14 arginine methylation, but only mutation of all arginine sites to lysine completed blocked METTL14 methylation. Ponceau S staining shows the loading of the recombinant proteins. The black triangles indicate arginine methylated-METTL14; open triangles indicate recombinant METTL14 proteins. METTL14 is methylated at multiple arginine residues in cells. HEK293 cells expressing Flag-tagged WT or various R-to-K METTL14 mutants were lysed and immunoprecipitated with an anti-Flag antibody. Arginine methylation of immunoprecipitated METTL14 was detected by Western blot analysis using an anti-ADMA antibody. Both short and long exposures of the chemiluminescence signals are shown. Arginine methylation of the METTL14 IDR enhances its interaction with RNA substrates. RNA pull-down assay was performed by incubating biotin-labeled RNA with WT, hypomethylated (MS023), and arginine methylation-deficient (RK) mutant METTL14. The pull-down samples were detected by Western blot analysis using an anti-Flag antibody. The methylation status of the recombinant proteins was confirmed by Western blot analysis using an anti-ADMA antibody. EMSA was performed to compare the interactions of WT, hypomethylated (MS023), and arginine methylation-deficient (RK) mutant METTL14 with 6-FAM-labeled RNA. Arrow indicates the shift of the RNA probe caused by the protein-RNA interaction. Coomassie staining shows the increasing amounts of recombinant proteins used in the assay. Fluorescence polarization assays were performed by incubating 6-FAM-labeled RNA with WT, hypomethylated (MS023), and arginine methylation-deficient (RK) mutant METTL14. Each point represents the average of three independent replicates and error bars represent standard deviation (SD). The dissociation constant values (Kd) were listed as mean ± SD. Arginine methylation of the C-terminal IDR enhances the RNA methylation activity of the METTL14/METTL3 complex in vitro. In vitro RNA methylation assays were performed by incubating biotin-labeled RNA substrates with METTL3/METTL14 methyltransferase complexes containing WT, hypomethylated (MS023), and arginine methylation-deficient (RK) mutant METTL14 in various concentrations (10-100 nM). The methylation status of the METTL3/METTL14 complex was confirmed by Western blot analysis using an anti-ADMA antibody. Coomassie staining shows the purification of the enzyme complex. Enzymatic activity was measured in counts per minute (c.p.m.) using a scintillation counter. Data from three replicates were analyzed by Student's t-test and shown as mean ± SD. *, p < 0.05; **, p < 0.01; ns, not significant.
Genes related to microtubules and microtubule-based processes were downregulated in sinhcaf mutant FG follicles and eggs.Figure 5(A and B) Volcano plots showing the relative abundances of transcripts in WT and MZsinhcaf -/- FG follicles (A) or mature eggs (B). Transcripts were differentially expressed if p< 0.05 and fold-change >2 in MZsinhcaf -/- FG follicles or mature eggs compared to WT. (C) Annotations for the gene ontology (GO) terms biological process (green), cellular component (blue), molecular function (red) terms were performed for differentially expressed genes in WT and MZsinhcaf -/- mature eggs. Ten GO terms (at least two genes in each term) with highest value of -log10P value are listed in each category. Red arrowheads point to GO term microtubule. (D) Significantly downregulated genes related to microtubule and microtubule-based process in sinhcaf mutant mature eggs, revealed by transcriptome analysis, are reconfirmed by independent qRT-PCR analysis. Data shown are mean ± SEM (n = 3, biological replicates). Statistical analysis was performed using 2-way ANOVA followed by Holm-Sidak post-hoc test, * p < 0.05. NS, no significant difference. (E) Maximal intensity projection of a confocal z-stack for α-tubulin and phospho-non-muscle myosin II (p-NMII) in 2-cell WT and MZsinhcaf -/- mutant embryos using whole-mount immunohistochemistry. White arrowheads point to cleavage furrows of 2-cell embryos. Scale bars, 100 μm.
Figure 2Protein lysates from 10-13-week old CTRL and FAKO (Mllt4-/-) mice following retro-orbital injection of insulin (1U) were immunoblotted and quantified. BAT; Immunoblotting of Afadin S1795 phosphorylation (n=4-5). Protein lysates from 10-13-week old CTRL and FAKO (Mllt4-/-) mice following retro-orbital injection of insulin (1U) were immunoblotted and quantified. BAT quantification of Afadin S1795 phosphorylation (n=4-5). Data are presented as means + SEM; ANOVA with Tukey or Dunnet's post hoc test: *p<0.05, **p<0.01, ***p<0.001, NS= no significance. Protein lysates from 10-13-week old CTRL and FAKO (Mllt4-/-) mice following retro-orbital injection of insulin (1U) were immunoblotted and quantified. sWAT; Immunoblotting of Afadin S1795 phosphorylation (n=4-5). Protein lysates from 10-13-week old CTRL and FAKO (Mllt4-/-) mice following retro-orbital injection of insulin (1U) were immunoblotted and quantified. sWAT; quantification of Afadin S1795 phosphorylation (n=4-5). Data are presented as means + SEM; ANOVA with Tukey or Dunnet's post hoc test: *p<0.05, **p<0.01, ***p<0.001, NS= no significance. Protein lysates from 10-13-week old CTRL and FAKO (Mllt4-/-) mice following retro-orbital injection of insulin (1U) were immunoblotted and quantified. Liver; Immunoblotting of Afadin S1795 phosphorylation (n=4-5). Protein lysates from 10-13-week old CTRL and FAKO (Mllt4-/-) mice following retro-orbital injection of insulin (1U) were immunoblotted and quantified. Liver quantification of Afadin S1795 phosphorylation (n=4-5). Data are presented as means + SEM; ANOVA with Tukey or Dunnet's post hoc test: *p<0.05, **p<0.01, ***p<0.001, NS= no significance. Protein lysates from 10-13-week old CTRL and FAKO (Mllt4-/-) mice following retro-orbital injection of insulin (1U) were immunoblotted and quantified. (G) Quantifications of phosphorylated Insulin Receptor β and phosphorylated Akt across the three tissues (n=4-5). Data are presented as means + SEM; ANOVA with Tukey or Dunnet's post hoc test: *p<0.05, **p<0.01, ***p<0.001, NS= no significance. Protein lysates from 10-13-week old CTRL and FAKO (Mllt4-/-) mice following retro-orbital injection of insulin (1U) were immunoblotted and quantified. (H) Quantifications of Total IRβ/tubulin in BAT, sWAT and Liver (n=5-6). Data are presented as means + SEM; ANOVA with Tukey or Dunnet's post hoc test: *p<0.05, **p<0.01, ***p<0.001, NS= no significance. Protein lysates from 10-13-week old CTRL and FAKO (Mllt4-/-) mice following retro-orbital injection of insulin (1U) were immunoblotted and quantified. (I) Insulin-induced lipogenesis in isolated mature adipocytes from CTRL and FAKO (Mllt4-/-) mice (n=5). Data are presented as means + SEM; ANOVA with Tukey or Dunnet's post hoc test: *p<0.05, **p<0.01, ***p<0.001, NS= no significance.
Figure 1Metaphysis region in the BM is rich in endomucin+ capillaries expressing HO-1. mp - metaphysis; gp - growth plate; scale bar 100 μm. The HO-1 positive small capillaries in metaphysis express endomucin and CD31. Shown maximum intensity projection, scale bar 20 μm. HO-1 is expressed by smaller endomucin+CD31+ capillaries (#) as well as in bigger endomucin-/lowCD31+ arteries (*). CD31- pericytes wrapping the artery also express HO-1 (*); scale bar 20 μm. HO-1 positive capillaries in the metaphysis expressed CD31 and Sca-1. The capillaries are enveloped by HO-1 expressing pericytes. Part of the HO-1+ pericytes express Sca-1 (#) while others show no or low Sca-1 signal (*); scale bar 20 μm. Flow cytometry analysis revealed the highest expression of HO-1 in CD31+Sca-1+ ECs. CAR and PαS populations also express HO-1, while most of non-hematopoietic CD45-Ter119- are HO-1-negative in steady-state conditions. BM macrophages (MQ) express HO-1. The MHCIIhigh MQ express higher levels of HO-1 than MHCIIlow MQ. Cells within whole HSPC compartment (LKS) express no or low levels of HO-1 in comparison to MQ. HO-1 expression on mRNA level quantified by qPCR qPCR analysis based on two independent experiments n = 10-11/group HO-1 expression on mRNA level quantified by RNA-seq. RNAseq analysis has n = 3-4/group, two-tailed t-test for two groups comparison, and one way ANOVA with Bonferroni post-test for multiple group comparison. *p< 0.05, ****p< 0.0001. Data are shown as mean ± SEM. ECs and CARs from middle-aged animals (11-12 months) express lower levels of HO-1 protein. Two independent experiments, n = 5-10/group. Data are shown as mean ± SEM. *p< 0.05, ***p<0.001, two-tailed unpaired t-test. Middle-aged animals have lower frequency of ECs. Two independent experiments, n = 10-11/group. Data are shown as mean ± SEM. **p<0.01, two-tailed unpaired t-test. The control staining of HO-1 on HO-1-/- bone marrow section is provided in Appendix Fig S1.
Distribution differences of polySer and polyGln proteins in SCA8 BAC mouse and human autopsy brains A Immunohistochemistry (IHC) of end-stage SCA8 BAC mouse cerebellum shows that polySer but not polyGln is found in the molecular layer and cerebellar white matter and that polyGln but not polySer accumulates in Purkinje cells.Figure 2A Immunohistochemistry (IHC) of end-stage SCA8 BAC mouse cerebellum shows that polySer but not polyGln is found in the molecular layer and cerebellar white matter and that polyGln but not polySer accumulates in Purkinje cells. Representative polySer aggregates are indicated by black arrows. Inset: higher magnification of molecular layer and white matter. B IF double staining shows no co-localization of polyGln and polySer in frontal cortex, pons or hippocampus of end-stage SCA8 BAC mice. C IF double staining of end-stage SCA8 BAC frontal cortex shows exclusive localization of polyGln (mouse α-Gln, red, bottom panel) in neurons (rabbit α-NeuN, green, bottom panel). In contrast, polySer (Rabbit α-SerCT, red, top panel) shows widespread accumulation in the frontal cortex including within neurons (mouse α-NeuN, green, top panel). D IHC of SCA8 human cerebellum shows that PolySer accumulates in the white matter but not in the Purkinje cells (left panels) while polyGln accumulates in Purkinje cells but not cerebellar white matter (right panels).
Figure 6(A) CSAG2 does not alter SIRT1 protein levels. HEK293FT cells were transfected with different amounts of Myc-CSAG2 for 48 hr before cells lysates were harvested and immunoblotted for indicated proteins. (B) CSAG2 does not alter SIRT1 subcellular location. U2OS cells were transfected with or without mcherry-CSAG2 for 48 hr before fixation, SIRT1 immunostaining, and imaging. Scale bar: 10 µm. (C) Summary of results mapping interaction region of SIRT1 recognized by CSAG2. HEK293FT cells stably expressing HA-CSAG2 were transfected with indicated Myc-SIRT1 constructs for 48 h before IP with anti-HA followed by SDS-PAGE and immunoblotting for anti-Myc. (D) CSAG2 directly binds SIRT1 catalytic domain in vitro. HA-tagged SIRT1 fragments were in vitro translated. In vitro binding assays with recombinant GST or GST-CSAG2 were performed followed by SDS-PAGE and immunoblotting for anti-HA. (E) CSAG2 enhances SIRT1 activity in vitro. In vitro SIRT1 activity towards p53 ac-K382 peptide was determined in the presence of GST or GST-CSAG2, n=3 biological replicates. Nicotinamide (1 mM) was added to the reaction were indicated to block SIRT1 activity. (F) CSAG2 Δ37 mutant that does not bind SIRT1 does not enhance SIRT1 activity in vitro. In vitro SIRT1 activity towards p53 ac-K382 peptide was determined in the presence of GST, GST-CSAG2 or GST-CSAG2 38-127, n=3 biological replicates. (G) CSAG2 enhances SIRT1 deacetylation of p53 ac-K382 peptide through increasing kcat, but not Km. not affecting the binding ability with its substrate p53. SIRT1 activity assays were performed as described above, except varying amounts of p53 ac-K382 fluorometric peptide were used in the presence of saturating NAD+ (3 mM), n=3 biological replicates. (H-I) U2OS cells were transfected with Myc-Vector or Myc-CSAG2 for 48 hr before being treated with or without 0.4 μM TSA for 6 hr. Cell lysates were blotted for the indicated proteins (H). Quantitation of expression levels of acetylated Histone relative to total Histone is shown as the mean ± SD, n=3 biological replicates. White circles indicate Myc-Vector and black circles indicate Myc-CSAG2. (J) HeLa, U2OS, or HCT116 cells were transfected with Myc-Vector or Myc-CSAG2 for 48 hr. Cell lysates were blotted for the indicated proteins. Quantitation of expression levels of acetylated Histone relative to total Histone is shown as the mean ± SD, n=3 biological replicates. White circles indicate Myc-Vector and black circles indicate Myc-CSAG2. (K) Knockdown of endogenous CSAG2 in A375 cells increased ac-H4K16 levels. Cell lysates were blotted for the indicated proteins. Quantitation of expression levels of ac-H4K16 relative to total H4 is shown as the mean ± SD, n=3 biological replicates.