EMBO/sd-character-level-ner huggingface dataset:
Figure 2Presentation of UTY246-254 on splenocytes after exposure to indicated concentrations of PRN1126. Data are presented as the mean absorbance ± s.d. of three replicate cultures. The experiment has been performed twice, yielding similar results. Flow cytometry analysis of H-2Kb surface expression on splenocytes derived from C57BL/6 mice treated with the indicated concentrations of PRN1126 overnight. Pooled data from three independent experiments (n=9) are shown as the means of median fluorescent intensity ± SEM. Splenocytes from C57BL/6 mice (C) were exposed (continuous treatment) to 300 nM PRN1126 or vehicle (DMSO) or 300 nM ONX 0914 and stimulated with LPS overnight. IL-6 concentrations in the supernatant were analyzed by ELISA. (C) IL-6 concentrations are presented as mean and individual data points from triplicate wells. The experiment has been performed twice, yielding similar results. Splenocytes from human PBMCs (D) were exposed (continuous treatment) to 300 nM PRN1126 or vehicle (DMSO) or 300 nM ONX 0914 and stimulated with LPS overnight. IL-6 concentrations in the supernatant were analyzed by ELISA. Data are presented as single dots from three independent donors. The highest cytokine concentration was set to 100%. *P < 0.05. Unpaired student´s t-test. Colitis was induced by oral administration of 3 % DSS for 5 d. Mice were treated daily (s.c.) with either PRN1126 (40 mg/kg), or ONX 0914 (10 mg/kg), or vehicle. Data points represent mean ± SEM of 15 mice pooled from three independent experiments. Mice were immunized with MOG35-55 peptide and were monitored daily for clinical symptoms of EAE. Mice were treated three times a week (s.c.) with either PRN1126 (40 mg/kg), or ONX 0914 (10 mg/kg), or vehicle. All data were statistically compared to the vehicle treated group. *P < 0.05, **P < 0.01, ***P < 0.001. Two-way ANOVA. Shown are the means of the clinical scores ± SEM (n = 6 per group).
Activation of mTORC1 by NO Is Dependent on TSC2 and IKKβ.Figure 4(A) Immunoblot analyses with anti-phospho-S6K and anti-phospho-S6 antibodies show that NO donors activated mTORC1 in Tsc2+/+ MEFs, but not in Tsc2−/−, MEFs. (B) NO donors reduced phosphorylation of IKKα/β and AMPKα in MEFs, as analyzed by immunoblotting with anti-phospho-IKKα/β and anti-phospho-AMPKα antibodies, respectively. (C) Immunoblot analyses with anti-phospho-S6K and anti-phospho-S6 antibodies show that NO donors activated mTORC1 in Ikkβ+/+ MEFs, but not in Ikkβ−/− MEFs. (D) Immunoblot analysis with anti-LC3 antibody shows that DETA NONOate reduced autophagosome synthesis in bafilomycin A1-treated Tsc2+/+ and Tsc2−/− MEFs. (E) DETA NONOate decreased EGFP-LC3 vesicles in Tsc2+/+ and Tsc2−/−MEFs transfected with EGFP-LC3, along with either pcDNA3.1 or AAA Myc-Bcl-2. Although it further reduced EGFP-LC3 vesicles in Tsc2+/+MEFs expressing AAA Myc-Bcl-2 compared to mock-transfected Tsc2+/+MEFs, this effect was not seen in Tsc2−/−MEFs. Tsc2+/+ and Tsc2−/−MEFs were analyzed separately. Graphical data denote mean ± SEM.
Figure 3A,B Seahorse analysis of basal (glucose injection) and maximal (oligomycin injection) ECAR or basal (no treatment) and maximal (FCCP injection) OCR in WT and Peli1-KO OT-I CD8 T cells that were either untreated (naïve) or activated with plate-bound anti-CD3 (1 μg/ml) plus anti-CD28 (1 μg/ml) for 16 h. Data are presented as a representative plot (A) or summary graph based on 6 wildtype and 6 KO mice (B). qRT-PCR analysis of the indicated genes using RNAs isolated from wildtype or Peli1-KO OT-I CD8 T cells stimulated with anti-CD3 and anti-CD28 for 6 h. D qRT-PCR analysis of the indicated genes using RNAs isolated from wildtype or Peli1-KO OT-I CD8 T cells stimulated with anti-CD3 and anti-CD28 for 6 h. E Immunoblot analysis of HK2 and Glut1 in wildtype or Peli1-KO OT-I CD8 T cells that were stimulated with plate-bound anti-CD3 and anti-CD28 for 24 h.
Inhibition of mTOR signaling increases survival of Phb2pko mice.Figure 7A Phb2-deficient mouse podocytes showed higher levels of phosphorylated S6 ribosomal protein than control podocytes, which could be blocked by treatment with rapamycin (n = 5, bars represent mean ± SEM, Student's t-test, **P = 0.0036). pS6RP, phosphorylated S6 ribosomal protein; S6RP, S6 ribosomal protein. B Immunohistochemistry for phosphorylated S6 ribosomal protein (pS6RP) on kidney sections revealed increased levels of pS6RP in glomeruli of Phb2pko compared to Phb2fl/fl mice (arrows point to cells showing a signal for pS6RP; scale bar: 20 μm). C Kaplan-Meier survival curve revealed that treatment of Phb2pko mice with rapamycin prolonged survival for several days compared to vehicle-treated Phb2pko mice (n = 11 for Phb2fl/fl + rapamycin, n = 15 for Phb2pko + vehicle, n = 16 for Phb2pko + rapamycin).D Statistical analysis comparing rapamycin-treated Phb2pko mice with vehicle-treated Phb2pko mice.
a) Miz1 activates the Rorc and Ambra1 promoters, and the POZ domain is required for activation.f4(a) Miz1 activates the Rorc and Ambra1 promoters, and the POZ domain is required for activation. HEK293 cells were transiently transfected with plasmids containing promoter sequences of Ambra1 or Rorc in front of luciferase and with expression vectors encoding Miz1 or MizΔPOZ. Empty vectors were used as control. Values were normalized with CMV-βGal. Error bars are s.d. of biological triplicates. (b) Direct target genes of Miz1 are downregulated in cerebella of Miz1ΔPOZNes mice. Microarray analysis was performed from cerebella of mice at the indicated ages. The panels show box plots (line: median, box: 50% of data points, whisker: 1.5 interquantile range, outliers are not shown) of relative gene expression (plotted as log2-fold change) of the 140 Miz1 direct target genes comparing control and Miz1ΔPOZNes mice. The P-value was calculated using a Mann-Whitney-Wilcoxon test. Microarrays were performed as technical duplicates from RNA pooled from two animals for each genotype. (c) Gene-set enrichment analysis documenting significant downregulation of Miz1 target genes in cerebella of Miz1ΔPOZNes mice. NES, normalized enrichment score. Target genes of Miz1 were the only significant gene set in this analysis. (d) qRT-PCR documenting the expression of selected Miz1 target genes in cerebella of control and of Miz1ΔPOZNes mice at the indicated ages. Data represent the mean of two independent biological experiments each with three technical replicates. Error bars are s.e.m.
Figure 2A. TetB model description including the effect of TetB to reduce cytoplasmic concentrations of drug to relieve the inhibition of translation. PA and PO describe an available and occupied pump, respectively. Drug can bind PA by a rate, k1, to create PO where two events can occur: 1. Drug disassociates from the pump at a rate, k-1 or 2. Drug is transported into the periplasm at a pump efficiency rate, r. B. Normalized fitness functions of cells expressing wild type tet(B) and their corresponding model fits in the presence of TET, DOX and MCN. Triplicate data is shown for each antibiotic C. Predicted concentrations of TET in the cytoplasm as a function of TET concentration in the media, κ, and γ. The κ and γ values held constant in the plots are those for wild-type TetB.
Figure 9(A) During DSB repair by a variety of mechanisms shown on the left, ssDNA gaps require DNA synthesis to restore dsDNA. ssDNA formed as a result of DSB processing is covered by Rad51. (B) In wild type cells, the Srs2 helicase displaces Rad51, thereby promoting RPA binding to the ssDNA. (C) Presence of RPA at ssDNA-dsDNA junction is required for PCNA loading (via RPA-RFC interaction) followed by recruitment of DNA polymerase. (D) Because DNA synthesis is faster than resection the ssDNA gap shortens. (E) When long stretches of DNA have to be synthesized, the likelihood of the replication machinery stalling and disassembly is increased. However, its re-assembly is efficient in wild type cells as Srs2 ensures that ssDNA is clear from Rad51 and covered with RPA. (F) Once loaded, the polymerase rapidly catches up with the resection machinery due to a higher speed of replication vs. resection. As a result, DNA processing is terminated. (G) Therefore, due to efficient initial loading as well as re-loading of PCNA SRS2 cells are able to restore dsDNA and complete repair. (H-I) In contrast to wild type cells, in srs2Δ mutants the replacement of Rad51 with RPA at the ssDNA-dsDNA junction is slow as it occurs either stochastically or relies on another, less efficient helicase. (J) As a result, recruitment of PCNA and initiation of DNA synthesis is delayed and the ssDNA gap becomes longer. (K) Once loaded, the replication machinery in srs2Δ mutants moves at the same rate as in wild type cells and shortens the gap, albeit more DNA synthesis is now required to fill in the gap. If uninterrupted, the replication machinery will eventually catch up with the processing nucleases and complete repair, like in wild type cells (see F-G). (L-M) Re-loading of disrupted PCNA-polymerase complexes in srs2Δ cells depends on inefficient replacement of Rad51 with RPA at the ssDNA-dsDNA junction which leads to an increase of the ssDNA gap and, depending on the balance between DNA synthesis and resection, may result in "runaway" resection and inability of srs2Δ to complete restoration of dsDNA.
Figure 2A. De novo protein synthesis measured by puromycin incorporation in shSCR, hTrmt13 knockdown cells (sh-1 and sh-2), and sh-1 with stable expression of wild type hTrmt13 (sh-1+hTrmt13) or enzymatically-inactive hTrmt13 (sh-1+E463A). Puromycin-labeled proteins were detected by immunoblotting. Representative images (left) and statistical analysis (right) are shown. Knockdown and overexpression efficiency of hTrmt13 in MDA-MB-231 verified by Western blots. Data information: Statistical analysis was performed using t-tests, and error bars represent mean ± SD for three independent experiments. B. Northern blot analysis of substrate tRNAs of hTrmt13 from control and the hTrmt13 knockdown MDA-MB-231 cells. C. The in vivo aminoacylation level of hTrmt13 substrate tRNAs assayed by Northern blot. The samples were also deacylated (DA) by incubating with alkaline buffer (pH 9.0) to obtain fully uncharged tRNA control. D. RT-qPCR using a stem-loop primer to quantify the tRFs levels in shSCR, sh-1, and sh-2. Data information: Statistical analysis was performed using t-tests, and error bars represent mean ± SD for three independent experiments. E. Quantification of the level of 5'-Gly-CCC in shSCR, sh-1, sh-2, sh-1+hTrmt13, and sh-1+E463A assayed by RT-qPCR. Data information: Statistical analysis was performed using t-tests, and error bars represent mean ± SD for three independent experiments. F. Quantification of the Cm/G ratios of endogenous HctRNAGly(CCC) isolated from shSCR, sh-1, sh-2, sh-1+hTrmt13, and sh-1+E463A cells by UPLC-MS/MS analysis. Data information: Statistical analysis was performed using t-tests, and error bars represent mean ± SD for three independent experiments. G. Quantification of de novo protein synthesis in cells treated with different concentration of tRFs H. Quantification of de novo protein synthesis in cells treated with different concentration of tRNAGly(CCC) Data information: Statistical analysis was performed using t-tests, and error bars represent mean ± SD for three independent experiments.
Figure 3(A) RNF114 substrate validation. HEK293 cells were co-transfected with constructs expressing FLAG-tagged substrates, wild-type or mutated His-RNF114 and EGFP, and western blot was performed with anti-FLAG and anti-His antibodies. The results show that the protein levels of CD74, TAB1, OPTN, TNIP1, APPL1, PSAT1, and RALGPS1 were reduced in the presence of wild-type RNF114 compared to co-transfecting with mutant RNF114. (B) RNF114-dependent in vitro ubiquitination of substrates. Substrate ubiquitination assays were performed with purified RNF114, the reaction products were immunoblotted using anti-His antibody to detect the ubiquitin (upper panel) or anti-FLAG antibody detecting the substrate protein (middle panel), and the lower panel showed the merged signals. The results show that clear polyubiquitin chains were formed in the systems containing recombinant CD74, TAB1, TNIP1, PSAT1 or RALGPS1.
NCC apoptosis, cell cycle and differentiation.Figure 4A. Homozygous ALX1165F/165F NCC (blue) showed an increase in sensitivity to apoptosis when compared to control ALX1165L/165L NCC (black). The data on the left represents the mean percentage of Annexin V positive cells, indicative of apoptosis, as determined by FACS analysis, with the data on the right being an example of one such experiment. Apoptosis was induced by immersion in a 55°C water bath for ten minutes. Representative experiment for each condition is shown. Data is represented as pooled mean ± SEM of three independent experiments. Data obtained of each clone from three independent experiments were pooled and the mathematical mean was calculated. SEM was used to determine the standard error. To test statistical significance, an ANOVA test was performed. A p-value <0.05 was considered to be statistically significant. . *: Significantly different from the basal apoptosis rate: p=3x10-12 between control NCC basal apoptosis and induced apoptosis, and between ALX1165F/165F NCC basal apoptosis and induced apoptosis. **: Significantly different from control NCC (p=0.0004). B. Expression levels of cyclins CCNA2 (blue) and CCND1 (orange) in NCC at passages 2 and 3 of ALX1165L/165L and ALX1165F/165F NCC. The RT-qPCR relative expression values were normalized to RPLP0 and GAPDH expression. Data is represented as pooled mean ± SEM of three experiments on three clones from each genotype. *: Significantly different from control NCC at passage 2 (p=0.001 between control and ALX1165F/165F NCC at passage 2 for CCNA2, p=0.0052 for CCND1). **: Significantly different from control NCC at passage 3 (p=0.0494 between control and ALX1165F/165F NCC for CCNA2, p=0.0008 for CCND1). C. Fluorescence Activated Cell Sorting (FACS) experiments showed that control ALX1165L/165L NCC (green) exhibited increased expression of mesenchymal markers CD90, CD105, and CD73 with culture time (passages 1 through 4), whereas homozygous ALX1165F/165F NCC (blue) showed a consistent expression of the markers expressed at passage 1 throughout. Further, control ALX1165L/165L NCC showed a downregulation of CD57 expression with culture time, while ALX1165F/165F NCC maintained the same level of CD57 across passages. Data are presented as the mean percentage of positive-stained cells across passage numbers. Data obtained of each clone from three independent experiments were pooled and the mathematical mean was calculated. SEM was used to determine the standard error. To test statistical significance, an ANOVA test was performed. A p-value <0.05 was considered to be statistically significant. *: Significantly different from control NCC. For CD90, p=0.0013 at passage 3 and p=0.0207 at passage 4. For CD105, p=0.0016 at passage 2, p=0.00004 at passage 3 and p=0.0021 at passage 4. For CD73, p=0.0060 at passage 2, p=0.00004 at passage 3 and p=0.0114 at passage 4. For CD57, p=0.0026 at passage 2, p=0.000003 at passage 3 and p=0.000007 at passage 4.
Figure 1Primary BMDMs were stimulated with recombinant murine TNF (100 ng/ml) in combination with SM Primary BMDMs were stimulated with recombinant murine TNF (100 ng/ml) in combination with TAK1i for 6 or 4 h respectively. Time-lapse confocal images (hour:min) of BMDMs stimulated with recombinant murine TNF (100 ng/ml) and SM (250 nM) stained with propidium iodide (red) for 6 h. Black arrowheads indicate membrane ballooning while white arrowheads indicate apoptotic bodies. immortalized BMDMs were stimulated with recombinant murine TNF (100 ng/ml) in combination with SM immortalized BMDMs were stimulated with recombinant murine TNF (100 ng/ml) in combination with TAK1i for 6 or 4 h respectively.
Figure 4U2OS cells stably expressing GFP-TFEB fusion protein were treated with indicated chalcones at 30 µM for 6 h. GFP intensities in nuclei and cytoplasm were measured and the ratio of GFP intensities in nuclei and cytoplasm were calculated to indicate TFEB translocation to nuclei (A). Data are means ± SD (* = p < 0.05;** = p < 0.01;*** = p < 0.001). U2OS cells stably expressing GFP-TFEB fusion protein were treated with indicated chalcones at 30 µM for 6 h. Representative images are shown in (B). Scale bar equals 10 µm. (C, D) U2OS cells were treated with 30 µM 3,4-DC for 6 h, and then endogenous TFEB translocation was assessed by immunostaining (C). TFEB intensities in nuclei and cytoplasm were measured and the ratio of TFEB intensities in nuclei and cytoplasm were calculated to indicate TFEB translocation to nuclei (D). Data are means ± SD (*** = p < 0.001). Representative images are shown in (C). Scale bar equals 10 µm. (E, F) U2OS cells were treated with 30 µM 3,4-DC for 6 h or were left untreated. Cytoplasmic and nuclear fractions were assessed for nuclear translocation of the transcription factors TFEB and TFE3 by SDS page. MEF wild type and TSC2 knockout (TSC2 KO) cells were treated with 3,4-DC for 6 h. The cells were fixed and immunofluorescence was performed to detect endogenous TFEB. The ratio of TFEB intensities between nuclei and cytoplasm was calculated in (H). Data are means ± SD (*** = p < 0.001 versus Ctr; ###=p<0.001 versus WT/3,4-DC). Representative images are shown in (G). Scale bar equals 10 µm. MEF wild type and TSC2 knockout (TSC2 KO) cells were treated with 3,4-DC for 6 h. (I) After the treatment, nuclei were isolated and western blot was performed to detect nuclear and cytosol TFEB protein levels. (J-L) MEF wild type and TSC2 KO cells were collected and lysed for western blot after the treatment with 3,4-DC. Antibodies against LC3, p62, TSC2, or GAPDH were administrated to detect protein levels, and phosphorylation of mTOR substrate p70 S6K at Threonine 389 (T389) was measured with the phosphorylation specific antibody (P-p70(T389)) and anti-p70 antibody. The bands intensities of LC3-II, p62, and GAPDH were measured with Image J, and the ratios of LC3-II/GAPDH and p62/GAPDH were calculated in (K) and (L). Data are means ± SEM of at least three independent experiments (** = p < 0.01, *** = p < 0.001 vs Ctr; #=p<0.05, ##=p<0.01 vs WT/3,4-DC) (M,N) U2OS cells treated with 3,4-DC or Torin for 6 h were collected and processed for western blot. TFEB phosphorylation at Serine 211 was checked with the phosphorylation specific antibody (P-TFEB(S211)) and total TFEB protein level was also detected with anti-TFEB. The bands intensities of P-TFEB and total TFEB were measured with Image J, and their ratio was calculated in (N). Data are means ± SEM of at least three independent experiments (* = p < 0.05 vs Ctr).
hst suppresses amiRSUL-silencing(A) HST genomic sequence spanning exons 11-to-13 and position of the hst-1/-25/-26 mutations.Figure 2HST genomic sequence spanning exons 11-to-13 and position of the hst-1/-25/-26 mutations. EMS-induced nucleotide transitions (arrows) in hst-25 and hst-26 are in red; upper-/lower-case fonts: exonic/intronic sequences. HST western analysis, in biological duplicates, in the indicated genotypes. Coomassie blue staining (Coom.) provides a control for total protein loading. Phenotype of pSUC2::amiRSUL plants in hst-1/-25/-26 backgrounds. RT-qPCR analysis of SUL mRNA levels in leaves with the indicated genotypes. Error-bars: SD. Welch's t-test p-values are indicated. n=5.
Figure 5(A) Analysis of nuclear Rad52-GFP foci formation. An illustrative image of the experimental setup is shown. DIC, differential interference contrast; yellow, Rad52-GFP; red, mCHERRY-Pus1 (nuclear compartment marker). Scale bar = 5 µm. (B) (C) Kinetic analysis of Rad52 foci formation. Wild-type and mus81∆ cells containing Rad52-YFP and mCHERRY-Pus1 were synchronized in G1 with α-factor, treated with DMSO or 100 µM CPT, let in G1 for 30 min, and released into S phase. Cells were collected at the indicated time points and visualized by fluorescence microscopy. Mean ± SEM of cells with Rad52 foci are shown for each time point. Flow cytometry profiles corresponding the experimental setup are shown. Four biological replicates have been performed. (D) Mms4-Flag10 phosphorylation analyzed by immunoblot in wild type cells exposed to CPT. Wild-type cells were synchronized in G1 with α-factor, treated with DMSO or 50 µM CPT, let in G1 for 1 h, and released into S phase. Cells were collected at the indicated time points and Mms4 was immunodetected with anti-Flag antibodies. Clb2 immunodetection serves as a marker for G2 phase entry. FACS profiles corresponding the experimental setup are also shown. Two biological replicates have been performed. (E) CPT sensitivity assayed by 10-fold serial dilutions of S-MUS81 and G2-MUS81 alleles compared to the wild type, mus81, mms4∆ and cdc5-2 mutants. Three biological replicates have been performed. (F) UV sensitivity assayed by 10-fold serial dilutions of S-MUS81 and G2-MUS81 alleles in combination with rad3-102 on YPAD plates after exposure to the indicated UV-C doses. Three biological replicates have been performed.
Figure 1(A) The TVKS/T-motif has been conserved throughout metazoan evolution. The phospho-acceptor sites are indicated in red, the DFG- and APE motif flanking the activation loop (AL) are indicated in blue. Residues differing from human B-Raf are shown in grey or in red/blue with reduced intensity. (B) B-RafE586K signals independently from Ras-GTP, but requires an intact AL. The MEK-ERK activation potential of the indicated HA-tagged B-Raf mutants was assessed by Western blotting using TCLs from transiently transfected Plat-E cells. (C) B-RafCAAX but not the B-RafV600E oncoprotein signals independently from Ras-GTP, but requires an intact AL. Same experimental set-up as in (B). (D) Quantification of experiments shown in (B) and (C). The signal elicited by the individual reference proteins (B-Rafwt, B-RafE586K, B-RafV600E and B-RafCAAX) was set in each analysis to 100%. n = 3, mean + SEM, t-test, * p < 0.05, ** p < 0.01
Figure 6WT or MARCO-/- C57BL/6 mice were inoculated s.c. in the rear feet with 5*104 PFU of CHIKV-E2-mCherry and the popliteal dLNs were collected at 2 hpi. (A) Frozen dLN sections were stained for Lyve-1+ LECs (white), MARCO (green), B220 (blue) and mCherry+ CHIKV particles (red). Scale bars: 200 μm. (B) Representative histograms of confocal images showing voxel intensities for Lyve1 and mCherry. (C) Percent of Lyve1+ voxels with mCherry signal; dots represent individual sections. Data are representative of two experiments, n=6-9. One-way ANOVA with Tukey's multiple comparisons test; ****P<0.0001. (D) Higher magnification confocal image of a WT lymph node section stained as in (A). Scale bar: 20 μm. (E) Higher magnification confocal image of a MARCO-/- lymph node stained for CD11b+ cells (white), mCherry+ CHIKV particles (red), B220+ B cells (blue), and CD169+ macrophages (green). Scale bar: 20 μm.