EMBO/sd-character-level-ner huggingface dataset:
Fig.Figure 4A FST expression in different AML cell lines was detected by Western Blotting. FST317 and FST344 overexpression resulted in significant increases of FST transcription by RT-qPCR and protein by Western Blot (B) Green, ML-2-GFP; Blue, ML-2-FST317; Red, ML-2-FST344. The RT-qPCR experiments were performed in triplicates (B). Data information: data were presented as mean ± SEM. *P<0.05, **P<0.01, ***P<0.001 (Student's t-test). ns: Not significant. FST317 and FST344 overexpression promoted ML-2 cell growth in vitro (C). Green, ML-2-GFP; Blue, ML-2-FST317; Red, ML-2-FST344. Data information: data were presented as mean ± SEM. *P<0.05, **P<0.01, ***P<0.001 (Student's t-test). ns: Not significant. D-E The clonogenicity of ML-2 overexpressing GFP, FST317, and FST344 in vitro for 14 days. The CFU experiments were performed in triplicates (E). Data information: data were presented as mean ± SEM. *P<0.05, **P<0.01, ***P<0.001 (Student's t-test). ns: Not significant. The engraftment of ML-2 (with luciferase gene) overexpressing GFP, FST317, and FST344 was quantified by bioluminescence imaging (F and G) Data information: data were presented as mean ± SEM. *P<0.05, **P<0.01, ***P<0.001 (Student's t-test). ns: Not significant. The engraftment of ML-2 (with luciferase gene) overexpressing GFP, FST317, and FST344 and the survival of ML-2-engrafted NSG mice in vivo was recorded (H). Survival curve in panel H was analyzed by Log-rank test. *P<0.05, **P<0.01. RNA-seq of upregulation of RET, IL2RA and CCL5 after FST344 overexpression in ML-2 cells. RT-qPCR validation of upregulation of RET, IL2RA and CCL5 after FST344 overexpression in ML-2 cells. RT-qPCR experiments were performed in triplicates (J). Data information: data were presented as mean ± SEM. *P<0.05, **P<0.01, ***P<0.001 (Student's t-test). ns: Not significant. Overall survival analysis of patients from TCGA-AML based on the differential expression of IL2RA Overall survival analysis of patients from TCGA-AML based on the differential expression of CCL5. M Activation of MAPK/ERK pathway in ML-2 by FST344 overexpression were validated by Western Blotting. Scale bar = 1 mm.
Figure 1A: Genome organization of SARS-CoV-2. B: Schematic representation of SARS-CoV-2 N protein domains. Three intrinsically disordered regions, i.e. N-arm, linker region (LKR) and C-tail, and the N-terminal domain (NTD) and C-terminal domain (CTD) are illustrated. The charged Ser/Arg (SR)-rich motif (coloured purple) is shown. C: Phylogenetic analysis of N proteins from seven human-infecting coronaviruses. SARS-CoV-2 is highlighted by a red asterisk. D: Analytical ultracentrifugation absorbance data analysis of N-NTD. The clear peak at ~15.4 kDa molecular weight (MW) corresponds to the N-NTD monomer. E: Analytical ultracentrifugation absorbance data analysis of N-CTD. The clear peak at ~28.7 kDa MW corresponds to the N-CTD dimer.
Figure 5A A simple regulatory model for DA signaling and proteostasis. When nematodes encounter a bacterial lawn, the lawn's viscosity is sensed by dopaminergic mechanosensory neurons, which then release the neurohormone dopamine (DA) into the pseudocoelomic body cavity in response. DA signaling activates the expression of xenobiotic detoxification genes in epithelial tissues. The products of these genes remove toxins and ROS, which otherwise would damage proteins. Arrows indicate positive relationships whereas T-bars indicate inhibitory relationships.B DA signaling, through activation of the xenobiotic response, protects most proteins (gray shapes) from proteotoxic stress from toxins and ROS. Protein quality control mechanisms like the UPS (particularly, E3 and E4 ubiquitin ligases, indicated by yellow circles) are fully available to act on unstable proteins (green oval), thereby triggering the rapid ubiquitination and degradation of such proteins. Individual ubiquitin molecules are indicated by small circles with the letter "u."C In the absence of DA signaling, there is less activation of the xenobiotic response and thus many proteins within epithelial cells become damaged by toxins and ROS (red shapes comprising dotted lines). Protein quality control mechanisms like the UPS (yellow circles) become overwhelmed ubiquitinating and degrading the large load of stressed proteins, leaving few of them available to process unstable proteins (green oval). The result is an observed stabilization of such proteins, many of which are not ubiquitinated or only partially ubiquitinated. Individual ubiquitin molecules are indicated by small circles with the letter "u."
FIP200 is required for membrane targeting and proper function of ULK.Figure 8.(A) FIP200+/− and FIP200−/− MEFs stably expressing GFP-ULK1 or -ULK2 were cultured in starvation medium for 120 min. The cells were fixed and examined by fluorescence microscopy. Bar, 20 μm. (B) FIP200+/+ and FIP200−/− MEFs were cultured in complete medium. The cell lysates were subjected to immunoblot analysis with antibodies against ULK1 or HSP90 (loading control). (C) Phosphatase sensitivity of ULK1. FIP200+/+ and FIP200−/− MEFs were cultured in complete medium. ULK1 was immunoprecipitated from cell lysates and treated with λ phosphatase for 30 min at 30°C in the absence or presence of phosphatase inhibitors (1 mM Na2VO4, 50 mM KF, 15 mM Na4P2O7, and 1 mM EGTA).
FLNAΔECS aortae and primary vSMCs show hypercontraction in response to U46619.Figure 3A,B) Treatment of aortic rings with thromboxane A2 receptor agonist U46619 leads to (A) hypercontraction and (B) increased contraction force in FLNAΔECS aortae in myograph chambers. Emax was higher in FLNAΔECS aortae without any difference in EC50 as compared to wt aortae. For each condition, 10-12 aortic rings from at least 4 wild-type (wt) and 4 FLNAΔECS mice were used. Data shown as mean ± SEM. * P<0.05, ** P<0.01, *** P<0.001 (Student's t test) (C) Graph shows the normalized cell contraction measured by Xcelligence Real time cell analyzer in wt (red) and FLNAΔECS (green) primary vascular smooth muscle cells (vSMC) indicating hypercontraction in FLNAΔECS cells in response to 10μM U46619. Data shown as mean ± SD from three independent experiments. (D) Quantification of cell index measurements plotted as percentage of contraction following different concentrations of U46619. Data shown as mean ± SD from three independent experiments. P value <0.05 (Student's t test) was considered significant. The difference between wt and FLNAΔECS was significant at every concentration of U46619.
Figure 4A. Representative immunofluorescence images of the association of MavQ with the LCV. BMDM cells infected with wild-type or Dot/Icm-deficient (dotA-) L. pneumophila were sequentially stained with antibodies against L. pneumophila and MavQ. Images were taken under a fluorescence microscope. Insets represent 3x magnification of regions defined by dash lines. Scale bar, 5 μm. B. The rates of of LCVs positively stained for MavQ 2 h post-infection. At least 100 LCVs (n≥100) were scored for each sample. C. Dynamics of MavQ association with LCV. BMDMs were infected with the wild-type L. pneumophila strain at an MOI of 2 for the indicated experimental durations. At least 100 phagosomes (n≥100) were counted for each sample to obtain the percentage of MavQ-positive LCVs. D. Intracellular growth of L. pneumophila in macrophages. BMDMs were infected with indicated L. pneumophila strains at an MOI of 0.05. At the indicated time points, CFUs were determined by plating the saponin-solubilized lysates of infected cells on CYE plates. E. Expression of mavQ in L. pneumophila strains used for infection. Total proteins of bacteria cultured to the post-exponential phase separated by SDS-PAGE were probed with MavQ-specific antibodies. ICDH was detected as a loading control.
Figure 3C57BL/6 mice were treated with G-CSF, CoPP or solvent controls (NaCl, DMSO) for 5 consecutive days. Samples were collected 6 hours after the last injection. (A) Representative flow cytometry plots show higher granularity (SSC) and Ly6G expression in CoPP-mobilized granulocytes than in G-CSF mobilized granulocytes. C57BL/6 mice were treated with G-CSF, CoPP or solvent controls (NaCl, DMSO) for 5 consecutive days. Samples were collected 6 hours after the last injection. Relative abundance of different granulocyte phenotypes in blood of mice treated with G-CSF and CoPP. Mice treated with CoPP have higher proportion of granulocytes with mature phenotype, than mice treated with G-CSF. C57BL/6 mice were treated with G-CSF, CoPP or solvent controls (NaCl, DMSO) for 5 consecutive days. Samples were collected 6 hours after the last injection. Relative abundance of different granulocyte phenotypes in BM (C) of mice treated with G-CSF and CoPP. Mice treated with CoPP have higher proportion of granulocytes with mature phenotype, than mice treated with G-CSF. C57BL/6 mice were treated with G-CSF, CoPP or solvent controls (NaCl, DMSO) for 5 consecutive days. Samples were collected 6 hours after the last injection. The percentage of cells isolated from C57BL/6xFVB mice treated with G-CSF or CoPP that are producing reactive oxygen species after incubation with indicated stimuli.
a, Volume density of early (Avi) and late (Avd) autophagic vacuoles.figf4a, Volume density of early (Avi) and late (Avd) autophagic vacuoles from four independent cultures. Examples of Avi and Avd are shown in b and c, respectively. Bars represent 0.4 µm. d, Labelling density of cathepsin-D as estimated in two independent experiments. e, Labelling density of LAMP-1. f, Degradation of long-lived proteins followed over 24 h in amino acid and fetal-calf-serum-containing medium. g, Effect of amino-acid (AA) deprivation and 3-methyladenine (MA) on protein degradation during 4 h of chase. The error bars in a, d, e and g represent s.e.m.
Figure 3A. Variant locations on the predicted TetB topology. A-helices are shown in blue, B-helices are shown in green, and C-helices are shown in grey. B. Model fits for E. coli BW25113 strains expressing a chromosomal copy of tet(B)WT and seven variants in TET, DOX, and MCN. Our model fit triplicate fitness function data for all strains (Figure S2). At low drug concentrations for all antibiotics, tet(B)G366C exhibits much slower growth rates than wild-type. Compared to wild-type, strains expressing tet(B)L9F exhibit similar growth rates at low and high drug concentrations but have faster growth rates at intermediate drug concentrations. C. Fitness landscapes with mutants overlaid for TET=35 µg/mL, DOX=10 µg/mL, and MCN=2.5 µg/mL corresponding to the dashed lines in 3B.
Diversity of Tap-1 among V.Figure 5Cartoon of vgrG-1, tap-1, and the effector protein-encoding gene in the auxiliary cluster 1 of three indicated V. cholerae strains. Same color indicates more than 90% identity of the amino acid sequences encoded in the genes compared. Different colors indicate < 30% identity, and mixed color indicates 30-90% identity. The presence or absence of the ACD encoded in vgrG-1 is indicated with + and −, respectively. Diversity of Tap-1. Phylogenetic analysis of Tap-1 encoded in 36 V. cholerae strains. Analysis was performed using Raxml (Stamatakis, 2014). The names of Tap-1-harboring strains are indicated in the tree. Three clades are highlighted with different colors. Three representative sequences that cluster to different clades are highlighted with an arrow and used for analysis. Cartoon of Tap-1 from the three different clades. Regions that share more than 80% identity in the three amino acid sequences are shown in the same color, and regions with < 45% identity are shown in different colors. The conserved sequences of the motifs of DUF4123 and their positions are indicated. Specificity of the C-terminal Tap-1 segment. Killing assay in which the lack of tap-1 in V52 is complemented by empty vector (control) or one of two different alleles of tap-1. The ability to kill in a TseL-dependent manner is indicated by the logarithm of surviving C6706ΔtsiV1. Specificity of N-terminal segment of Tap-1. Killing assay in which the lack of tap-1 in 1587 is complemented by empty vector (control) or one of two different alleles of tap-1. The ability to facilitate killing by the effector A55_1502 is indicated by the logarithm of surviving 1587ΔA55_1501-03.
Figure 1(A-B) Western-Blotting (WB) of p53-negative lung cancer H1299 cells stably expressing (A) or transiently expressing (B) the indicated constructs. Post-translationally modified Δ133p53 and Δ160p53 isoforms are indicated with *. (C) The same WB membrane containing lysates from R273Hp53-expressing A431cells was incubated with rabbit polyclonal CM-1 antibody and mouse monoclonal 1801 antibody against the N-terminus of p53. Detection using anti-rabbit IRDye 680LT (red) and anti-mouse IRDye 800CW (green) secondary antibodies confirmed that the Δ160p53 band corresponds to a C-terminal isoform of p53. (D) WB of H1299 cells transiently expressing the indicated constructs. (E) WB of cell lines expressing wild-type (HCT116, U2OS, A549) or mutant (SW480 [R273H/P309S], MDAMB231 [R280K], A431 [R273H], HT29 [R273H] and SKBR3 [R175H]) p53. (F) Cell lines harbouring endogenous wild-type (A549) or R273H mutant (A431) p53 were treated or not with control siRNA (ctl) or siRNA targeting both p53 transcripts (ex6 and ex7), full-length mRNA only (ex2/3) or Δ133p53 mRNA only (in4) before lysis and WB. (G) WB of H1299 cells stably expressing R273Hp53 and treated with DMSO, proteasome inhibitor MG132 or mRNA translation inhibitor cycloheximide (CHX). Shown are representative data of three independent experiments. The numbers in parenthesis indicate the amounts of protein for the indicated bands according to WB quantifications and normalization against α-tubulin or GAPDH and relative to the value indicated in bold and set to 1.0. In WBs for endogenous p53 cell lines the Δ160 lane is used as a marker lane showing Δ160p53 as transiently expressed in p53-null H1299 cells.
Figure 9(A) Complex IV activity was assessed by oxidation of cytochrome c in digitonized cellular extracts from the immortalized patient-derived skin fibroblasts and control healthy fibroblasts The results of three biological replicates were quantified, and normalized to control fibroblasts. Data are expressed as a mean ± SEM (n=3, *p=0.01). (B) Complex IV activity was assessed by oxidation of cytochrome c in digitonized cellular extracts from immortalized patient-derived skin fibroblasts treated with MG132 (1 μM), bortezomib (20 nM), or carfilzomib (50 nM) for 12 h and recovered for another 6 h. The results of three biological replicates were quantified and normalized to DMSO treated samples and the data are expressed as a mean ± SEM (n=3, *p=0.04 [DMSO vs MG132], **p=0.0025 [DMSO vs Bortezomib], *p=0.02 [DMSO vs Carfilzomib]). (C) Immortalized patient-derived skin fibroblasts transiently expressing wildtype or mutant COA7 (COA7-Y137C and COA7-Ex2∆) for 48 h were treated with bortezomib (20 nM) during the last 12 h. The fibroblasts were harvested, and complex IV activity was measured in digitonized cellular extracts. The results of three biological replicates were quantified and normalized to empty vector-bortezomib treated samples and the data are expressed as a mean ± SEM (n=3, *p=0.01 [Empty vector vs COA7- Wt], *p=0.005 [Empty vector vs COA7 - Y137C]). (D) Mitochondria isolated from control and patient fibroblast were solubilized in digitonin buffer and analyzed by 4-13 % gel BN-PAGE and Western blot. SC, supercomplexes. (E) Immortalized patient-derived skin fibroblasts transfected with plasmid encoding wtCOA7HIS and COA7-Y137CHIS were solubilized in DDM buffer and analyzed by 4-13 % gel BN-PAGE and Western blot. (F) Mitochondria were isolated from immortalized patient-derived skin fibroblasts treated with bortezomib (10 nM) for 12 h and recovered for another 6 h. Mitochondria were solubilized in digitonin buffer and analysed by 4-13 % gel BN-PAGE and Western blot. SC, supercomplexes.
| PenG exposure is associated with ROS production.Figure 8(A) RNA expression profiles of first line ROS defense systems in V. cholerae monitored by S1 nuclease mapping assay. Quantified average values (normalized to WT before PenG exposure) from three independent experiments are presented under the signals of each gene. White demarcation line between time points indicates that these data points were not adjacent in the original experiment but were cropped for presentation purposes. Source data is available as supplemental material. Note that the loading control is the same as in Fig. 2D, as these S1 assays were done on the same RNA preparations. (B and C) Quantification of the SOD and catalase activity values from the gel shown in Appendix Figure S9. Data are average values from three independent experiments normalized to untreated wild type. Data information: Data are mean of three independent biological replicates and error bars represent standard deviation. (D) Detection of protein oxidation after PenG treatment. To measure total cellular protein oxidation, OxyBlot Protein Oxidation Detection Kit (Sigma-Aldrich) was used. For positive protein oxidation control, oxidized standard marker (OXM) was used. Both DNP-derivatized or nonderivatized (negative control) protein samples were separated by polyacrylamide gel electrophoresis followed by Western blotting with-DNP specific antibody. (E) Time-dependent killing of respiratory chain mutants (∆nqrA, encoding NADH dehydrogenase and ∆ubiA, encoding an early step of ubiquinone synthesis) in the presence of PenG (100 µg/ml, 10xMIC). All strains were grown in LB 0.2 % glucose. Data information: Data are mean of three independent biological replicates and error bars represent standard deviation. (F) Growth-phase dependent killing assay of ROS detoxification systems (∆fur, ∆sodB, and ∆oxyR1∆katG∆katB). At designated time-points, a 5 ml aliquot was withdrawn and exposed to 100 µg/ml PenG for 3 h. CFU/mL for each time point was assessed before (solid lines) and after addition of PenG (dotted lines). Data information: Data are mean of three independent biological replicates and error bars represent standard deviation.
Figure 2(A) The vasculature in P5 retinas from (A) mTmG reporter mice with and without EC cilia was visualized by EGFP fluorescence.(B) The fractional distance migrated to the edge of the retina was quantified. There was no statistically significant difference (p=0.305, Student's 2-tailed t-test, n=6 mice for each group). The average value of 8 measurements (4 retina leaflets measured per eye on both eyes) was used for each mouse.(C) The number of vascular branchpoints was counted. There was no statistically significant difference (p=0.25, Student's 2-tailed t-test, n=6 for control and n=5 for mutant). (D) Control and mutant adult mouse aortas were stained for GM130 (green) to mark the Golgi, CD144 (red) to mark ECs and DAPI (blue) to label DNA.(E) The angle between the Golgi, nucleus and tissue axis was measured and binned as 'left', 'right', 'upstream' or 'downstream'.(F) Six areas in the aorta were analyzed.(G) There was no difference in Golgi distribution at any single region or overall, nor was there a difference in the variance of the individual un-binned angles. Error bars are +1 S.E.M. Scale bars are 500μm. n=3 control and mutant mice for each region except region 2 where n=3 control and n=2 mutant mice.
AGR2 status alteration impacts on client protein biogenesis.Figure 4A) The ERMIT-based validation of the complex formation between AGR2 and LYPD3 or CD59, but not OS9. The monomeric AGR2 E60A was used as bait, while LYPD3, CD59 and OS9 were used as prays (n=6). B-C) Expression and subcellular localization of CD59-GFP WT and C94S. D and E) Co-immunoprecipitation of CD59-GFP WT or CD59 C94S mutant with AGR2 WT (D) or AGR2 AA mutant (E) in HEK293T cells. F-I) FACS analysis of CD59 WT (F and G) or CD59 C94S (H and I) total (F and H) and membrane (G and I) expression in HEK293T cells silenced for AGR2 (siAGR2) and TMED2 (siTMED2) when compared to control (siGL2) cells (F and H) or silenced for TMED2 (siTMED2) and overexpressing AGR2 WT or AGR2 AA mutant (G and I). Data are representative of three to four independent experiments. Membranous CD59 expression was detected with anti-GFP APC. ns: non-statistically significant; for F (*): p=0.0385, (***): p=0.0003 (left panel) and p=0.0002 (right panel), respectively; for G (*): p=0.0334, (**) p=0.0036 and (***) p=0.0003; for H (*): p=0.0171 and (**): p=0.0084; and for I (**) p=0.0086 (left panel) and p=0.0076 (right panel), respectively.
a, b, Lysates from Δapg12 cells carrying only vector or 3 × HA-APG12 (a), and Δapg5, apg7-1, apg10-1 and Δapg1 cells carrying 3 × HA-Apg12 plasmid (b) were immunoblotted using anti-HA antibody.figf2a, b, Lysates from Δapg12 cells carrying only vector or 3 × HA-APG12 (a), and Δapg5, apg7-1, apg10-1 and Δapg1 cells carrying 3 × HA-Apg12 plasmid (b) were immunoblotted using anti-HA antibody. The positions of 3 × HA-Apg12 and the larger product (asterisks) are indicated. c, Immunoblot analysis of wild-type and Δapg12 cells harbouring HA-APG5 plasmid. d, Δapg5 Δapg12 cells were co-transformed with Myc-APG12 and HA-APG5. Their lysates were immunoprecipitated with anti-Myc or anti-HA antibodies and detected by immunoblotting using anti-Myc antibody. The position of the crossreacting IgG heavy chain is indicated
The rate of glucose uptake (a,e), lactate released after 4 h of incubation (b,f), glycolytic flux (c,g) and pentose–phosphate flux (d,h) were assessed in MEF and mouse cortical neurons in primary culture, as indicated, obtained from either WT or Pink1 KO (Pink1−/−) mice.f1The rate of glucose uptake (a,e), lactate released after 4 h of incubation (b,f), glycolytic flux (c,g) and pentose-phosphate flux (d,h) were assessed in MEF and mouse cortical neurons in primary culture, as indicated, obtained from either WT or Pink1 KO (Pink1−/−) mice. Blood concentrations of glucose (i) and lactate (j), and the rate of 14C-lactate appearance in the blood from acutely injected [U-14C]glucose in the tail vein (k) of WT and Pink1 KO mice were analysed. Data are expressed as mean±s.e.m. *P0.05 (Student's t-test; n=3-4 independent experiments).
Correlation of AR and UPR gene expression in prostate cancer cohorts.Figure 1Possible correlation between AR- and UPR-associated gene expression was assessed in the global gene expression data available in the TCGA Prostate Adenocarcinoma cohort (n = 190) (http://www.cbioportal.org/public-portal/index.do). Tumors were stratified according to AR status into three groups, that is ARlow (n = 60), ARmedium (n = 70), and ARhigh (n = 60). The levels of UPR gene expression in the three groups were compared using Pearson's correlation analysis by the R software and presented as a heatmap. There were significant differences between the three groups (Supplementary Table S2). The expression profiles of some prominent UPR genes from the data in (A), including ERN1 (IRE1), EDEM1, ATF6, and DNAJC3 (P58IPK), are presented. P-values of the different genes are given.
Figure 4(A,B) (A) Transient expression of PAP8NLSm5-GFP mutated in the NLS as described in Fig. EV4A. The circle marks the position of the nucleus (N) as observed with DIC in (B); yellow arrowheads show stromules (str). Scale bars equal 20 µm. C-G Pictures of representative genotypes obtained in the functional complementation test of pap8-1 using pP8::PAP8-NLSm5 (C, F) or pP8::PAP8ΔcTP (G) both constructions without GFP tags; ⌀ indicates pap8-1 without any PAP8 transgene. (D-G) Pictures using Keyence technology of plants with genotypes as labelled; transgenes expressed using the 1.1-kb PAP8 promoter. Scale bars equal 1 mm.