EMBO/sd-character-level-ner huggingface dataset:
Figure 1(A) γδ T cell concentration in the peripheral blood from healthy donors and indicated metastatic cancer patients. (B) RT-PCR analysis of the expression of NKG2D in γδ T cells isolated from indicated samples. (C) RT-PCR analysis of the expression of IFN-γ and TNF-α in γδ T cells isolated from the peripheral blood of healthy controls and metastatic cancer patients. (D) RT-PCR analysis of the expression of miR-125b-5p and miR-99a-5p in γδ T cells isolated from indicated samples. (E) RT-PCR analysis of the expression of miR-181a(-5p and -2-3p) in γδ T cells isolated from indicated samples.
Figure 5Puf3 is phosphorylated in WT cells and is hyper-phosphorylated in ρ0 cells. Puf3-FLAG was immunoprecipitated from the indicated strains and treated with/without λ-phosphatase, and analyzed by regular and Phos-tag SDS-PAGE. Puf3 phosphorylation in response to OXPHOS inhibitors and in OXPHOS mutants. WT cells were treated with antimycin (10 μM), KCN (10 μM) or oligomycin (10 μM) for 3 hours, or shifted from SCD to SCEG medium for 3 hours. Whole cell lysates were extracted and equal amount of proteins was loaded for western blot. Treatments and strains with Puf3 hyper-phosphorylation was highlighted in red. Glucose depletion was used as a positive control. Asterisk indicates a non-specific band. Puf3-15A mutation inhibits Puf3 hyper-phosphorylation in ρ0 cells. A cassette expressing WT or mutant Puf3-FLAG (11A or 15A) under the control of PUF3 promoter was inserted into the HO site in puf3∆ cells. The 15A mutant that blocks Puf3 hyper-phosphorylation is highlighted in red. Puf3-FLAG was immunoprecipitated from the indicated strains and analyzed by regular and Phos-tag SDS-PAGE. Puf3-15A mutation inhibits Puf3 hyper-phosphorylation upon oligomycin treatment and in ∆V and ρ0 cells. WT cells were treated with antimycin (10 μM), KCN (10 μM) or oligomycin (10 μM) for 3 hours, or shifted from SCD to SCEG medium for 3 hours. Whole cell lysates were extracted and equal amount of proteins was loaded for western blot. Asterisk indicates a non-specific band. Puf3-15A mutation downregulates Puf3-associated mitochondrial transcripts in ρ0 cells. ρ0 and ρ0 puf3-15A cells were cultured to log phase. RNAseq was performed with three biological replicates per genotype (Dataset EV7). All the transcripts and the Puf3-associated transcripts encoding mitochondrial proteins (gene lists in Table EV1D) are shown. The significantly changed transcripts (P value < 0.05; fold change > 1.5 or < 0.67) are highlighted with dashed lines; numbers of significantly changed transcripts are indicated. Puf3-15A mutation inhibits the upregulation of Mia40 in ∆V and ρ0 cells. Mia40 was endogenously tagged with FLAG. The selection markers were removed by the Cre-loxP system. Whole cell lysates were extracted and equal amount of proteins was loaded for western blot. Puf3-15A mutation decreases ∆ψm in ρ0 cells. Cells were cultured to mid-log phase and stained with 125 nM TMRM for FACS analysis. The red ] highlight two cell lines for comparison. Puf3-15A mutation has no effect on the proliferation of WT cells.Cartoon illustration that Puf3-hyperphosphorylation in ∆V and ρ0 cells upregulates Puf3-associated mitochondrial transcripts, especially Mia40, to enhance mitochondrial biogenesis and ∆ψm maintenance.
Figure 5A non-heme oxidase UndA is required for the production of a 11-carbon olefin, 1-undecene, a volatile. This volatile induces calcium signaling in AWB odor sensory neurons leading to aversion response (FLIGHT) as well as upregulation of pathogen-specific immune response genes (FIGHT) and resistance in C. elegans.
Centriole maturation and localization to the apical cell membrane are defective in Ccno‐deficient MCCs A, B IF analysis of mTEC cultures at ALI day 7 using antibodies against the centriole marker Centrin and CEP164, which marks maturing centrioles.Figure 6A, B IF analysis of mTEC cultures at ALI day 7 using antibodies against the centriole marker Centrin and CEP164, which marks maturing centrioles. At ALI day 7, centrioles of wild‐type MCCs are decorated with CEP164 and are mostly localized to the apical cellmembrane. The fewer centrioles of Ccno‐deficient MCCs do not colocalize with CEP164 and fail to reach the apical cell surface as seen in z‐projection. Lack of CEP164 indicates immature centrioles in Ccno‐deficient MCCs. (B) Total centriole numbers per MCC were counted in n = 11 MCCs for both genotypes using Imaris 7.7.2 software. C, D Double IFstaining using Centrin‐ and CP110‐specific antibodies shows an almost complete absence of CP110 from Centrin‐positive centrioles in the majority of wild‐type MCCs at day 7 of ALI cultures. Most Ccno‐deficient MCCs exhibit pronounced CP110 staining that colocalizes with Centrin as seen in z‐projection. (D) CP110‐positive MCCs were counted in wild‐type and Ccno‐deficient MTEC cultures and the percentages of CP110‐positive MCCs from total MCCs calculated.
BAG3 specifically directs Hsp70 substrates to the aggresome by loading misfolded proteins on to dynein.Figure 2(A) GST pull‐down analysis as in Fig 1I, but performed in cells transfected for 36 h with full‐length FLAG‐BAG3 (I) or the BAG3 deletion mutants FLAG-(Δ421-498)-BAG3 (II), FLAG-BAG‐domain (III), and FLAG-PxxP-BAG‐domain (IV). Asterisk, nonspecific band. (B) HEK cells were transfected for 18 h with SODG85R‐GFP, along with the indicated FLAG‐tagged BAG3 constructs (shown in A) and HA‐tagged Hsp70 (HA‐Hsp70). SODG85R‐GFP showed three distinct localization patterns: cytoplasmic, pre‐aggresomal and aggresomal (see representative pictures on the right and supplementary Fig S2F online). The percentages of cells bearing pre‐aggresomal (upper panel) and aggresomal (lower panel) SODG85R‐GFP are shown. Scale bar, 10 μm. (C) HEK cells transfected with SODG85R‐GFP, BAG3 and Hsp70 were additionally transfected with FLAG‐p50, as indicated. Diagrams show the percentages of cells with pre‐aggresomal and aggresomal SODG85R‐GFP. Right panel: representative image showing pre‐aggresomal localization of FLAG‐p50 and SODG85R‐GFP. Scale bar, 5 μm. (D) GST pull‐down analysis as in (A), but in HEK cells transfected for 18 h with SODWT‐GFP or mutant SODG85R‐GFP, along with FLAG‐BAG3. (E) HEK cells were transfected with the indicated plasmids with nonsense or bag3 siRNA. At 6 h after transfection, lactacystin (10 μM) or GST as vehicle control was added for GST and GST pull‐down analysis was performed as in (D). (F) Upper panel: sucrose‐density gradient analysis of extracts from HEK cells transfected with SODG85R‐GFP and GST‐DIC, along with FLAG‐BAG3 or vector control. Note the immunoreactive bands in high‐density fraction 14 on BAG3 overexpression (arrow). Migration of a 20S marker protein is indicated. Lower panel: GST‐DIC pull‐down analyses of dynein complexes present in sucrose fraction 6 and 14. (G) GST pull‐down analysis of SODG85R‐GFP as in (D) but in cells transfected with FLAG‐tagged BAG3 constructs (I-IV; indicated as in A). (H) GST pull‐down analysis of SODG85R‐GFP as in (D) but in cells treated with PYR41 (10 μM; 12 h), as indicated. DIC, dynein intermediate chain; DMSO, dimethyl sulphoxide; GST, glutathione‐S‐transferase; HA, haemagglutinin; Hsp70, heat‐shock protein 70; NS, not significant; nons, nonsense; PxxP, proline‐rich repeat; siRNA, short‐interfering RNA; SOD, superoxide dismutase; SODWT, wild‐type SOD.
Figure 5Total RNA isolated from untreated (UT), PMA-treated, Vs-treated and Vs+PMA treated U1 was examined by NanoString technology to assess the expression of genes responsive to oxidative stress and HIV. (A) Differentially expressed genes (DEGs) under the indicated conditions. (B) The Venn diagram of DEGs significantly perturbed under different comparison conditions. Total RNA isolated from untreated (UT), PMA-treated, Vs-treated and Vs+PMA treated U1 was examined by NanoString technology to assess the expression of genes responsive to oxidative stress and HIV. (C Heat map showing functional categories of DEGs under PMA/UT, Vs/UT and PMA+Vs/UT comparisons. mRNA counts were normalised using the internal control β2 microglobulin (B2M), and fold change (FC) was calculated using the nSolver 4.0 software. Genes showing an absolute FC >1.5, and P <0.05 were considered as significantly altered. Total RNA isolated from untreated (UT), PMA-treated, Vs-treated and Vs+PMA treated U1 was examined by NanoString technology to assess the expression of genes responsive to oxidative stress and HIV. D) Heat map showing functional categories of DEGs under PMA/UT, Vs/UT and PMA+Vs/UT comparisons. mRNA counts were normalised using the internal control β2 microglobulin (B2M), and fold change (FC) was calculated using the nSolver 4.0 software. Genes showing an absolute FC >1.5, and P <0.05 were considered as significantly altered. E, F. Vs-treated or untreated U1 cells were exposed to Bay11-7082 (7.5 µM). PMA mediated HIV reactivation at 12 h was monitored by gag RT PCR and p24 ELISA.
Figure 3A Schematic graph of structural domains of ATRX protein. Arrows indicated target sites of individual sgRNAs. B Western blots of extracts from U87 cells infected with Lenti-CRISPR(puro) encoding individual ATRX-targeted sgRNAs. The extracts were isolated after puromycin selection, and assayed for ATRX depletion levels using antibody against ATRX or ACTIN control. The asterisks (*) denote the two sgATRXs (sgA#10 and sgA#19) used in following experiments. C ATRX immunofluorescence staining of U87 cells infected with lentiviral control sgRosa, sgA#10, or sgA#19. D, DAPI. Scale bar, 50 µm. D Schematic showing competition-based proliferation assay of individual sgRNAs. As sgRNA expression in Lentiviral LRG vector is linked to GFP, percentage changes of GFP+ cells reflect the fitness induced by CRISPR-based gene targeting. Bar graphs show time-course analyses of U87 (E) transduced with lentiviral LRG vector encoding sgRosa control or ATRX-targeted sgA#10 or sgA#19. The graph plots the percentage of GFP+ cells over time following transduction with indicated sgRNAs. Data are expressed as means ± s.d., * denotes ns (not significant); **, p < 0.01; ***, p < 0.001. N = 3, unpaired t test. Bar graphs show time-course analyses of U2OS cells (F) transduced with lentiviral LRG vector encoding sgRosa control or ATRX-targeted sgA#10 or sgA#19. The graph plots the percentage of GFP+ cells over time following transduction with indicated sgRNAs. Data are expressed as means ± s.d., * denotes ns (not significant); **, p < 0.01; ***, p < 0.001. N = 3, unpaired t test. G, H 53BP1/TRF2 co-immunofluorescence (G) revealed strongly increased TIF formation in lentiviral sgA#10 or sgA#19 transduced U87 cells assayed at day 45 but not day 15 post infections (H). Scale bar, 15 µm. (H) Percentages of cells containing > 4 TIFs are expressed as means ± s.d., N = 3, unpaired t test. I, J 53BP1/TelG immuno-FISH (I) revealed strongly enhanced TIF formation in lentiviral sgA#10 or sgA#19 transduced IMR90 cells assayed at day 35 but not day 10 post infections (J). Scale bar, 10 µm. (J) Percentages of cells containing > 4 TIFs are expressed as means ± s.d., N = 3, unpaired t test. K Bar graphs show time-course analyses of IMR90 cells transduced with lentiviral sgRosa, sgA#10 or sgA#19. Data are expressed as means ± s.d., * denotes ns (not significant); **, p < 0.01; ***, p < 0.001. N = 3, unpaired t test.
Figure 5(A, B) NADPH levels (A) and NOX (B) level was measured in WT and MCK-KO muscles at rest and after a bout of low-intensity exercise for 50 min (n = 6, means ± SEM, * p < 0.05, two-tailed student's t-test and two-way ANOVA followed by Bonferroni post-hoc testing). (C-G) L6 myotubes were transduced with lentiviral expression plasmids for Flag-ALDH1L1 and GFP. NADPH levels (C), H2O2 levels (D, E) , Mitochondrial respiration (F, G) were measured from these cells in the presence and absence of NADPH oxidase inhibitor apocynin (n = 3 per group, for (C-E) and n = 5 for (F, G), means ± SEM, * p < 0.05, two-tailed student's t-test and two-way ANOVA followed by Bonferroni post-hoc testing). Single and double knockdowns of Dnmt3a and Aldh1l1 in L6 myotubes were achieved by lentiviral transduction. NADPH levels (H) and NOX activity (I) were measured in single and double knockdowns of Dnmt3a and Aldh1l1 in L6 myotubes. (n = 3, means ± SEM, * p < 0.05, two-tailed student's t-test and two-way ANOVA followed by Bonferroni post-hoc testing). (J, K) H2O2 levels were measured in single and double knockdowns of Dnmt3a and Aldh1l1 in L6 myotubes. (J: n = 3 and K: n = 6 for Control, Aldh1l1 KD, Aldh1l1 KD + Dnmt3a KD, n = 5 for Dnmt3a KD, means ± SEM, * p < 0.05, two-tailed student's t-test and two-way ANOVA followed by Bonferroni post-hoc testing).
Figure 1A Lysates from S2 cells expressing HA-tagged versions of MBP or AGO1 (wild-type or pocket mutants) were immunoprecipitated using an anti-HA antibody. Inputs (1% for the HA-tagged proteins and 2.5% for GW182) and immunoprecipitates (20% and 35%, respectively) were analyzed by western blotting using an anti-HA antibody. Endogenous GW182 was detected using anti-GW182 antibodies. The association between HA-AGO1 and endogenous miR-2a was analyzed by northern blotting. tRNAAla served as a loading control. B Interaction of HA-tagged AGO2 (wild-type or pocket mutants) with endogenous TNRC6A in HEK293T cells. C,D Interaction of HA-tagged AGO2 (wild-type or pocket mutants) with GFP-tagged TNRC6B and TNRC6C in HEK293T cells. HA-tagged MBP served as a negative control. Inputs (1.5% for the HA-tagged proteins and 2% for the GFP-tagged proteins) and immunoprecipitates (20% and 35%, respectively) were analyzed by western blotting using the corresponding antibodies. The presence of endogenous miR-16 in the immunoprecipitates was determined by northern blotting (panel D). tRNAAla served as a loading control. AGO mutants are described in Appendix Fig S1; Appendix Table S1.
Hsp70 inhibits inflammation through Siglec‐5 in THP1 cellsSecretion of pro‐inflammatory cytokine TNFα by THP1 cells transfected with an empty vector (EV) or Siglec‐5 (Sig5) expression plasmid was evaluated by ELISA after concurrent exposure with LPS and 10 μg/ml Hsp70 or LPS alone.Figure 5Secretion of pro‐inflammatory cytokine TNFα by THP1 cells transfected with an empty vector (EV) or Siglec‐5 (Sig5) expression plasmid was evaluated by ELISA after concurrent exposure with LPS and 10 μg/ml Hsp70 or LPS alone. Secretion of TNFα was reduced only in THP1 Sig5 cells exposed to both LPS and Hsp70 but not LPS alone. Similarly, IL‐8 secretion from THP1 EV and Sig5 cells was evaluated by ELISA after concurrent exposure with LPS and 10 μg/ml Hsp70. Secretion of IL‐8 was also reduced in only THP1 Sig5cells exposed to both LPS and Hsp70 but not LPS alone. THP1 Sig5 cells were incubated with Hsp70, and the levels of SHP‐1 recruitment to Siglec‐5 were evaluated by immunoprecipitation of Siglec‐5 and Western blot for SHP‐1. Higher levels of SHP‐1 were co‐immunoprecipitated with Siglec‐5 when the cells were incubated with Hsp70. Western blots demonstrate phosphorylated p38 (p‐p38) was reduced in THP1Siglec‐5cells when exposed to both LPS and Hsp70 in comparison with LPS alone. This reduction was also greater in THP1Siglec‐5cells in comparison with the control THP1cells. Numbers below immunoblots indicate densitometric analysis of each band normalized to the respective cell line's unstimulated control group, divided by the respective loading control (total p38). Secretion of pro‐inflammatory cytokine IL‐8 by PMA‐differentiated THP1 EV or THP Sig5 cells was evaluated by ELISA after concurrent exposure with 10 ng/ml LPS with an anti‐Hsp70 antibody or isotype‐matched antibody. Secretion of IL‐8 was increased in only THP1 Sig5 cells exposed to LPS and an anti‐Hsp70 antibody in comparison with the isotype control antibody, but this increase was not replicated in THP1 EV cells.
Figure 4Cells were infected with a moi 10 of influenza (Flu) (A) or SFV (B) for 4, 8 or 24h. Levels of EGOT (A-E) were calculated by qRT-PCR in RNA isolated from the control, treated or infected HuH7 cells. GAPDH expression was also evaluated in all cases and used as a reference to calculate the relative levels of each transcript. The experiment was performed twice in triplicate (n=6). Each value shows the average and error bars indicate standard deviations. Asterisks mark significant differences (ns, non-significant; * denotes p<0.05, ** p<0.01) obtained with a two-tailed non-parametric Mann-Whitney U-test. Cells were infected with HCV for 72 or 96h (C). Some infected cells were treated with inhibitors of replication (DAA) the last 24 or 48h of infection (C). All HCV infections were done with a moi of 0.3. Levels of EGOT (A-E), or genome equivalents (GE) of HCV RNA per μg of total RNA (C-D) were calculated by qRT-PCR in RNA isolated from the control, treated or infected HuH7 cells. GAPDH expression was also evaluated in all cases and used as a reference to calculate the relative levels of each transcript. The experiment was performed twice in triplicate (n=6). Each value shows the average and error bars indicate standard deviations. Asterisks mark significant differences (ns, non-significant; * denotes p<0.05, ** p<0.01) obtained with a two-tailed non-parametric Mann-Whitney U-test. Cells were infected with HCV for 5, 12 (D). All HCV infections were done with a moi of 0.3 except the one examined at 12 hpi, which was performed with a moi of 1 of control HCV virus or UV-irradiated virus (UV-HCV). Levels of EGOT (A-E) or genome equivalents (GE) of HCV RNA per μg of total RNA (C-D) were calculated by qRT-PCR in RNA isolated from the control, treated or infected HuH7 cells. GAPDH expression was also evaluated in all cases and used as a reference to calculate the relative levels of each transcript. The experiment was performed twice in triplicate (n=6). Each value shows the average and error bars indicate standard deviations. Asterisks mark significant differences (ns, non-significant; * denotes p<0.05, ** p<0.01) obtained with a two-tailed non-parametric Mann-Whitney U-test. E. Cells were treated with oncostatin M (OSM), LPS or Imiquimod (Imi) for 24h. Levels of EGOT (A-E), CXCL10 mRNA (E) were calculated by qRT-PCR in RNA isolated from the control, treated or infected HuH7 cells. GAPDH expression was also evaluated in all cases and used as a reference to calculate the relative levels of each transcript. The experiment was performed twice in triplicate (n=6). Each value shows the average and error bars indicate standard deviations. Asterisks mark significant differences (ns, non-significant; * denotes p<0.05, ** p<0.01) obtained with a two-tailed non-parametric Mann-Whitney U-test.
T cell factor-4 is responsible for TDG controlling neural differentiation (a) Diagram of the TCF-4 promoter region.Figure 7(a) Diagram of the TCF-4 promoter region. (b) TDG ChIP-qPCR revealed that TDG mainly binds to TCF-4 CpG islands. Seven regions (from -5 kbp to -200 bp) of the promoter were analyzed. -500 bp and -200 bp regions were located in the CpG island of the TCF-4 promoter. (n=4 biological repeats). Data information: Error bars represent the means ± S.E.M. Two tailed unpaired T-tests were used to analyze the data, n.s. (no significant difference), p<0.05(*), p<0.01(**). and p<0.001(***). (c-f) MeDIP-qPCR showed that 5fC, 5caC, and 5mC are increased in the TCF-4 promoter, while 5hmC is decreased when TCF20 is insufficient. The MeDIP assay measure the region -500 bp from the TCF-4 promoter. (n=4 biological repeats) Data information: Error bars represent the means ± S.E.M. Two tailed unpaired T-tests were used to analyze the data, n.s. (no significant difference), p<0.05(*), p<0.01(**). and p<0.001(***). (g) NSCs were isolated from C57B6/J E13.5 embryonic brains and then were infected with a lentivirus containing a TCF20 shRNA or with a control plasmids. Immunostaining of 5fC shows that TCF20 depletion increased 5fC levels. White dashed lines show the cells infected with lentivirus. Scalebar 25 μm (h) Statistical analysis of 5fC fluorescence level. n=20 cells, and P<0.0001 Data information: Error bars represent the means ± S.E.M. Two tailed unpaired T-tests were used to analyze the data, n.s. (no significant difference), p<0.05(*), p<0.01(**). and p<0.001(***). (i) Immunostaining of 5caC shows that TCF20 depletion increased 5caC level. White dashed lines show the cells infected with lentivirus. Scalebar 25 μm. (j) Statistical analysis of the 5caC fluorescence level. n=20 cells, and P<0.0001. Data information: Error bars represent the means ± S.E.M. Two tailed unpaired T-tests were used to analyze the data, n.s. (no significant difference), p<0.05(*), p<0.01(**). and p<0.001(***). (k, l) Overexpression of TDG and TCF-4 rescued the TCF20 knockdown phenotype in brains; n=3 brains. Scalebar 50 μm Data information: Error bars represent the means ± S.E.M. Two tailed unpaired T-tests were used to analyze the data, n.s. (no significant difference), p<0.05(*), p<0.01(**). and p<0.001(***).
Lysosomal fusion is impaired in LSD cells.Figure 1(A) EGFR degradation was followed in MSD, MPS‐IIIA and WT MEFs by treating the cells with EGF for the indicated time to stimulate EGFR internalization. The cells were then immediately lysed and subjected to anti‐EGFR blotting. The amount of remaining EGFR was quantified by densitometry analysis (Image J) of the blot and expressed in the chart as % of the EGFR amount present at time T0 (100%). The values in the chart represent the mean±s.e.m. values of three independent experiments. (B) MSD, MPS‐IIIA and WT MEFs cells loaded with dextran (alexafluor‐594 conjugated) were labelled with anti‐LAMP1 antibody and the percentage of dextran co‐localizing with LAMP1 was evaluated. The chart displays merge values (means±s.e.m.) that represent the percentage of dextran co‐localizing with LAMP1 measured in 15 different cells of triplicate experiments. (C) The rate of lysosome fusion with autophagosomes was monitored in MSD, MPS‐IIIA and WT MEFs transfected with a tandem fluorescently tagged LC3 ( Kimura et al, 2007). The rate of autophagosome maturation reflected the percentage of the LC3 'unfused' (green/red fluorescence ratio) at each time (1 and 3 h) after bafilomycin removal (T0). The percentage of LC3 'unfused' was displayed versus the value at T0 (assumed to be 100%). Values are represented as means±s.e.m. of triplicate experiments. *P0.05, Student's t‐test: (A): WT versus MSD and WT versus MPS‐IIIA; (B, C): WT versus MSD and WT versus MPS‐IIIA at each time point. Scale bar: 10 μm (B, C).