EMBO/sd-character-level-ner huggingface dataset:
Figure 3Bronchial epithelial (BEAS-2B) cells were challenged with PBS (as control) or with 2 µg/mL Poly(I:C) (PIC) or with influenza A/Scotland/20/74 (H3N2) virus (IAV) at MOI=1 for 4 h and treated or not with succinate (Suc) for 20 h. (a) Levels of IL-6 and IL-8/CXCL8, as measured by ELISA in the supernatants of cells stimulated with (PIC) and subsequently treated or not with succinate. Data information: Data are represented as the mean ± SEM of 4 (a) independent experiments. Statistical analysis was performed using the Kruskal-Wallis test with Dunn's post-test (*P < 0.05). Bronchial epithelial (BEAS-2B) cells were challenged with PBS (as control) or with 2 µg/mL Poly(I:C) (PIC) or with influenza A/Scotland/20/74 (H3N2) virus (IAV) at MOI=1 for 4 h and treated or not with succinate (Suc) for 20 h. A neuraminidase (NA) activity test (b) were used to assess the production of physical (b viral particles in the supernatants of IAV-infected cells, treated or not with succinate. Data information: Data are represented as the mean ± SEM of 6 (b, c, e) independent experiments. Statistical analysis was performed using the Kruskal-Wallis test with Dunn's post-test (*P < 0.05). Bronchial epithelial (BEAS-2B) cells were challenged with PBS (as control) or with 2 µg/mL Poly(I:C) (PIC) or with influenza A/Scotland/20/74 (H3N2) virus (IAV) at MOI=1 for 4 h and treated or not with succinate (Suc) for 20 h. a Plaque-Forming Units assay (c) were used to assess the production of infectious (c) viral particles in the supernatants of IAV-infected cells, treated or not with succinate. Data information: Data are represented as the mean ± SEM of 6 (b, c, e) independent experiments. Statistical analysis was performed using the Kruskal-Wallis test with Dunn's post-test (*P < 0.05). Bronchial epithelial (BEAS-2B) cells were challenged with PBS (as control) or with 2 µg/mL Poly(I:C) (PIC) or with influenza A/Scotland/20/74 (H3N2) virus (IAV) at MOI=1 for 4 h and treated or not with succinate (Suc) for 20 h. scanning electron microscopy (d) were used to assess the production of physical viral particles in the supernatants of IAV-infected cells, treated or not with succinate. IAV particles budding are colored in purple. Scale bar: 1 µm. Bronchial epithelial (BEAS-2B) cells were challenged with PBS (as control) or with 2 µg/mL Poly(I:C) (PIC) or with influenza A/Scotland/20/74 (H3N2) virus (IAV) at MOI=1 for 4 h and treated or not with succinate (Suc) for 20 h. (e) A neuraminidase activity test was also applied in cells infected or not by IAV and treated or not with malonate (a succinate dehydrogenase inhibitor), in comparison with succinate. Data information: Data are represented as the mean ± SEM of e) independent experiments. Statistical analysis was performed using the Kruskal-Wallis test with Dunn's post-test (*P < 0.05).
Figure 8A, B, E, F Immunoblot analysis with indicated antibodies of protein extracts prepared from root tips of 10-d-old Arabidopsis WT heat-stressed seedlings (39ºC) 0, 20, 40 and 60 min after the onset of HS. For CHX treatment in B, the seedlings were pre-treated with 200 ng μL-1 CHX for 30 min before HS. The charts show SnRK1 activity, expressed as the ratio of phosphorylated to total SnRK1 protein. The data represent mean ratios of integrated band intensities (for both isoforms) normalized to 0 min ± SD from at least four different experiments. P values denote statistically significant differences for comparisons to 0 min (two-tailed t-test). C, Localization of GFP-SnRK1α1 and GFP-SnRK1α2 in root cells of 5-d-old Arabidopsis WT seedlings incubated at 39°C and imaged at the indicated time points. Scale bars = 10 μm. D, Quantification of GFP-SnRK1α1 and GFP-SnRK1α2 foci in the experiment shown in C. Data represent means ± SD of at least 16 replicate measurements. The experiment was repeated three times with similar results. ***P < 0.05 (two-tailed t-test). E, F, Immunoblot analysis with indicated antibodies of protein extracts prepared from root tips of 10-d-old Arabidopsis tsn1 tsn2 (E) or tsn1 tsn2 expressing ProTSN2:GFP-TSN2 (F) heat-stressed seedlings (39ºC) 0, 20, 40 and 60 min after the onset of HS. The charts show SnRK1 activity, expressed as the ratio of phosphorylated to total SnRK1 protein. The data represent mean ratios of integrated band intensities (for both isoforms) normalized to 0 min ± SD from at least four different experiments. P values denote statistically significant differences for comparisons to 0 min (two-tailed t-test). G, Expression levels of DIN2 and DIN6 in Arabidopsis WT, tsn1 tsn2, tsn1 tsn2;TSN2 and snrk1α1-/- snrk1α2-/+ 10-d-old heat-stressed seedlings relative to unstressed controls. For CHX treatment, the WT seedlings were pre-treated with 200 ng μL-1 CHX for 30 min before HS. Upper and lower box boundaries represent the first and third quantiles, respectively. Horizontal lines mark the median of five replicate measurements, and whiskers mark the highest and lowest values. Means with different letters are significantly different at P < 0.05 (one-way ANOVA).
Alvocidib reduces tumour growth in vivo.Figure 6Tumour growth of in vivo xenografts of H3122 CrizR1 cell lines in response to crizotinib and correspondent tumour weights (P.O. control n=5; I.P. control n=4; crizotinib n=8 Tumour weights, crizotinib P= 0.44 Tumour growth of in vivo xenografts of H3122 CrizR1 cell lines in response to alvocidib and correspondent tumour weights (P.O. control n=5; I.P. control n=4 and alvocidib n=6). Tumour weights, , alvocidib P=0.03. C) Tumour growth of in vivo xenografts of Ste-1 AlectR cell lines in response to alvocidib and correspondent tumor weights (I.P. control n=6; alvocidib n=4). Tumour weights alvocidib P=0.004. D) Number of cleaved caspase-3 positive nuclei in alvocidib treated tumours compared to controls. E) Ki67 and p21 staining of CrizR1 xenograft tumours. Scale bar = 200μm. Quantitative analysis of IHC staining is presented on the right. Ki67 P=0.0004; p21 P=0.02. F) qPCR of Survivin (P=0.0009), MCL-1 (P=0.04), MYC (P=0.007) and BIM (P=0.03) in Ste-1 AlectR cells xenografts treated with alvocidib (Control n=6; alvocidib n=4). G) Proliferation assay of H3122 parental and isogenic drug-resistant cell lines, treated with DMSO, 100nM lorlatinib, 200nM alvocidib or 50nM dinaciclib for 72h (n=4). H) Growth curve of CrizR1 xenograft tumors in response to lorlatinib (Control n=7; lorlatinib n=7). I) Model suggesting the clinical sequencing of CDK inhibitors post-ALK inhibition failure as a potential alternative to chemotherapy.
Figure 5a) Heat shock proteins, HSP90AA1 and HSP90AB1, were destabilised during SARS-CoV-2 infection of Caco-2 cells (circular plots, left side (n=3); see 1A for circular plot legend). Significant protein changes (abundance and thermal stability) are represented as circle plots for individual proteins, as previously described (Becher et al, 2018). Inner circle corresponds to protein abundance and the outer circle corresponds to stability changes (n=3). Asterisk denotes significant regulation (see methods). b) The corresponding HSP90 inhibitor, Tanespimycin, effectively suppressed SARS-CoV-2 proliferation (n=2). Calu-3 cells were pre-incubated with the compound, followed by the addition of SARS-CoV-2 and subsequent co-incubation in the presence of the compound for 20-24 hours. Viral load was quantified in parallel using antibody-based detection of double stranded RNA (orange). Compound toxicity in Calu-3 cells was assessed (purple) treated with the compound alone for 20-24 hours. c) The aryl hydrocarbon hydroxylase (Cyp1a1) increased in abundance between 4-12 hours post infection and was then reduced between 24-48 hours post infection (circular plot, left, (n=3); see 1A for circular plot legend). Circular plots displayed as described in panel a. Asterisk denotes significant regulation (see methods). d) The Cyp1a1 inhibitor suppressed SARS-CoV-2 proliferation (n=2). Dose response curves were performed as described in 4A. See Appendix Figure S7 for additional tested drugs. Data displayed as described in 5B.
Figure 5GFP-Parkin stably expressing HeLa cells were mock-transfected (Mock) or transfected with siRNA (KD) for Stx17 or PGAM5. At 48 h after siRNA transfection, the cells were transfected with a plasmid encoding FLAG-DFCP1, incubated for 24 h, treated with 20 μM CCCP for 2 h and then immunostained for FLAG. Scale bars, 5 μm. The bar graph below shows the Manders' coefficients for the colocalization of GFP-Parkin and FLAG-DFCP1. Values are means ± SEM (n = 3). **P<0.01 as compared with Mock (paired Student's t-test). GFP-Parkin stably expressing HeLa cells with mock treatment or depleted of Stx17 or PGAM5 were incubated with 20 μM CCCP for 2 h and immunostained for LC3. Scale bar, 5 μm. The bar graph below shows the Manders' coefficients for the colocalization of GFP-Parkin and LC3. Values are means ± SEM (n = 3). **P<0.01 as compared with Mock (paired Student's t-test). GFP-Parkin stably expressing HeLa cells with mock treatment or depleted of Stx17 or PGAM5 were incubated in the absence (Vehicle) or presence of 20 μM CCCP (+CCCP) for 2 h and analyzed by IB using the indicated antibodies.
Figure 5Hela cells were co-transfected with an RFP-based TDP-NLS reporter and GFP or GA175-GFP. 24 h after transfection, cells were treated with rolipram (30 µM) for 16h. In the immunofluorescence GFP is not shown because diffuse GFP expression would hide the cytoplasmic RFP-reporter. White arrows indicate cells with cytoplasmic TDP-43. (B) Automated quantification of cells with cytoplasmic TDP-NLS reporter in GFP and GA175-GFP positive cells. n=4 biological replicates. In total 345 GFP and 386 GA175-GFP cells treated with vehicle, 371 GFP and 404 GA175-GFP cells treated with rolipram were analyzed. Scatter plot with bar-graphs of mean ± SD. One-way ANOVA with Tukey's multiple comparisons test. Hela cells were co-transfected with an RFP-based TDP-NLS reporter and GFP or GA175-GFP. 24 h after transfection, cells were treated with rolipram (30 µM) for 16h. (C) RFP-NLSTDPWT mRNA expression levels were measured by qPCR. n=3 biological replicates. Bar-graphs of mean ± SD. One-way ANOVA with Tukey's multiple comparisons test. Hela cells were co-transfected with the RFP-based TDP-NLS reporter, GFP or GA175-GFP, as well as PSMD11 or empty vector. Image analysis as in (A). n=4 biological replicates. Scatter plot with bar-graphs of mean ± SD. One-way ANOVA with Tukey's multiple comparisons test. 354 GFP and 330 GA175-GFP cells with vector, 367 GFP and 369 GA175-GFP cells with PSMD11 in total were analyzed. Hela cells were co-transfected with the RFP-based TDP-NLS reporter, GFP or GA175-GFP, as well as PSMD11 or empty vector. F) RFP-NLSTDPWT mRNA expression levels were measured by qPCR. n=3 biological replicates. Bar-graphs of mean ± SD. One-way ANOVA with Tukey's multiple comparisons test. Immunofluorescence of Hela cells transfected with GFP or GA175-GFP showing reduced poly-GA aggregation upon rolipram treatment (30 µM, 16 h). (H) Automated quantification of poly-GA aggregate number per cell. n=3 biological replicates. In total 223 cells treated with vehicle and 286 cells treated with rolipram were analyzed. Scatter plot with bar-graphs of mean ± SD. Unpaired two-tailed t-test with Welch's correction. (I) GA-GFP mRNA expression levels were measured by qPCR. n=3 biological replicates. Bar-graphs of mean ± SD. Unpaired two-tailed t-test with Welch's correction.
Figure 2(A) Schematic representation of AFR, consisting of N-terminal VHH fused via a 20xGGS linker to an engineered TNF (e-TNF) variant and C-terminal affinity tag. (B,C) Viability of L929 parental or mCD20-expressing cells after 72h stimulation with mTNF or sc mTNF mutant Y86F fused to a BcII10 or mCD20 VHH (B) and viability of MCF7 parental or hCD20-expressing cells after 72h stimulation with hTNF or sc hTNF mutant Y87F fused to a BcII10 or hCD20 VHH (C). Cell viability was measured via an ATP luminescence assay. Each point is the mean of three replicates and error bars are SEM. (D) IL-8 concentration in HUVEC supernatant after 24h stimulation with the indicated concentration of stimulus, measured via ELISA.
Figure 7A. C4-2 PCa cells infected with virus expressing EV, SPOP Q165P or F133V were harvested for western blot analysis. B MTS was performed to compare the cell growth between EV, Q165P, F133V C4-2 groups after the treatment with indicated inhibitors (2 µM of JQ1, 2 µM of CPI-637, 2 µM NEO2734) for 72 h. All data shown are means ± SEM. The P value was calculated by the unpaired two-tailed Student's t-test; n.s., not significant, * P < 0.05, ** P < 0.01, *** P < 0.001. C. Clonogenic survival assay was performed to determine the sensitivity of NEO2734 in SPOP mutant C4-2 cells. The survival curve showed IC50 for EV (1.01 µM), SPOP Q165P (0.621 µM) and SPOP F133V cells (0.395 µM). D, E. C4-2 cells expressing EV, Q165 or F133V constructs were treated with 1 µM of NEO2734 for 4 days and cultured for another 8 days before harvesting. The number of colonies with more than 50 cells was counted. Representative images of colonies are shown in (D) with quantification data in (E). All data shown are means ± SEM. The P value was calculated by the unpaired two-tailed Student's t-test; * P < 0.05, *** P < 0.001. F - H. A schematic depicts the procedure of the establishment of SPOP mutant xenograft models and inhibitor administration (F). When the tumor reached 100 mm3, mice were treated with vehicle (40% polyethylene glycol), JQ1 (50 mg/kg) or NEO2734 (30 mg/kg) five days a week for three consecutive weeks. Tumors isolated from mice at day 21 of treatment were photographed (G) and tumor growth are shown in (H). All data shown are means ± SEM. The P value comparing the tumor volume at day 21 post- treatment was calculated by the unpaired two-tailed Student's t-test; * P < 0.05, ** P < 0.01; *** P < 0.001.
Figure 4(A) Immunofluorescence microscopy analysis for colocalization of M.tb with Lamp-1 in WT BMMs pre-treated with EVs from macrophages infected with WT, ∆secA2 or secA2-complemented (∆secA2-C) M.tb CDC 1551 strains. The cells were pre-treated with EVs supplemented with recombinant mouse IFN-γ for 5 hr and subsequently infected for 24 hr with GFP-expressing M.tb. (B) quantitative analysis for the colocalization of M.tb with Lamp-1. Data shown are the mean ± SD of three independent infections and all data shown are representative of at least three independent experiments. Scale bar, 5 μM ; * p < 0.05 and ** p < 0.01 by two-tailed Student's t-test. (C) M.tb CFU in WT BMMs pre-pretreated with recombinant mouse IFN-γ and EVs from macrophages infected with WT, ∆secA2 or secA2-complemented (∆secA2-C) M.tb CDC 1551 strains. Data shown are the mean ± SD of three independent infections and all data shown are representative of at least three independent experiments. n.s., not significant; * p < 0.05 and ** p < 0.01 by two-tailed Student's t-test. (D) and (E) liposomes containing RNA isolated from EVs released from BMMs infected with WT M.tb were also added to cells 5 hr prior to infection. Data shown are the mean ± SD of three independent infections and all data shown are representative of at least three independent experiments. Scale bar, 5 μM n.s., not significant (F) liposome-encapsulated RNA isolated from EVs released from BMMs infected with WT M.tb was included during EV and IFN-γ pre-treatment. Data shown are the mean ± SD of three independent infections and all data shown are representative of at least three independent experiments. n.s., not significant; * p < 0.05 and ** p < 0.01 by two-tailed Student's t-test.
Genome-wide analysis of BrdU incorporation in cells exposed to MMS.Figure 1Wild type, rad9∆ and mrc1∆ cells were arrested in G1 with α-factor and were released synchronously into S-phase with the addition of pronase, in medium containing BrdU and 0.1% or 0.033% MMS. Cells were collected at the indicated time points. Analysis of DNA content by flow cytometry. Analysis of BrdU incorporation by BrdU-IP-chip 45 minutes after release into S phase in cells treated with 0.1% MMS as described in (A). A representative region on chromosome VII is shown. The position of replication origins is indicated above the map. Asterisks: late origins firing in mrc1∆ cells. Quantification of the BrdU incorporation (signal log ratio) at 384 early and late replication origins in wild type, rad9∆ and mrc1∆ strains exposed to 0.1 or 0.033% MMS. Red lines indicate median values. Median replication times (Trep) are indicated for bins of 96 origins.
a) Intracellular ATP in fresh lysates of Atg5f/f and Atg5f/fCD19-Cre splenic B cells activated for 3 d with LPS (3) or left untreated (0).figf5(a) Intracellular ATP in fresh lysates of Atg5f/f and Atg5f/fCD19-Cre splenic B cells activated for 3 d with LPS (3) or left untreated (0). (b) Death of Atg5f/f and Atg5f/fCD19-Cre splenic B cells activated for various times (horizontal axis) with LPS, quantified by flow cytometry by propidium iodide (PI) staining. (c) Quantitative PCR analysis of genomic DNA in differentiating B cells from Atg5f/f and Atg5f/fCD19-Cre mice, assessing nonrecombinant (Undeleted; left) and recombinant (Deleted; right) loxP-flanked Atg5 alleles; results are presented relative to those of unstimulated Atg5f/fCD19-Cre B cells. (d) Quantitative RT-PCR analysis of Atg5 mRNA in differentiating Atg5f/f and Atg5f/fCD19-Cre B cells at 0 and 3 d after stimulation with LPS, presented relative to that in untreated Atg5f/fCD19-Cre B cells. *P 0.05, **P 0.01 and ***P 0.001 (Student's t-test).
Figure 2A DPH anisotropy of B. subtilis WT or Δbkd cells supplemented either with MB, IB or depleted for precursor (IBPF) for the times indicated. High DPH anisotropy indicates low membrane fluidity. B DPH anisotropy of E. coli WT cells grown steady state at 30°C, 37°C or shifted from 30°C to 37°C followed by immediate measurement. In addition, DPH anisotropy of fabA(Ts) cells grown steady state at 30°C or shifted from 30°C to 37°C followed by measurement at the times indicated. D
Figure 1A. Ovaries from control heterozygous Rpp3018.2 flies (left), homozygous Rpp3018.2 or transheterozygous Rpp3018.2/Rpp30PE flies (middle) and homozygous Rpp3018.2 carrying a copy of ubiRpp30GFP transgene (right). Scale bar, 100µm. B. Rpp30 is a subunit of RNase P (red), a ribozyme involved in pre-tRNA processing by cleaving the 5' tail. The RNase Z (blue) cuts the 3' tail of the pre-tRNA, and CCA motif is added, forming mature tRNA ready to bind an amino acid (aa). C. Gene region corresponding to Rpp30 (RNAse P protein 30, adapted from Flybase). The P-element insertion (Rpp30PE, dark grey) and the point mutation Leucine to Proline (Rpp3018.2, arrow) are shown. D. Ovaries were dissected, lysed and protein extracts analyzed by Western Blot using anti-Rpp30, anti- GFP or anti-tubulin antibodies. Orange arrows indicate overexpressed and endogenous Rpp30 signals. Genotypes are numbered as 1,2,3. E. Rpp30-dependent early oogenesis arrest is restored by expression of Rpp30GFP transgene in two different genetic backgrounds: Left. Homozygous Rpp3018.2 ovariole expressing ubiquitous ubiRpp30GFP (st, stage). Right. Transheterozygous Rpp3018.2/Rpp30PE ovarioles overexpressing germline specific nanosGal4,UASRpp30GFP. Note follicular somatic cells with no Rpp30GFP expression (arrowhead). Magnifications show nuclear Rpp30 localization, Rpp30 perinuclear foci (arrows), and Orb (a specific oocyte marker). Scale bar, 10µm. F. flp/FRT clones mutant for Rpp3018.2specifically in the germline (star) and/or the follicular cells (arrows) are detected by the absence of GFP. Magnifications show mutant oocytes with Orb mislocalization next to the karyosome (dotted circle) instead of being localized to the posterior, as seen in stage 3 (st.3) wild-type clones. Scale bar, 10µm.
Figure 7U2OS-H2B-EGFP cells were transfected with indicated siRNAs for 48 h or were treated with PF-670462 and filmed in 5 min intervals for 24 h. Time from nuclear envelope breakdown to MAT was quantified in 100 cells. Shown is mean of the medians ± SD (n=3). Statistical significance was determined by ANOVA (**** p<0.0001). RPE cells or RPE cells stably expressing indicated EGFP-FAM110A variants were transfected with control (siNC) or FMA110A siRNA. Full arrow indicates migration of the EGFP-FAM110A-WT, empty arrow of endogenous FAM110A. Staining for TFIIH was used as a loading control. RPE cells or RPE cells stably expressing the wild-type or mutant EGFP-FAM110A were transfected with control or two various FAM110A siRNAs and were analyzed by time-lapse microscopy. Plotted are means of the medians of the time from NEB to MAT ±SDs. A hundred cells were quantified per condition (n=3). Statistical significance was determined by ANOVA (**** p<0.0001, ** p<0.005). RPE cells or RPE cells stably expressing indicated EGFP-FAM110A variants were transfected with control (siNC), FAM110A or CSNK1D siRNA and grown for 48 h. Alternatively, cells were incubated with PF670462 for the last 12 h. Cells were treated with MG132 for 30 min prior fixation. Impaired chromosomal alignment was scored in >30 metaphase cells per condition. Error bars indicate median ±SD. Statistical significance was determined by ANOVA (n=3) (**** p<0.0001, *** p<0.001). Representative immunoblot analysis of samples from (D). Full arrow indicates migration of the EGFP-FAM110A-WT, empty arrow of endogenous FAM110A. RPE cells or RPE cells stably expressing indicated mutants were transfected with control or FAM110A siRNAs, fixed after 48 h and stained for γ-tubulin. Metaphase cells were imaged by confocal microscopy (Z-stacks 0.5 μm) and spindle angle between the spindle axis and growth axis was determined. Plotted is a mean of medians from three repeats where 30 cells were imaged for each condition. Error bars indicate median ± SD. Statistical significance was determined ANOVA (n=3) (*** p<0.001, ** p<0.005). RPE cells or RPE cells stably expressing the wild type FAM110A, -252-255A or -252-255E mutants were transfected with control, FAM110A or CSNK1D siRNAs. Alternatively, cells were treated with PF670462 (1 μM) for 12 h. Spindle angle was determined as in (F). Statistical significance was determined by ANOVA (n=3) (*** p<0.001, ** p<0.005).
The Spectrin cytoskeleton represses interommatidial cell number and Hippo target gene expression in parallel with Kibra.Figure 2A-F The eyeless FLP MARCM system was used to generate mutant eyes that also express UAS.RNAi targeting the Spectrin cytoskeleton. Pupalretinas were examined at 42-46 h after puparium formation (APF). Cellmembranes were marked with Dlg staining. (A) Control pupalretina showing cone cells surrounded by interommatidial cells. (B) kibra mutant pupalretina displaying additional interommatidial cells. (C) Pupalretina expressing α-spectrin RNAi showing additional interommatidial cells. (D) kibra mutant pupalretinas expressing α-spectrin RNAi showing many additional interommatidial cells. (E, F) Clones of α-spectrin mutant cells (GFP negative) (E) and βH-spectrin/karst mutant cells (GFP negative) (F) show extra interommatidial cells. Scale bars, 20 μm. G-K UAS.RNAi lines were driven with hh.Gal4 UAS.GFP for expression in the posterior compartment and contained the ex.lacZ reporter transgene. (G) Control wing showing a normal ex.lacZ expression pattern, which is low at the dorsal-ventral boundary but high in the proximal regions of the wing disc. (H) RNAi knock-down of α-Spectrin results in a mild up-regulation of ex.lacZ in the posterior compartment. (I) RNAi knock-down of kibra results in a mild up-regulation of ex.lacZ in the posterior compartment. (J) Dual RNAi knock-down of both α-spectrin and kibra results in enhanced up-regulation of ex.lacZ in the posterior compartment. (K) RNAi knock-down of hippo results in an up-regulation of ex.lacZ in the posterior compartment. Scale bars, 50 μm.