EMBO/sd-character-level-ner huggingface dataset:
Figure 3The calcium-induced differentiation protocol used in this study. Up until day 0 cells were maintained in low calcium media and then induced to differentiate by growing them in high calcium medium for six days. Cells were harvested for immunoblot at day 0, day 1, day 3 and day 6 of differentiation. Immunoblot showing increase in expression levels of differentiation markers Involucrin (IVL) and Transglutaminase-1 (TGase-1) as differentiation progressed indicating that keratinocyte differentiation protocol was successful. Protein levels on day 0 (D0), day 1 (D1), day 3 (D3) and day 6 (D6) of differentiation are shown. Immunoblot for JARID2 during D0, D1, D3 and D6 of differentiation (as in B) are shown. Effect of JARID2 removal on levels of differentiation markers Involucrin (IVL), Keratin-1 (KRT1) and Keratin-10 (KRT10) mRNAs as measured in qPCR experiment relative to 18S rRNA and the rescue using exogenous expression of ΔN-JARID2. The effect of exogenous expression of full-length JARID2 (FL-JARID2) in JARID2 knockout is also shown. Data from wildtype HaCaTs, two independent JARID2 knockout (KO1 and KO2) HaCaT lines as well as KO cells exogenously expressing empty vector control, ΔN-JARID2 and FL-JARID2 are shown. Levels of Involucrin (IVL), Keratin-1 (KRT1) and Keratin-10 (KRT10) mRNAs as above but measured on day 3 of differentiation.
EGFR inhibition decreases tumor progression of human OS cells 143b after orthotopic injection.Figure 7Western Blot analysis showing EGFR and c-Fos protein levels in human OS cell lines cultured under standard conditions Cell viability of 143b or LM7 cells cultured for 24h in medium (+10% FCS) with DMSO (1:2000) or Erlotinib (10µM) (n=3, representative result from 3 independent experiments, shown as fold change, normalized to DMSO) Cell viability of 143b or LM7 cells cultured for 24h in medium (+0.5% FCS) containing 1xPBS (contr., 1:200) or EGF (50ng/ml) (n=3, representative result from 3 independent experiments, shown as fold change, normalized to control) Treatment scheme: 143b OS cells (106 in 25µl PBS) were intratibially injected; mice were treated for 14 days with vehicle or Erlotinib (50mg/kg) 7 days after injection, when the tumors started to grow 143b xenograft growth curve during therapeutic regime (n=8 vehicle, 6 Erlotinib; two independent experiments) 143b tumor weight at endpoint (n=8 Vehicle, 6 Erlotinib; two independent experiments)Treatment scheme: LM7 OS cells (106 in 25µl PBS) were intratibially injected; mice were treated for 21 days with vehicle or Erlotinib (50mg/kg) 42 days after injection, when the tumors started to grow LM7 xenograft growth curve during therapeutic regime (n=7 Vehicle, 5 Erlotinib; two independent experiments) LM7 tumor weight at endpoint. n=7 vehicle, 5 Erlotinib, two independent experiment IHC analysis of PCNA and cleaved Caspase 3 in 143b-derived tumors (n=4). Scale bars: 50µm IHC analysis of pCREB and c-Fos in 143b-derived tumors. (n=5 for pCREB and n=4 for c-Fos analysis). Scale bars: 50µm Western Blot analysis of pCREB/CREB and p-c-Fos/c-Fos protein expression in lysates directly isolated from 143b xenografts at endpoint, after 14 days of vehicle or Erlotinib treatment
Figure 6(A) Representative confocal micrographs of BMMs infected with either wild type (2308), ∆bspF or or complemented ∆bspF (∆bspF::bspF) bacteria (red) for 24 h and immunostained for the TGN vesicular marker Stx6 (green) and GM130 (blue). Scale bars, 10 µm and 2 µm (insets). Magnified insets show the association between Stx6-positive vesicles and rBCVs. (B) Recruitment of Stx6-positive vesicles to rBCVs (expressed as percentage of Stx6-positive BCVs) in BMMs infected for 24 h with either wild type (2308), ∆bspF or complemented ∆bspF (∆bspF::bspF) bacteria. Data are means ± SD from n=3 independent experiments, in which at least 300 BCVs were analyzed per experiment via CellProfiler image analysis. Asterisks indicate statistically significant differences compared to 2308-infected BMMs as determined by one-way ANOVA with Tukey's multiple comparisons test (P < 0.05). (C) Representative Western blot analysis of Arf6, Rab8a, Rab6a/a' and Stx6 depletions in BMMs following siRNA-mediated knockdowns, compared to non-targeting siRNA (siNT) treatments. β-actin was used as loading control. (D-F) Recruitment of Stx6-positive vesicles to rBCVs in BMMs treated with non-targeting siRNAs (siNT), siRNAs against Arf6 (siArf6) (D), Rab8a (siRab8a) (E) or Rab6a/a' (siRab6a/a') (F) and infected for 24 h with either wild type (2308), ΔbspF, or complemented ∆bspF (ΔbspF::bspF) bacteria. Data are means ± SD of n=3 independent experiments, in which 200 BCVs were analyzed per experiment. Asterisks indicate statistically significant differences (P<0.05, one-way ANOVA followed by Dunnett's multiple comparisons test) between test and control conditions; ns, not significant. (G) Quantification of CTxB retrograde transport in BMMs following siRNA-mediated depletion of Stx6 (siStx6) after AlexaFluor™488-CTxB binding on ice followed by 30 min incubation at 37˚C. Data are means ± SD from n=3 independent experiments, in which 100 cells were analyzed per experiment. The asterisk indicates a statistically significant difference (P=0.0114, unpaired Student's t test) compared to the siNT control. (H) Brucella replication in BMMs treated with either non-targeting siRNAs (siNT), or siRNAs against Stx6 (siStx6) and infected for 24 h with either wild type (2308), ΔbspF, or complemented ∆bspF (ΔbspF::bspF) bacteria. Data are means ± SD of n=3 independent experiments, in which at least 100 cells were analyzed per experiment. Grey dots represent individual cells analyzed (n>300); black dots indicate means of individual experiments. Asterisks indicate statistically significant differences (P<0.05, one-way ANOVA followed by Dunnett's multiple comparisons test) between test and control conditions. (I) Model of BspF remodeling of TGN-RE membrane traffic. Bacterially delivered BspF targets RE membranes where it binds ACAP1 and promotes inactivation of Arf6. Increased turnover of active Arf6 results in inhibition of the Arf6/Rab8a cascade and retrograde RE-TGN transport, which alters TGN-derived vesicular traffic and redirects Stx6-positive vesicles to rBCVs in a process that promotes intravacuolar bacterial growth.
Clustering of vDNA/vRNA and nuclear RT in primary human macrophages The left panel shows the timeline of infection and/or drug exposure experiments.Figure 7The left panel shows the timeline of infection and/or drug exposure experiments. (A-D) Multicolor images of vDNA, vRNA, DAPI and/or GFP in monocyte derived macrophages (MDMs) from two different donors, infected with HIV1. Cells from donor 1 were infected with VSV-G-pseudotyped HIV-1ΔEnv Vpx carrying a GFP reporter (A) and cells from donor 2 were infected with a VSV-G-pseudotyped HIV-1ΔEnv without Vpx (B-D). (A,B) Infected cells were left untreated and fixed at 6 d p.i. (C) Infected cells were treated with 10 µM Nevirapine (NVP) throughout the experiment and fixed at 6 d p.i. (D) Infected cells were exposed to NVP for 3 d p.i., then NVP was washed out and cells were cultured for another 4 d and fixed at 7 d p.i. Arrows in (A-D) show the position of selected vRNA foci. (E) Boxplot shows the size of DNA foci in untreated infected cells from donor 2 imaged at 7 d p.i. (B), measured by the FWHM of n=24 foci. The red line defines the median, top and bottom limits define upper and lower quartiles, respectively. Whiskers show the full data range, except for outliers. Grey dots are individual data points. (F) Boxplot shows Pearson's r, measuring colocalization of DNA with RNA foci in the same cells (n=20). Center line defines the median, top and bottom limits define upper and lower quartiles, respectively. Whiskers show the full data range, except for outliers. Grey dots are individual data points. (G) FACS analysis of Gag positive cells from donor 2 in different conditions. Red: uninfected cells. Blue: untreated infected cells at 7 d p.i. as in B. Orange: cells were treated with NVP for 7 d as in C. Green: infected cells were exposed to NVP for 3 d, then NVP was washed out and cells were cultured for another 4 d as in D. Median of fluorescence intensities (MFI) for each sample are shown in the table.
Figure 1(A) OI-RD image of a small molecule microarray (SMM). Each compound was printed in duplicate in adjacent vertical positions. (B) The categories of the compound library. 3,375 compounds were printed on the chip, including 1,527 FDA-approved drugs, 1,053 natural products and 795 known inhibitors. (C) Representative image showing the binding of recombinant STING protein to compounds printed on HTS chip. The SMM chip was titrated to purified STING protein observing emission from 300 to 400 nm at 1nm intervals (excitation 290 nm). Scans were done in triplicates per sample. Excitation and emission slits were set to 5 nm. A2 = 8J19; A3 =10A9; A4 =9O17; A5 =3C17; A6 =7H8; A7 =8G10; A8 =9H14; A9=10G10; A10 =7D21; A11 =9J12; B2 =9L12; B3 =7H21; B4 =2B8; B5 =9D14; B6 =9P7; B7 =2E22; B8 =7P6; B9 =7I21. (D-E) qRT-PCR measurement of Ifnb1 transcripts in mouse peritoneal macrophages that were left unstimulated or stimulated with 2'3' cGAMP (0.1 μg/mL) (D) or ISD (1 μg/ml) (E) for 4 h in the presence of DMSO or indicated compounds (10 μM). The data are presented as the relative fold induction of Ifnb1/Gapdh transcripts. A 90% reduction of IFNB1 transcripts in mouse peritoneal macrophages in comparison to the DMSO group was selected as the cutoff value for follow-up study, which has been indicated by the red dashed line. The arrow indicates the group that meets the cutoff value. The data shown are mean ± SEM from n=3 biological replicates.
The distribution of LAMP‐2A in discrete lysosomal membrane regions changes with changes in CMA.Figure 6(A) Fed ratliverlysosomes were incubated in an isotonic buffer without additions, or in the presence of ratlivercytosol (25 μg), glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH) (25 μg), GST‐hsc70 (10 μg/ml), ATP (5 mM) or the indicated combinations for 15 min at 37°C. Lysosomes incubated directly with apyrase or with cytosol treated with apyrase are shown in the right bottom panel. At the end of the incubation, lysosomes were subjected to Triton X‐114 extraction, apyrase density gradient centrifugation and immunoblot as in Figure 2C. Graph shows changes in LAMP‐2A distribution expressed as times the value in untreated lysosomes and are the mean of the densitometric quantification of 3-6 experiments as the one shown here. Because all the incubations were performed in the presence of protease inhibitors, total levels of LAMP‐2A in lysosomes remained constant throughout the incubation. (B) Lysosomes from livers of fed rats, untreated or treated with paraquat (+PQ), were processed as in (A). Graph shows changes in LAMP‐2A distribution induced by the PQ treatment, expressed as times the value in untreated animals and are the mean of three experiments. (C) Lysosomes with high (CMA+) and low (CMA−) ability for CMA isolated from livers of fed or 48 h starved rats were processed as in (A). Top left: Levels of two CMA components (LAMP‐2A and hsc70) and of an unrelated lysosomalmembrane protein (LAMP‐1) in each of the four groups of lysosomes. Bottom left: Percentage of total lysosomal LAMP‐2A present in each fraction, determined by densitometric quantification of the immunoblots.
Figure 1A-D) Confocal image analysis of cryostat sections from adult CX3CR1+/GFP;Sfrp1+/βgal (A,B); Sfrp1+/βgal (C) and CX3CR1+/GFP;Sfrp1-/- (D) and mouse brains three days after intra-cortical infusion of saline or LPS. Sections were immunostained for βgal (magenta, green in C) and Iba1 (green in A; red in C) or Sfrp1 (magenta) and GFP (green, B and D), and GFAP (cyan in A;B, D, red in C). Arrowheads indicate βgal/GFAP (A) and Sfrp1/GFP co-localization (B). No Sfrp1 protein is detected in the null mice independently of the treatment (two bottom lines). Scale bar 25μm. E) ELISA determination of Sfrp1 levels in brain extracts from 3 months-old wt and Sfrp1-/- mice. WT mice were injected either with saline or LPS. Three days after injection the region around the injected side (10 mm3 cortical cube) was isolated and SFRP1 content compared with that present in similar region of non-injected or Sfrp1-/- mice used as negative control (n=5 mice for each group). Error bars represent Standard Error. Statistical significance: ns P>0.5 ****P<0.0001; One-way ANOVA followed by Bonferroni multiple comparisons test.
Figure 3(A) Excerpts of complexome profiling data showing the distribution of CIV subunits and assembly factors HIGD1A, HIGD2A, CMC1 and COA3 in control (143B), COX1∆ and COX2∆ cybrids. The relative abundance and distribution of detected proteins is shown in the lower graphs. The localization of SC I+III2plus in COX2Δ cybrids is highlighted with a red line. The approximate molecular weights of CIV-containing structures are indicated on the top (in kDa). (B) 2D-BN/SDS-PAGE analysis from control (143B) and COX2Δ cybrids followed by immunoblotting with the indicated antibodies. I+III2+IV1, SC containing CI, CIII, and CIV; I+III2plus, SC containing CI, CIII and a COX1-submodule; III2+IV, SC containing CIII and CIV; IV2, CIV dimer; IV, CIV monomer. The presence of a COX1-CMC1-COA3-containing subcomplex is indicated.
Model of the enzymatic pathway for the Apg12p–Apg5p conjugation system.Figure 6Model of the enzymatic pathway for the Apg12p-Apg5p conjugation system. Apg12p is activated by Apg7p with ATP hydrolysis; the carboxy group of the C‐terminal glycine of Apg12 is conjugated to the thiol group of the cysteine‐507 residue of Apg7p via a high‐energy thioester bond (∼). Subsequent transfer of Apg12p to the thiol group of the cysteine‐133 residue of Apg10p results in the formation of the Apg12p-Apg10p thioester. Finally, the C‐terminus of Apg12p is covalently attached to the lysine‐149 residue of Apg5p via an isopeptide bond, which may require hypothetical Apg5p‐recognizing protein(s). Model of the enzymatic pathway for the Apg12p-Apg5p conjugation system. Apg12p is activated by Apg7p with ATP hydrolysis; the carboxy group of the C‐terminal glycine of Apg12 is conjugated to the thiol group of the cysteine‐507 residue of Apg7p via a high‐energy thioester bond (∼). Subsequent transfer of Apg12p to the thiol group of the cysteine‐133 residue of Apg10p results in the formation of the Apg12p-Apg10p thioester. Finally, the C‐terminus of Apg12p is covalently attached to the lysine‐149 residue of Apg5p via an isopeptide bond, which may require hypothetical Apg5p‐recognizing protein(s).
Figure 11Spot tests of co-transformants of either Bd0367 (DgcA; GGDEF protein) response regulator domain or Bd3125 (CdgA; a degenerate GGDEF protein likely involved in c-di-GMP binding and signalling) with full length Bd1971, or the N-terminal (1-145), or C-terminal (145-400) portions of Bd1971 in either pUT18c or pKT25 as indicated. Positive control was co-transformants with pUT18c-zip and pKT25-zip, negative control was the empty vectors. The co-transformants were plated on LB-X-Gal medium with positive interaction resulting in blue colouration, which can be seen for all of the interactions in both plasmid combinations, but not for the negative control. Images are representative of at least 4 independent experiments. Confirmation of BTH interactions by β-Galactosidase activity assays. The interaction is significantly stronger between Bd3125 and the N-terminal domain of Bd1971 than the interaction with the C-terminus of Bd1971. The response regulator domain of Bd0367 showed significant interaction with Bd1971, but the full length Bd0367 did not. *p>0.05 by Student's t-test N/S- not significant. Data are from at least three independent experiments, error bars are the standard error of the mean
Figure 3(A) FBW7 mutations across medulloblastoma molecular subgroup as identified by exome sequencing. In the SHH medulloblastoma subgroup, FBW7 mutations occurred approximately 11 % in a cohort containing 133 SHH medcases (Kool et al., 2014) and can be categorized as stop-gain (n=3), non-synonymous (n=7). One case of splicing error mutation is detected in the Group 3 medulloblastoma. (B) Immunoblotting of HA-SOX9-WT protein following expression of various FLAG-FBW7α constructs including wild type (WT), or those with R465A or R479A mutation in Daoy. The cells were transfected with equal amount of HA-SOX9-WT and FLAG-FBW7α and the whole cell lysates were collected 24 hours following the transfection. GAPDH proteins were shown to indicate total protein loading for each sample. The set of blots shown is representative from 2 independent experiments. (C) Co-immunoprecipitation of HA-SOX9 with either wild type (WT) or R465A- FLAG-FBW7α. Unlike the wild type protein, FLAG-FBW7α containing arginine 465 mutation did not significantly co-immunoprecipitate with HA-SOX9 (IP with anti HA antibody) and vice versa (IP with anti FLAG antibody). The expressions of HA-SOX9 and FLAG- FBW7α- WT or R465A mutant were assessed in the whole cell extract (WCE) of the transfected HEK293 with GAPDH protein used to indicate total protein loading. (D) Comparison of FBW7 mRNA expression across the medulloblastoma subgroups (WNT, SHH, Group 3, and 4) in a cohort containing 423 medulloblastoma cases. Expression of FBW7 in both normal fetal (n= 5) and adult cerebellum (n = 13) were used for comparison against the medulloblastoma. (E) Overall survival analysis of medulloblastoma patient based upon FBW7 expression level. Analysis was performed on 383 out of the 423 cases from which the survival data were available. Cohort was divided into high- (blue line) and low- FBW7 (red line) expressing subgroup using FBW7 median expression as group classifier. Statistical analysis was determined by using a LogRank test. (F) Correlation analysis between SOX9 protein/mRNA and FBW7 mRNA expression level from medulloblastoma tissue microarray consisting of 142 tissue samples. SOX9 protein level was scored following immunohistochemistry staining, while SOX9 and FBW7 mRNAs expression by the RNA Scope as described in Materials Methods. A modest negative correlation was determined by Kendall's correlation test (τ = - 0.223, p0.001) between SOX9 protein/mRNA and FBW7 expression. Affiliation of samples with medulloblastoma subgroups was indicated by different colored circle (blue=WNT, red=SHH, yellow=Group 3, green=Group 4, grey=Undetermined).
Figure 2(A) CRISPR-SAM workflow. Cell library was exposed to DMSO (solvent of BRAFi) or BRAFi 2 µM (vemurafenib (Vem) or PLX8394 (PB, Paradox Breaker)) during 14 days. 40x106 cells per arm. Experiments were done in duplicate. (B) Plot showing sgRNAs detected in BRAFi-resistant cells versus in control cells (cell library exposed to DMSO) with a fold change >1.5. Raw data are available in Table EV5. (BRAFi for PBV). (C) sgRNAs counts in BRAFi-resistant cells versus in DMSO-exposed cells for EGFR and BIRC3. (BRAFi for PBV, (D) Determination of differentiation states of 12 human BRAF(V600) melanoma cell lines from CCLE (E) BRAF(V600) melanoma cell lines from CCLE were distributed in two groups (Sensitive and "intrinsically" Resistant, n=6 cell lines per group) according to their BRAFi IC50 (µM, half maximal inhibitory concentrations) (F) Volcano plot showing the expression levels of BRAFi-resistant genes (identified in 501Mel by CRISPR-SAM screen) in 12 human melanoma cell lines. The fold change corresponds to the ratio of expression levels found in resistant and sensitive cell lines. In red: selected genes. EGFR is considered as positive control and SMAD3, BIRC3 and SLC9A5; selected as favorite genes for the next steps. SMAD3, BIRC3, SLC9A5 and AFAP1 are BRAFi-resistance genes and potent tumor-promoting genes (BRAFi for PBV (G) Heatmap recapitulating the expression levels of the selected hits (red dots in Fig. 2F) in BRAFi-resistant and -sensitive cell lines. Markers of differentiation (MITF, MLANA, OCA2, TYRP1, TYR). In red: resistance genes already published (NRP1, AXL, ZEB1, LPAR1).
Figure 2(A) Western blot analysis of Parp1 and DNA damage markers in Wild-type (WT) and Ews-/- mBA cells. Cells were treated with MMS (0.02%) in a time dependent manner. Proteins were fractionated into two groups, chromatin-bound (Chro) proteins and soluble (Solu) proteins. (B) After treatment with MMS (0.02%, 1hour), the media was replaced to release the DNA damage. The proteins were fractionated and the specific protein kinetics at chromatin was analyzed by western blot. (C) GFP-PARP1 U2OS cells were transfected with siControl and siEWS. Local DNA damage was induced by micro-irradiation using a 405nm laser. Data represented as mean ±SEMs and more than six cells were analyzed from six independent experiments. The statistical significance determined by One-way ANOVA, ***P< 0.001. Scale bar indicates 5 μm. (D) Fluorescence correlation spectroscopy (FCS) was measured after 15 minutes later following micro-irradiation (Upper) or under normal condition in GFP-PARP1 U2OS cells with either siControl or siEWS. Data represented as mean ±SEMs, more than 50 cells were analyzed, and significance determined by One-way ANOVA, ***P< 0.001. Scale bar indicate 5 μm. (E) AsiSI endonuclease-integrated U2OS cells were transfected with siControl and siEWS. Cells were incubated with doxycycline for 4 hours to induce DSBs. With or without changing the Dox-added media to fresh media for 2 hours (for release samples), the amount of chromatin-associated PARP1 was measured using ChIP assay. (N.T: Non-treat, T: AsiSI treat, Release: Damage released samples). Data represented as mean ±SEMs, and technical repeats (n=3), significance determined by two-way ANOVA, ***P< 0.001.
Figure 6HEK293 cells were transfected with empty pcDNA3 vector (-) or Flag-DDX5 (+). Whole cell lysates (WCE) and anti-Flag immunoprecipitations (IP) were immunoblotted with anti-Flag and anti-XRN2 antibodies. Untransfected U2OS cells were lyzed and subjected to immunoprecipitation with control immunoglobulin G (IgG) or anti-XRN2 antibodies. The bound proteins were separated by SDS-PAGE followed by Western blotting with anti-DDX5 or anti-XRN2 antibodies. The migration of DDX5 and XRN2 is depicted with an arrow. U2OS cells were transfected with empty pcDNA3 vector (pcDNA3) or expression vectors encoding Flag-DDX5 WT (1-614) or truncated Flag-DDX5 proteins (60-614; 1-554; 1-435). WCE of the transfected cells (left panel) were Western blotted with anti-Flag and anti-XRN2 antibodies to confirm equivalent expression. The transfected cells were lysed and anti-Flag immunoprecipitations (right panel) performed for the presence of co-immunoprecipitating endogenous XRN2 by Western blotting with anti-XRN2 antibodies. M denotes molecular mass markers in kDa. Untransfected and U2OS stably expressing Flag-DDX5 WT or Flag-DDX5-RK were subjected to immunoprecipitation with anti-Flag antibody. The WCE and the anti-Flag immunoprecipitated proteins were Western blotted with anti-monomethylarginine (MeR), anti-Flag and anti-XRN2 antibodies, respectively. M denotes molecular mass markers in kDa. U2OS cells transfected with siCTL, siDDX5, or siXRN2 were subjected to DRIP-qPCR analysis with anti-IgG, and anti-S9.6 antibodies with or without RNaseH treatment. The average and S.E.M. from 3 independent experiments is shown. Statistical significance was assessed using Student's t-test. *: p < 0.05; **: p < 0.01.
Figure 7Canonical inflammasomes act as sensors for a variety of pathogens and cellular insults. Assembly of these types of inflammasomes involves multimerization of the receptor (purple), which initiates filament formation of the adaptor protein ASC (green). ASC filaments act as activation platforms for caspase-1 (blue). In the noncanonical pathway, LPS is directly bound by caspase-11 (yellow), resulting in its activation. Caspase-1 processes interleukin IL-1β (red). Both caspase-1 and caspase-11 process GSDMD (orange), which results in release of the GSDMDNterm fragment. GSDMDNterm forms a large pore in the plasma membrane. Pore formation results in rapid loss of membrane integrity, the dissipation of the electrochemical gradient and ultimately in cell death.