EMBO/sd-character-level-ner huggingface dataset:
Open chromatin at the ACE2 and TMPRSS2 loci in AT2, ciliated and secretory cells in the lung and airways (a,c) Single-cell ATAC-seq of lung samples from primary carina (1C) and subpleural parenchyme (RPL) (n=1 patient, k=3 samples, 3,366 cells from 1C, 8,340 cells from RPL).Figure 2.(a,c) Single-cell ATAC-seq of lung samples from primary carina (1C) and subpleural parenchyme (RPL) (n=1 patient, k=3 samples, 3,366 cells from 1C, 8,340 cells from RPL). Uniform Manifold Approximation and Projection (UMAP) embedding of scATAC-seq profiles (dots) colored by (a, left) cell types, (a, right) cells with at least 1 fragment (indicating accessibility, open chromatin) mapping to the ACE2 gene locus (defined as −2kb upstream the Transcription Start Site to Transcription End Site), or by sample location (b) Inferred gene activity of ACE2, TMPRSS2, CTSL across cell types. Log normalized mean "scATAC activity score" (quantified from accessibility, open chromatin) (dot color) and proportion of cells with active scores (dot size) for ACE2, TMPRSS2, and CTSL (columns) across different cell types (rows) from the primary carina (1C) and subpleural parenchyme (RPL). (a,c) Single-cell ATAC-seq of lung samples from primary carina (1C) and subpleural parenchyme (RPL) (n=1 patient, k=3 samples, 3,366 cells from 1C, 8,340 cells from RPL). Uniform Manifold Approximation and Projection (UMAP) embedding of scATAC-seq profiles (dots) colored by (a, left) cell types, (a, right) cells with at least 1 fragment (indicating accessibility, open chromatin) mapping to the ACE2 gene locus (defined as −2kb upstream the Transcription Start Site to Transcription End Site), or by sample location (c). Grey shaded area: epithelial cell types. (d) Some AT2, ciliated and secretory cells have accessible chromatin at both ACE2 and TMPRSS2 loci. Proportion of cells (x axis) in each cell type (y axis) with accessible chromatin (at least 1 fragment) at both the ACE2 and TMPRSS2 loci (defined as −2kb upstream of the Transcription Start Site to Transcription End Site).
Figure 2Single-cell RNA sequencing was performed to profile cell populations in normal mouse pancreas (n = 2), early KIC (40-day-old, n = 2) and late KIC (60-day-old, n = 3) pancreata. Samples from the same stage were pooled. Violin plots of expression of Il6, Tgfbr1 and Tgfbr2 in distinct cell populations is shown. Expression of TGFβR1 and TGFβR2 in cell lysates harvested from KIC (mPLRB8, mPLRB9), KPC (KPC-M01, KPC-M09) and KPfC (BMFA3, CT1BA5) mouse cancer cells, mouse macrophages (RAW 264.7), and mouse fibroblasts (NIH 3T3 and pancreatic stellate cells). RAW 264.7 cells were induced into M1 (30 ng/ml LPS for 18 hours) or M2 (20 ng/ml IL-4 for 18 hours) macrophages. Tubulin was used as a loading control. Pearson and Spearman correlation of the expression of IL6 and TGFBR2 in PDA patients from TCGA (n = 223) using R.
Figure 1A Topology of GlpG, Rhom7, and the artificial substrate with a maltose-binding protein (MBP) domain, triple-FLAG tag (3xFLAG), the TMD of P. stuartii TatA, and a thioredoxin domain (Trx). B Western blot analysis (probing with an anti-FLAG mAb) to detect cleavage of the artificial substrate by wild-type (WT)/inactive (S201A) GlpG encoded on pBAD33 with/without arabinose (Ara.). rhomboid substrates that are uncleaved, cleaved by GlpG are marked by black, red, arrows, respectively. Controls, empty pBAD33 (empty) C Western blot analysis to detect cleavage of the artificial substrate by wild-type (WT)/modified (S133A or H187A) Rhom7 encoded on pBAD33 with/without arabinose (Ara.). , rhomboid substrates that are uncleaved, cleaved by Rhom7, are marked by black, and blue arrows, respectively. Controls, empty pBAD33 (empty) and wild-type S. sonnei (Ss). D Activity of Rhom7 with/without its 7th TMD and/or C-terminal domain. rhomboid substrates that are uncleaved, cleaved by Rhom7, are marked by black, , and blue arrows, respectively. Controls, empty pBAD33 (empty) and wild-type S. sonnei (Ss).
Figure 6Representative images of open field test showing tracking pattern (open arena) of WT (females, n=7, males n=6) and AAV-mWwox-injected KO mice (females n=7, males n=5) at 8-10 weeks. Dot plot graphs represent the velocity (B), total distance travelled (C) the frequency to enter into the center (D) and periphery (E) in an open arena from the open field tracking as shown in A. F. Mice tracking images of elevated plus maze test from WT and KO+AAV-mWwox mice (age 8-10 weeks). O. A. (open arm) C.A. (closed arm) are shown with dotted white boxes on the left mice tracking image. G-I. Dot plot graphs represent the duration (sec) in closed arm (G), open arm (H) and movement velocity (I) from WT (females, n=7, males n=6) and KO+AAV-mWwox (females n=7, males n=5). J, K. Graphs represent the latency (sec) of the mice (WT and rescued mice at 8-10 weeks) to fall from the rota rod. WT (females, n=7, males n=6) and KO+AAV-Wwox (females n=7, males n=5). L. Representative images of open field test showing tracking pattern (open arena) of WT (females, n=6) and KO with AAV injected (females mWwox n=3, hWWOX n=3) at 8-9 months. M-O. Dot plot graphs represent the represent the velocity (M), total distance travelled (N), the frequency to enter into the center and periphery (O) in an open arena from the open field tracking of mice shown in L.
Figure 2(A) Experimental setup of fear conditioning paradigm. Mice placed in a fear- conditioning box received no shock (NS), an immediate shock (IS) or delayed shock (DS) and were decapitated 30 min after returning to the home cage. Dorsal and ventral hippocampi were subjected to cellular fractionation (P2). DS and IS significantly increased the levels of phosphorylated ERK1/2, a hallmark of ERK activation.(B) Activity values across three different training paradigms to ascertain that foot shock was received in the IS and DS group. (C) Simplified schematic representation of dorsal and ventral hippocampal connections and their main functions (see, Fanselow Dong, 2010). (D) Western Blot analysis of P2 synaptosomal fractions of ventral hippocampi from the fear conditioning paradigm stained for pM18S241, total Munc18-1, pSynapsin, total synapsin, pERK and GAPDH as loading control.(E) Quantification Munc18-1 phosphorylation in dorsal hippocampus expressed as ratio pM18S241 over total Munc18-1 levels. No shock was set to 1 (NS: 1.0±0.19, n=5; DS: 4.01±0.39, n=5; IS: 2.88±0.37, n=5; **p<0.01; ***p<0.001).(F) Quantification of Munc18-1 phosphorylation in ventral hippocampus expressed as ratio pM18S241 divided by total Munc18-1 levels. (NS: 1.0±0.32, n=5; DS: 4.39±0.55, n=5; IS: 3.62±0.50, n=5; **p<0.01; ***p<0.001).(G) Quantification of synapsin phosphorylation in dorsal hippocampus expressed as ratio pSynapsin divided by total synapsin levels. (NS: 1.0±0.31, n=5; DS: 1.88±0.43, n=5; IS: 1.18±0.34, n=5; n.s.). Inset shows quantification of ERK2 phosphorylation in dorsal hippocampus expressed as ratio pERK2 divided by total ERK2 levels. (NS: 1.0±0.11, n=5; DS: 3.51±0.41, n=5; IS: 3.98±0.24, n=5; **p<0.01).(H) Quantification of synapsin phosphorylation in ventral hippocampus expressed as ratio pSynapsin divided by total synapsin levels. (NS: 1.0±0.20, n=5; DS: 3.32±0.46, n=5; IS: 3.30±0.60, n=5; **p<0.01).
Figure 6A. RT-qPCR analysis of relative mRNA expression of thermogenic genes in cultured primary human brown adipocytes in response to chronic treatments (8 hours) with DMSO (control), MP-10 (1nM and 100nM), cyclic AMP (cAMP - 10µM), cyclic GMP (cGMP - 10µM) and cAMP + cGMP (cA + cG - 10µM (n = 4 separate cultures). * P = 0.0278 vs (100nM MP-10 vs control; Pgc1alpha), **P = 0.0097 (10µM cA + cG vs control; Pgc1alpha), ** P = 0.0063 (1nM MP-10 vs control; Ucp1), **** P < 0.0001 (100nM MP-10 vs control; Ucp1), ## P = 0.0017 (1nM MP-10 vs 100nM MP-10; Ucp1), §§§§ P < 0.0001 (10µM cAMP vs 100nM MP-10; Ucp1), §§§§ P < 0.0001 (10µM cGMP vs 100nM MP-10; Ucp1), §§§§ P < 0.0001 (10µM cA + cG vs 100nM MP-10; Ucp1), * P = 0.0112 vs (100nM MP-10 vs control; Cidea), *** P = 0.0007 (1nM MP-10 vs control; Dio2), **** P < 0.0001 (100nM MP-10 vs control; Dio2), # P = 0.0267 (1nM MP-10 vs 10µM cAMP; Dio2), §§§ P = 0.0001 (10µM cAMP vs 100nM MP-10; Dio2), §§§ P = 0.0007 (10µM cGMP vs 100nM MP-10; Dio2), * P = 0.0232 (10µM cA + cG vs control; Dio2) §§ P = 0.0058 (10µM cA + cG vs 100nM MP-10; Dio2), * P = 0.0416 (10µM cGMP vs control; Prdm16) and * P = 0.0105 (10µM cA + cG vs control; Prdm16) using one-way analysis of variance (ANOVA) with Tukey's post-hoc test.B. RT-qPCR analysis of relative mRNA expression of thermogenic genes in cultured primary human white adipocytes in response to chronic treatments (8 hours) with DMSO (control), MP-10 (1nM and 100nM), cyclic AMP (cAMP - 10µM), cyclic GMP (cGMP - 10µM) and cAMP + cGMP (cA + cG - 10µM (n = 4 separate cultures). * P = 0.0186 vs (1nM MP-10 vs control; Ucp1), **** P < 0.0001 (100nM MP-10 vs control; Ucp1), ## P = 0.0016 (1nM MP-10 vs 100nM MP-10; Ucp1), *** P = 0.0003 (10µM cAMP vs control; Ucp1), §§§ P = 0.0002 (10µM cGMP vs 100nM MP-10; Ucp1) and *** P = 0.0003 (cA + cG vs control; Ucp1) using one-way analysis of variance (ANOVA) with Tukey's post-hoc test.
a) Cartoon illustration of myosin VI domain structure.figf4(a) Cartoon illustration of myosin VI domain structure. Myosin VI contains a catalytic motor domain, a unique insert (green), an IQ calmodulin-binding motif and a cargo-binding tail domain. The cargo-binding tail domain contains multiple protein interaction motifs (RRL and WWY) as well as regions for ubiquitin and PIP2 binding. In addition, two alternative splicing events give rise to two insertions in the cargo-binding tail region. (b) RPE cells with stable expression of cherry-LC3 were transiently transfected with the GFP-myosin VI cargo-binding tail domain containing various mutations in the protein-interaction (ΔWWY and ΔRRL) and ubiquitin-binding motifs (A1013G), followed by treatment with 250 nM Torin1 for 3 h to induce autophagy. Immunofluorescence microscopy was performed either in the absence or presence of saponin extraction. The arrowheads indicate areas of co-localization. Scale bar, 20 μm. (c) A Pearson's coefficient was calculated on the basis of the degree of co-localization between the different GFP-myosin VI mutant tails and cherry-LC3 from confocal immunofluorescence micrographs. The graph represents data from more than 20 transfected cells from n = 2 independent experiments.
Figure 7A. Amplex Red-based detection of H2O2 emission, a surrogate for respiratory chain-derived ROS, during cI and cII-linked state 3 respiration (n=6/group) Bar graphs represent mean±SD Statistics one-way ANOVA followed by Tukey" B. Gene expression related to ROS scavenging C. Total glutathion in heart and liver tissue (n=5/group Bar graphs represent mean±SD Statistics one-way ANOVA followed by Tukey" D. Urinary isoprostanes per creatinine as a measure of oxidative stress (n=4/group) Bar graphs represent mean±SD Statistics: Mann-Whitney U test (graph D E and F. Representative images (D) of tissue sections immunostained with an antibody against 4-hydroxynonenal (4-HNE), a lipid peroxidation marker. (E) Quantification of the 4-HNE immunostaining.
Figure 5A. Immunohistochemical analysis of cortical and hippocampal regions of the brains in wild-type (Wt) and APP/PS1mice after administration of Nec-1 (6.25 mg/kg) or vehicle (2.5% DMSO in PBS). Diffuse plaques in the brain sections were stained by anti-Aβ antibody (6E10 clone, green colour) and anti-GFAP antibody (red colour). Hoechst 33342 (blue colour) was applied for nuclear counterstaining. Scale bars = 200 μM. B-D. Dot blot analysis of total Aβ (anti-Aβ: 6E10 clone, also recognizes APP) and protein oligomer (anti-amyloidogenic protein oligomer A11) in the cortical (B) and hippocampal (C) region of indicated groups. (D) Quantification analysis of total Aβ and protein oligomer in the cortical and hippocampal region of indicated groups. Relative intensity of each band was determined by densitometry of dot blots (B and C) using ImageJ software and normalized to vehicle-administered APP/PS1mouse group. B-D. Dot blot analysis of total Aβ (anti-Aβ: 6E10 clone, also recognizes APP) and protein oligomer (anti-amyloidogenic protein oligomer A11) in the cortical (B) and hippocampal (C) region of indicated groups. (D) Quantification analysis of total Aβ and protein oligomer in the cortical and hippocampal region of indicated groups. Relative intensity of each band was determined by densitometry of dot blots (B and C) using ImageJ software and normalized to vehicle-administered APP/PS1mouse group. E. Sandwich ELISA of Aβ-soluble and -insoluble fractions from cortical and hippocampal lysates of APP/PS1mice. F. Western blot analyses of cortical and hippocampal lysates from APP/PS1mice for APP and sAPP expressions. G. ThT assays for the inhibition of Aβ aggregation. Fluorescence intensity was normalized to Aβaggregates (100%, day 5). H. SDS-PAGE analysis of PICUP cross-linked Aβaggregates visualized by silver staining.
Figure 2(A, B) BHP1 or PC3 cells with stable expression of either control (shCont) or CHD1 shRNA (shCHD1) were treated with γ-radiation (3 Gy) and after 1 h and 24 h cells were immunostained for γH2AX (A) and the number of γH2AX foci per cell were determined for each time point, more than 50 cells were counted in each condition (B). Scale bar 5 µm. The mean values of three independent experiments (± SD) are shown. p-values (0.0008 and 0.006, **p ≤ 0.01, ***p ≤ 0.001) were calculated using ANOVA. (C) CHD1 depleted cells show prolonged γH2AX accumulation. PC3 cells with shCont or shCHD1 were treated with γ-radiation as in (A) and total protein was analyzed by Western blot. (D) CHD1-depleted cells show increased sensitivity to γ-radiation. For colony formation assay, both BHP1 and PC3 cells with shCont or shCHD1 were treated with the indicated doses of γ-radiation and surviving fractions were measured by counting colonies after 3 weeks, mean values are represented in the plot (n=3, ± SD). Data were normalized to the plating efficiency. p-values (0.0009 and 0.002, **p ≤ 0.01, ***p ≤ 0.001) were calculated using ANOVA. See also Figure EV2A-EV2B.
The side chain structure of CY enhances nitrogen mustard activity in cancer cells.Figure 1Chemical structures of bendamustine, CY, compound A, and compound B (referred to as Cpd A and Cpd B hereafter). Growth inhibitory concentration (GI50) of CY and bendamustine in the NCI60 cell line panel. Nontoxic dose of Cpd A enhances bendamustine cytotoxicity in cancer cells. Images were taken at 48 h posttreatment. Cpd A augments DNA damage caused by bendamustine. H1650 cells were treated with 100 μM Bend, 50 μM Cpd A, 100 μM Bend plus 50 μM Cpd A, or 10 μM CY for 12 h, and cell lysates were blotted for γ-H2AX. Actin was used as a loading control.
Figure 6A model of how phosphorylation of STAT2 on T387 regulates signaling in response to IFN-β.
Figure 4(A) Representative gel of the In organello translation analysis of heart mitochondria. De novo synthetized proteins are isolated after labeling with 35S-met (1 hour Pulse) or after cold chase (3h Chase). Positions of individual proteins are indicated on the left. Position of full-length proteins (Proficient) and low molecular weight polypeptides (Abortive) are indicated on the right. Note that in WT and ClpP KO low molecular weight polypeptides include ATP8/ND4L proteins. (B) Relative overall protein synthesis and turnover rate. (C) Western blot analysis of DARS2 levels. HSC70 is used as a loading control (CTRL). (D) Relative levels of the individual de novo synthesized OXPHOS subunits in DKO mitochondria normalized to the corresponding Dars2 KO polypeptide level. (E) Relative levels of protein synthesis of Proficient and Abortive polypeptides. (F) Quantification of the turnover rate of full-length (Proficient) de novo synthesized proteins. Bars represent mean ± S.E.M. (Student's t test, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001), (n=3-4).
Figure 4A) infliximab (mAb) and rhTNF-α (Ag) were mixed at different ratios (17 µg total protein, calculated from monomer molarity) and analysed via AF4. sIC size is maximal at a 1:3 ratio of mAb:Ag and reduced when either mAb or Ag are given in excess. <M>w = mass-weighted mean of the molar mass distribution. Three independent experiments. Error bars = SD. Data taken from Table 1. B) sICs of different size were generated by cross-titration according to the AF4 measurement. Reporter cells were incubated with fixed amounts of either mAb (infliximab, left) or Ag (rhTNF-α, right) and titrated amounts of antigen or antibody, respectively. X-Axis shows titration of either antigen or antibody, respectively (TNF-α calculated as monomer). Two independent experiments performed in technical duplicates. Error bars = SD. C) Purified primary NK cells from three different donors were incubated with cross-titrated sICs as in B. NK cells were measured for MIP-1β expression (% positivity). Incubation with PMA and Ionomycin served as a positive control. Incubation with medium alone served as a negative control. Each dot represents one experimental replicate, the dashed line represents the mean values.
Figure 2Confocal microscopy of YFP-ATG8e-labeled autophagic bodies in root cells of Col-0/YFP-ATG8e and VISP1OE/YFP-ATG8e plants. 7-day-old seedlings were inoculated in liquid ½MS medium with 1 µM ConA for 12 h in dark. Scale bar = 20 μm.   Puncta numbers of YFP-ATG8e-labeled autophagic bodies per 10 cells in panel A. Data points represent means of three biological repeats. Error bars indicate SD. ***p < 0.001 (Student's t-test).   Western blotting analysis of the vacuolar delivery of the free YFP protein in the plants as shown in panel A. YFP-ATG8e/Col-0 plants were treated with N starvation as a positive control of autophagy induction. Total proteins were analyzed with GFP antibodies. Actin served as loading controls. Western blotting analysis of accumulation of ATG8a and ATG8a-PE in leaves of 4-week-old Col-0, VISP1OE, VISP1mUIM/OE and VISP1mARM/OE plants. Western blotting detecting accumulation of VISP1-Flag in 7-day-old Col-0 and VISP1OE seedlings. Seedlings were treated with 3-MA (10 mM) or buffer for 18 h at dark prior to protein detection. Representative TEM images of autophagic structures in inoculated leaves of Col-0 and VISP1OE plants treated with mock buffer or CMV-2blm infection at 4 dpi. The leaves was treated with 1 µM ConA at 4 dpi for 12 h in dark before TEM sampling. Magenta arrowheads indicate autophagic bodies inside the vacuoles. Scale bar = 2 μm. Cp, chloroplast; CW, cell wall; V, vacuole.   Autophagic structure numbers per 10 cells in panel F. Error bars indicate SD. Letters indicate significant differences (ANOVA, P < 0.05).
L.Figure 5(A) Enlarged (EM) and swollen (WM) mitochondria seen by transmission electron microscopy (TEM) in Δatg5 promastigotes under standard growth conditions. WT is shown at bottom right. Scale bar, 500 nm. (B) Fluorescent intensity from MitoTracker Red (MTR, 0.1 µM) and MitoTracker Green (MTG, 0.2 µM) in 2×106promastigotes after 30 min incubation at 26°C. Values shown are the means ± SD from three independent experiments. * and **, fluorescence was significantly different between WT and Δatg5 (p<0.05). (C) Types of mitochondrial morphology observed by fluorescence microscopy of Δatg5 promastigotes expressing the mitochondrial marker protein MUP-GFP. Scale bar, 10 µm. (D) Differential staining of promastigotes with both MTR (0.1 µM) and MTG (0.2 µM). Scale bar, 10 µm. (E) Viability, as measured by Alamar Blue reduction, of promastigotes. All data are means ± SD from three independent experiments. *, Alamar blue reduction was significantly different (p<0.05). (F) Spectrometric analyses of the DCF fluorescence intensity resulting from incubating promastigotesat 2×106/ml with H2DCFDA at 0.1 mM for 2 h at 26°C. Values shown are the means ± SD from three independent experiments. *, DCF fluorescence was significantly different (p<0.05).
Figure 2(A) Immunostaining of IFT54 mutated proteins in various deletion mutants as indicated; wild-type (WT) cells were used as control. Specimens were stained with anti-HA and anti-α-tubulin antibodies followed by imaging using an epifluorescence microscope. Arrows indicate protein accumulation at ciliary tip (left) or proximal end of cilia (right). Scale bars, 5 μm. (B, C) Immunostaining analysis of IFT43 and IFT38 in the deletion mutants as indicated. Cells were fixed and stained with antibodies against IFT43 (an IFT-A subunit) (B) or against IFT38 (an IFT-B subunit) (C) followed by imaging using both DIC and epifluorescence microscopy. IFT43 and IFT38 accumulated at the ciliary tip in IFT54Δ261-275 mutant but at proximal end of cilia in IFT54Δ342-356 mutant. Arrows indicate ciliary bulges. WT, wild-type cells. Scale bars, 5 μm.
Figure 8Analysis of TFEB binding to DLEU2 and SMC4 promoters in human ECs. ChIP was performed using digested chromatin from control ECs and TFEBS142A ECs incubated with IgG (indicated in the bar graph as "+IgG") or with Ab anti-TFEB (indicated in the bar graph as "+Ab anti-TFEB"), followed by qPCR for DLEU2 and SMC4. DLEU2 expression is regulated by TFEB. qPCR of DLEU2 in human scr-shRNA, sh-TFEB or control and TFEBS142A ECs (left panel) and lung ECs obtained from control and TfebiEC-/- mice (right panel). Data are expressed as relative fold-change compared with the expression in scr-shRNA and control cells after normalization to the housekeeping gene TBP Human miR-15a-5p and miR-16-5p are regulated by TFEB. qPCR of miR-15a-5p (left panel) and miR-16-5p (right panel) in sh-TFEB or TFEBS142A ECs. Data are expressed as relative fold-change compared with the expression in scr-shRNA and control cells after normalization to the housekeeping gene RNU44 VEGFR2 expression is regulated by TFEB through a miR-dependent mechanism. qPCR of VEGFR2 in human scr-shRNA and sh-TFEB ECs and in control treated with a specific miR-control, miR-15a-5p and miR-16-5p mimics Data are expressed as relative fold-change compared with the expression in control cells after normalization to the housekeeping gene TBP VEGFR2 expression is regulated by TFEB through a miR-dependent mechanism. VEGFR2 in human scr-shRNA and sh-TFEB ECs and in control treated with a specific miR-control, miR-15a-5p and miR-16-5p mimics Representative western blot of VEGFR2 expression under the same experimental conditions previously reported. The bar graph shows the densitometric analysis expressed as the ratio between VEGFR2 and α-tubulin VEGFR2 expression is regulated by TFEB through a miR-dependent mechanism. qPCR of VEGFR2 in control and TFEBS142A ECs treated with a specific miR-control, miR-15a-5p and miR-16-5p inhibitors. Data are expressed as relative fold-change compared with the expression in control cells after normalization to the housekeeping gene TBP VEGFR2 expression is regulated by TFEB through a miR-dependent mechanism. VEGFR2 in control and TFEBS142A ECs treated with a specific miR-control, miR-15a-5p and miR-16-5p inhibitors. Representative western blot of VEGFR2 expression under the same experimental conditions previously reported. The bar graph shows the densitometric analysis expressed as the ratio between VEGFR2 and α-tubulin