EMBO/sd-character-level-ner huggingface dataset:
Figure 5A. Enhanced recruitment of DDX11 to chromatin associated PCNA following exposure to 4 µM PDS for 24 hours. PCNA was precipitated from cross-linked chromatin and the immunoprecipitate blotted for FLAG-DDX11. B. Fluctuation analysis for Bu-1a loss in wild-type DT40 cells, two independent ddx11 clones generated by CRISPR-Cas9 targeting (clones 1 and 2) and one ddx11 clone generated by conventional homologous recombination gene targeting (clone 3). Each symbol represents the percentage of cells in an individual clone expanded for 2-3 weeks that have lost Bu-1ahigh expression. C. Fluctuation analysis for Bu-1a loss variant generation in wild-type cells, ddx11 (clone 1) cells, ddx11 (clone 1) complemented by expression of chicken DDX11 WT cDNA, ddx11 (clone 1) complemented by expression of helicase-dead form of chicken DDX11 (K87A) cDNA, and a ddx11 clone generated in cells in which the endogenous +3.5 G4 has been deleted (ΔG4). D. Fluctuation analysis for Bu-1 loss in two independent timeless/ddx11 double-mutant clones (#1 and #2), ddx11 expressing DDX11KAK and fancj and fancj/ddx11 double-mutants. Fluctuation analyses for wild type, timeless #1 and ddx11 #1 are shown for comparison.
Shp2 ablation induces senescence in mammary gland tumors in PyMT mice Shp2 inhibition prevents tumor formation at early states and inhibits tumor growth at late stages in PyMT mice.Figure 8Immunohistochemistry analysis of EYFP (top) and SA‐β‐gal staining (bottom) on cryosections of mammary glandtumors from control MLN4924;Shp2fl/fl;EYFP−/fl, MLN4924;MMTV‐Cre;Shp2+/fl;EYFP−/fl, and MLN4924;MMTV‐Cre;Shp2fl/fl;EYFP−/flmice. Note that SA‐β‐gal activity was present in the EYFP‐positive (Shp2‐negative) areas (right, arrows) and was absent in the EYFP‐negative (Shp2‐positive) areas (right, arrowhead) of mammary glandtumors of MLN4924;MMTV‐Cre;Shp2fl/fl;EYFP−/flmice. Consecutive sections of EYFP and SA‐β‐gal staining for each group are shown. Scale bar: 100 μm. n = 5. Immunofluorescence analysis of Ki67, EYFP and CK8 (pan‐epithelial marker) (top panel), p27 and EYFP (middle panel), and p53 and EYFP (bottom panel) on cryosections of mammary glandtumors from control MLN4924;Shp2fl/fl;EYFP−/fl, MLN4924;MMTV‐Cre;Shp2+/fl;EYFP−/fl, and MLN4924;MMTV‐Cre;Shp2fl/fl;EYFP−/flmice. DAPI was used for nuclear counterstain. Ki67 was absent in EYFP‐positive cells (top right, arrows) and was present in EYFP‐negative cells (top right, arrowheads); p27 was present in EYFP‐positive cells (middle right, arrows) and was absent in EYFP‐negative cells (middle right, arrowhead); and p53 was present in EYFP‐positive cells (bottom right, arrows) and was absent in EYFP‐negative cells (bottom right, arrowhead). Scale bar: top panel, 100 μm; middle and bottom panel, 50 μm. n = 5. qRT-PCR analysis of mRNA levels of Shp2, Skp2, Aurka, Dll1, and Hey1 in EYFP+ and EYFP−cells isolated by FACS from MLN4924;MMTV‐Cre;Shp2fl/fl;EYFP−/fltumors. Error bars represent SEM (n = 5). Kaplan-Meier analysis of tumor formation in control (blue, n = 8) and GS493‐pretreated (red, n = 8) MLN4924;MMTV‐Cre;Shp2fl/flmice. The bar indicates the duration of treatment. Tumorgrowth curves for control (blue, n = 5) and GS493‐treated (red, n = 5) MLN4924 mice. Note that after day 24, all mice in the control group were sacrificed due to the tumor size. Error bars represent SEM (n = 5).
Figure 5H2S levels in DMSO- and R406-treated reprogramming intermediates detected by monobromobimane method. n = 4. H2S levels in reprogramming intermediates treated with DPBS, HA and NAC. n = 3. Immunofluorescence of Sall4 in reprogramming intermediates treated by R406 + HA and R406 + DPBS on d12. n = 3. Scale bar, 100 μm. Immunofluorescence of Sall4 in NAC- and DPBS-treated cells on d12. n = 5. Scale bar, 100 μm. RT-qPCR analysis of Sall4, Cdh1, Epcam and Esrrb gene expression in DPBS- and HA-treated cells on d12. n = 3. RT-qPCR analysis of Sall4, Cdh1, Epcam and Esrrb gene expression in DPBS- and NAC-treated cells on d12. n = 3. H2S levels in reprogramming intermediates treated with shNC and shCbs. n = 3. Diagram showing the procedure of reprogramming infected with shRNA for Cbs. RT-qPCR analysis of Cbs expression in MEFs infected with shNC and shCbs. n = 3. Immunofluorescence of Sall4 in shNC- and shCbs-infected cells with and without R406 treatment on d12. n = 5. Scale bar, 100 μm. Diagram showing the procedure of reprogramming infected with Cbs-OE (pSin-Cbs) virus. RT-qPCR analysis of Cbs expression in MEFs infected with pSin-GFPN and pSin-Cbs viruses. n = 3. Immunofluorescence of Sall4 in WT cells and Cbs overexpressing cells on d12. n = 4. Scale bar, 100 μm.
Figure 1A. SILAC-based strategy for multidimensional analysis of TNF-α signaling. The "medium" and "heavy" labeled cells were stimulated with TNF-α for 5 and 15 minutes, respectively, and mock-treated "light" labeled cells served as control. For site-specific quantification of phosphorylation and ubiquitylation, equal amounts of proteins from three SILAC populations were mixed and phosphorylated and di-Gly modified peptides were enriched in parallel from the same samples. To investigate the dynamics of TNF-RSC-associated proteins, cells were treated with FLAG-TNF-α, and the ligand-bound TNF-RSCs were affinity-purified using anti-FLAG antibody, proteins were mixed before proteolysis and the relative abundance of proteins was quantified with LC-MS/MS. B. Quantification of phosphorylation events in response to TNF-α stimulation. The bar chart shows the fraction of phosphorylation sites that were differentially regulated upon TNF-α stimulation. C. Sequence motif analysis of TNF-α-upregulated phosphorylation sites. The logo plots show enrichment of amino acids at positions flanking TNF-α-upregulated phosphorylation sites.
Figure 7A. Analysis of TCR nanoclustering in lymphoblasts from healthy WT and ccr5Δ32 homozygous donors by EM. Top, representative small field image showing gold particle distribution in cell surface replicas of anti-CD3ε-labeled cells; bottom, quantification (mean ± SEM) of gold particles in clusters of the indicated size in the WT (gray bars; n = 5 cells, 17689 particles) and Δ32/Δ32 cells (light red; n = 4 cells, 16938 particles). Insets show the distribution between clusters of one, two, three, four or more than four particles, and statistical analysis. B. Normalized SM, Cer and dhCer levels in lymphoblasts obtained as in A. A representative experiment is shown (n = 3 donors/genotype; n = 2 independent experiments) C. Relative CerS2 mRNA levels in day 8 WT and ccr5Δ32 lymphoblasts. Each data point is the average of a technical triplicate from 3 donors in two independent experiments (n = 6)
Figure 3A) U2OS cells were grown on glass coverslips and synchronized in G2 after thymidine release. Cells were fixed at the indicated times after adriamycin-induced DNA damage and stained for DAPI and γH2AX. Scale bar indicates 10 μm. B) Quantification of cells in 3A. Each dot represents the total signal of γH2AX in the nucleus of a single cell. Bars indicate the mean and error bars indicate the SD of the data points in each condition. Whiskers represent 5-95% of data points. Statistical significance was tested using an unpaired two-tailed t-test (NS for P>0.05, **** for P≤0.0001). C) U2OS cells were synchronized in G2 and harvested at the indicated times after induction of adriamycin-induced DNA damage with or without Tlk2-depletion. Samples were analyzed by western blotting for the indicated proteins. D) U2OS cells were synchronized in G2 and damaged with 6 Gy of IR. Samples were taken at the indicated times after induction of damage with or without Tlk2-depletion. Samples were analyzed by western blot for the indicated proteins. Band signal intensity for Asf1A and Asf1B was measured and corrected for Ponceau S staining. E) U2OS cells were transfected with the indicated siRNA's and treated as in 2A. Mitotic index was analyzed by FACS. Error bars represent SD, n=3. Statistical significance was tested using a paired two-tailed t-test (NS for P>0.05, * for P≤0.05). F) U2OS cells were synchronized with a single thymidine block, released into G2 and damaged with 0.5 μM Adriamycin for one hour. After a 16 hour G2 arrest cells were harvested for western blot analysis and analyzed for the indicated protein levels. Lanes were all present on the same blot but pasted together for comparison as indicated by the lines. G) Cells were synchronized in S- or G2-phase by a single thymidine block. Damage was induced by 0.5 μM Adriamycin for one hour and cells were allowed to incubate for 16 hours afterwards. Samples were fixed at the indicated cell cycle stages and stained for Asf1A-pS166. Nuclear fluorescence intensity was measured relative to DAPI. Whiskers represent 5-95% of data points. H) Cells were treated and analyzed as in 2E. Induction of FLAG-Asf1A-4D was induced using doxycycline. Error bars represent SD, n=5. Statistical significance was tested using a paired two-tailed t-test (NS for P>0.05, * for P≤0.05). I) U2OS cells were synchronized in G2 by a single thymidine block and where indicated treated with 0.5 μM Adriamycin for one hour and after the indicated time points were pre-extracted and fixed. Cells were stained for total levels of Histone H3 and quantified relative to DAPI. Scale bar indicates 10 μm. Whiskers represent 5-95% of data points. J) FLAG-Asf1A-4D inducible cells were treated as in 3I and expression was induced using doxycycline where indicated. Whiskers represent 5-95% of data points. Statistical significance was tested using an unpaired two-tailed t-test (**** for P≤0.0001).
Figure 1A. Quantification of the nociceptive responses in mice within 5 min after intraplantar injection of control saline or 500 ppm iodine. In this and subsequent similar figures, the number of mice is indicated. B. Averaged intracellular Ca2+ signals in cultured mouse DRG neurons in response to consecutive applications of 0.25 ppm iodine, 30 μM AITC and 2 μM capsaicin. All of the iodine responsive neurons (n = 332) from 6 adult mice were included in the analysis. C. Quantification of the nociceptive responses in mice within 5 min after intraplantar injection of control saline or 500 ppm iodine, following the intraperitoneal injection of HC030031 (HC) (C) or vehicle. D. Quantification of the nociceptive responses in mice within 5 min after intraplantar injection of control saline or 500 ppm iodine, following the intraperitoneal injection of AMG517 (D) or vehicle. E. Quantification of the nociceptive responses within 5 min after intraplantar injection of control saline in WT mice or 500 ppm iodine in WT and TRPA1-/-mice. F. Quantification of the nociceptive responses in TRPA1-/- mice within 5 min after intraplantar injection of control saline or 1,000 ppm iodine, following intraperitoneal injection of saline or vehicle.
The Dsc complex mediates degradation of Orm2 at cytosolic proteasomes.Figure 4WT cells and the indicated mutants were left untreated (0 min) or were treated with cycloheximide (CHX) to block protein synthesis for 90 min and 180 min at 26°C or at non-permissive temperature (37°C) where indicated. Total cell lysates were analyzed by SDS-PAGE and Western blotting with the indicated antibodies. (A) Graphs display the FLAG-Orm2 protein levels determined by densitometric quantification of Western blots from cell lysates of WT cells and the indicated mutants 0, 90 and 180 min after the addition of CHX. Each experiment was repeated at least three times, FLAG-Orm2 levels were normalized to Pgk1 loading controls, and each time-point was related to t=0 min (set to 1). Data are presented as mean ±standard deviation. WT cells and the indicated mutants were left untreated (0 min) or were treated with cycloheximide (CHX) to block protein synthesis for 90 min and 180 min at 26°C or at non-permissive temperature (37°C) where indicated. Total cell lysates were analyzed by SDS-PAGE and Western blotting with the indicated antibodies. Each experiment was repeated at least three times, FLAG-Orm2 levels were normalized to Pgk1 loading controls, and each time-point was related to t=0 min (set to 1). WT cells and the indicated mutants were left untreated (0 min) or were treated with cycloheximide (CHX) to block protein synthesis for 90 min and 180 min at 26°C or at non-permissive temperature (37°C) where indicated. Total cell lysates were analyzed by SDS-PAGE and Western blotting with the indicated antibodies. (C) SEC13 WT and sec13-4 cells were preincubated for 30 minutes at 37°C before CHX was added. (D) Epifluorescence and phase contrast microscopy of living WT cells and cdc48-3 mutants expressing GFP-Orm2 incubated for 5h at the non-permissive temperature (37°C). Data information: Scale bars 5µm. (E) Epifluorescence and phase contrast microscopy of living tul1∆ and tul1∆ sec13-4 mutants expressing GFP-Orm2 (green) and dsRed-HDEL (red) incubated at 26°C or shifted for 90 minutes to the non-permissive temperature (37°C). Data information: Scale bars 5µm.
Expression of mitochondrial functions-related genes is higher in Black than White HKCs and upregulated by HSD17B7 overexpression.Figure 7(A) Top bar: gradient of population admixture of the indicated HKC strains of Black versus White individuals, on the basis of the SNP genotype analysis shown in Figure 1B, with the simplest assumption of two ancestries (K = 2).Gradient ranges from black to white colors for Black and White individuals with no population admixture, respectively. Individuals with increasing proportions of White population admixture are represented with degrading shades of grey. Left: heat map of expression levels of the indicated genes from the OXPHOS gene signature of Fig 3C, in Black versus White HKC strains ordered according to genome admixture calculations and in three strains of White HKCs with HSD17B7 overexpression. Modified Z scores of the individual genes, as median-centered log2 Intensity values divided by standard deviation, are shown by color gradient variations. Right: heat map of expression levels of the same genes in three White HKC strains infected with a HSD17B7 overexpressing lentivirus versus empty vector control as in Fig 5A (strains: 260, 275, 249). Expression levels are derived from global transcriptomic analysis (Clariom D hybridization; GEO accession number GSE172288) and expressed as logFold Change values of treated vs control samples. (B) Gene Ontology (GO) analysis of genes analyzed in the previous panel. Shown is a list of process networks with statistical significance of enrichment in Black HKCs. The complete list is provided in Dataset EV1.
Fig.Figure 6Protoplasts from 12-day old rice (ZH11 background) leaf sheath tissue were collected. and protoplasts were transfected with pM999-GID1 and mutation constructs and imaged 12 hours after transfection. All confocal images were scanned using similar laser gain and offset settings. Bars = 10 μm. Subcellular location of GID1 in WT and pldα6 protoplasts with or without GA3 treatments. Cells for 12 hours after transformation were treated with or without 10 μM GA3 for two hours. Bars = 10 μm. Immunoblotting of subcellular fractions of GID1 and mutations expressed in rice protoplasts. Total (T), soluble (S) and nuclear (N) protein fractions were isolated from protoplasts for 12 hours after transformation. Equal amounts of proteins from each sample were loaded. Immunoblotting of subcellular fractions of GID1 expressed in WT and pldα6 protoplasts. After 2-hour treatment with 10 μM GA3, total (T), soluble (S) and nuclear (N) protein fractions were isolated from 10 samples of protoplasts. Equal amount of each sample was loaded for SDS-PAGE and immunoblotting.
Figure 5A) Immunostaining of anterior horn of lumbar spinal cord for ChAT (in green) and p62 (in red) at 130 days. Scale bar, 10 µm. B) Quantification of the percentage of ChAT-positive motor neurons (MN) containing p62-positive inclusions at 130 days. PKO PKO/G93A MN have less SOD1 aggregates than G93A, while Non Tg and PKO MN have none. Data were collected from n= 4 (2 males and 2 females) mice for Non Tg and PKO and n= 6 (3 males and 3 females) for G93A and PKO/G93A (males and females). Number of images recorded for each genotype: n= 9 for Non Tg and PKO, n= 16 for G93A and n= 12 for PKO/G93A. Results are expressed as mean ± SEM; *p= 0.032, by unpaired one-tailed Student's t test. C) Representative Western blots of OPTN, p62 and VCP in spinal cord mitochondrial fractions at 130 days. Protein levels are normalized by Complex V. The quantifications show that mitochondria of PKO/G93A mice have less mitophagy adaptor proteins than G93A relative to Complex V. D) Quantification of OPTN relative to Complex V at 130 days. Results are expressed as mean ± SEM and as percent of Non Tg; n= 8 (4 males and 4 females); *p= 0.039 by paired Student's t test. E) Quantification of p62, relative to Complex V at 130 days. Results are expressed as mean ± SEM and as percent of Non Tg; n= 8 (4 males and 4 females); **p= 0.0029 by paired Student's t test. F) VCP was quantified using Complex V as loading reference at 130 days. Results are expressed as mean ± SEM and as percent of Non Tg; n= 8 (4 males and 4 females); *p= 0.044 by paired Student's t test.
Figure 1(A) Immunoblots for LC3 using whole cell lysates of Huh7 cells before and after treatment with iron chelators (DFP, DFX or DFO). β-actin was used as loading control. Bafilomycin (-): without Bafilomycin, Bafilomycin (+): with 100 mM of Bafilomycin. (B) Dual excitation (550/488 nm) of mt-mKeima transfected-Huh7 cells shows red fluorescence indicating mitophagy with DFP treatment at 24 h and green fluorescence without DFP treatment. Scale bar: 10 μm. (C) The high ratio signal cells were quantified by flow cytometry with dual excitation of mt-mKeima at 633/488 nm for untreated (left panel) and DFP-treated (24 h, right panel) Huh7 cells. (D) Untreated Huh7 cells or those treated with DFP (10 μM), DFX (10 μM) or DFO (10 μM) were quantified for mitophagy using mt-mKeima transfection and FACS analysis (n=4, biological replicates). A high signal (633/488) area/ cell area indicates the proportion of cells undergoing mitophagy. The central horizontal bar and the error bards indicate mean ± SD. The Tukey's honestly significant test was used for statistical analysis. **: P<0.01. (E) Electron microscopic images of untreated (left panel) and DFP-treated (right panel) Huh7 cells. Arrows indicate mitophagosome-like structures. Scale bar: 500 nm. The number of mitophagosome-like structures/100 μm2 was compared for four randomly selected areas (technical replicates) between cells treated with DFP (1.0 mM) and those without DFP. The central horizontal bar and the error bards indicate mean ± SD. The Tukey's honestly significant test was used for statistical analysis. *: P<0.05. (F) Quantification of mitophagy using mt-mKeima transfection and FACS analysis for Huh7 cells treated with DFP (0, 0.01. 0.5 or 1.0 mM as indicated) in the absence or presence of bafilomycin (n=4, biological replicates). The central horizontal bar and the error bards indicate mean ± SD. *: P<0.05, **: P<0.01. (G) Immunoblots for Parkin, PINK1, phosphorylated p-62 (p-p62), HSP60, LC3, p62/SQSTM, and Atg5 using the mitochondrial fraction and whole cell lysates of Huh7 cells. HSP60 and β-actin were used as loading controls for mitochondria and whole cell lysates, respectively.
Figure 2(A) Localization of chromosomally expressed GFP-Pgc1 from the constitutive ADH1 promoter in wt and doa10Δ cells. Arrowheads indicate GFP-Pgc1 labeling at the ER, which is stained by Sec63-Cherry. LDs were visualized upon staining with the neutral lipid dye MDH. On the bottom, box plot with quantitation of GFP-Pgc1 fluorescence intensity at the nuclear envelope in wt and doa10Δ. GFP-Pgc1 measurements were performed as described in the Materials and Methods section. Scale bar: 5 µm. (B) Schematic representation of Pgc1. The location of the predicted hydrophobic domain is indicated. (C) Crude membranes from wt and doa10Δ cells expressing 3HA-Pgc1 were subjected to the indicated treatments and subsequently fractionated into membrane pellet (P) and supernatant (S).(D) Crude membranes from wt and doa10Δ cells expressing 3HA-GFP-Pgc1275-321 were analyzed as in (C).
Figure 5(A) Graphic depiction of the bacterial outer membrane composition and LPS structure of S. flexneri. Arrow indicates the LPS truncation site in the O-antigen deficient rfaL mutant. O-antigen oligosaccharide subunit composition of S. flexneri serotype 2a and E. coli serotypes O8 and O25 are shown. (B) Time-lapse microscopy of fixed GFP+ co-isogenic S. flexneri wild type and rfaL mutant after adding 10 µM Alexa-Fluor647-hGBP1F and 2 mM GTP. Bacteria bound by hGBP1F at 5 min and hGBP1F-enclosed bacteria after 60 min were quantified. Combined data from three independent experiments are shown as mean ± SEM. Significance was determined by unpaired t-tests, two-tailed. n.s., not significant; ****, P ≤ 0.0001. (C) Scanning-electron micrographs of live wild type and rfaL mutant strains incubated with no protein or with 5 µM of hGBP1F in the presence of 2 mM GTP for 4 min. Arrowheads point to unattached hGBP1 polymers, arrows point to hGBP1 polymers attached to bacteria, and asterisks mark polymeric structures that appear to fuse with bacterial surfaces (D) Model: O-antigen drives the transition of surface-docked hGBP1F polymers into bacteria-enveloping hGBP1F protein sheets. (E) Confocal images of formaldehyde fixed, co-isogenic GFP+ wild type and mutant S. flexneri strains harboring rfb regions of E. coli serotypes O8 and O25 after 60 min of incubation time in the presence of 10 µM Alexa-Fluor647-hGBP1F and 2 mM GTP.
Figure 2Liver morphology of WT and Cideb-/- liver under chow diet and HFLF diet. H/E staining, oil red-O staining, immunohistochemistry and ultra-structure (Electron Microscope) were performed. Scale bar represented 50 µm in the upper three rows of images, and 2 µm in the bottom row of images. LD: lipid droplet, ER: endoplasmic reticulum, M: mitochondrial, N: nucleus. Total liver TAG levels (B) of WT and Cideb-/- mice under chow diet and HFLF diet (n = 8 per group). Data information: Data represent Mean ± SEM; NS: not significant, *: p < 0.05, **: p < 0.01, ***: p < 0.001, by 2-tailed Student's t test. Liver cholesterol ester levels (C) of WT and Cideb-/- mice under chow diet and HFLF diet (n = 8 per group). Data information: Data represent Mean ± SEM; NS: not significant, *: p < 0.05, **: p < 0.01, ***: p < 0.001, by 2-tailed Student's t test. D Loss of Cideb inhibits diet-induced SREBP activation. IB of hepatic SREBP processing and lipogenic enzyme levels (Fasn, Scd1) of WT and Cideb-/- mice under chow diet and HFLF diet. E Serum AST and ALT levels of WT and Cideb-/- mice under chow diet and HFLF diet (n = 8 per group). Data information: Data represent Mean ± SEM; NS: not significant, *: p < 0.05, **: p < 0.01, ***: p < 0.001, by 2-tailed Student's t test. F Cideb deficiency alleviates the whole body inflammation response. Serum TNFα, MCP1 and IL6 levels of WT and Cideb-/- mice under chow diet and HFLF diet (n = 8 per group). Data information: Data represent Mean ± SEM; NS: not significant, *: p < 0.05, **: p < 0.01, ***: p < 0.001, by 2-tailed Student's t test. Serum fed glucose levels (G) of WT and Cideb-/- mice under chow diet and HFLF diet (n = 8 per group). Data information: Data represent Mean ± SEM; NS: not significant, *: p < 0.05, **: p < 0.01, ***: p < 0.001, by 2-tailed Student's t test. Serum fed insulin levels (H) of WT and Cideb-/- mice under chow diet and HFLF diet (n = 8 per group). Data information: Data represent Mean ± SEM; NS: not significant, *: p < 0.05, **: p < 0.01, ***: p < 0.001, by 2-tailed Student's t test. I Glucose tolerance test (GTT) of WT and Cideb-/- mice under HFLF diet (n = 5 per group). Data information: Data represent Mean ± SEM; NS: not significant, *: p < 0.05, **: p < 0.01, ***: p < 0.001, by 2-tailed Student's t test. J Insulin tolerance test (ITT) of WT and Cideb-/- mice under HFLF diet (n = 5 per group). Data information: Data represent Mean ± SEM; NS: not significant, *: p < 0.05, **: p < 0.01, ***: p < 0.001, by 2-tailed Student's t test.