EMBO/sd-character-level-ner huggingface dataset:
Figure 6A The immunoblot analyses of MCF-7 ENTREP-KO and parent MCF-7 cells. Note that the amount of CXCR4 protein was not changed. The immunoblot analyses were carried out using six independent samples, and their blot bands were semi-quantified using ImageJ software. CXCR4/GAPDH ratio was caluculated as a ratio of GAPDH-adjusted CXCR4 expression. CXCR4/GAPDH ratios shown as mean ± SD from at least four biological replicates. P-values were obtained by Student's t-tests and P < 0.005 was considered as statistically significant. No significant difference was observed. B Cell proliferation analysis. Cells were cultured in DMEM supplemented with 10% FBS and counted at day1 to day3. Data are shown as mean ± SD from six biological replicates. P-values were obtained by Student's t-tests and P < 0.005 was considered as statistically significant. NS, not significant. C The expression of endogenous CXCR4 in lentivirally-transduced MCF-7 ENTREP-KO cells. CXCL12 (+) and (-) indicate the presence and absence of CXCL12 treatment. In CXCL12-treated ENTREP-EGFP expressing cells, a non-negligible amount of CXCR4 was observed in the cytoplasm, in which CXCR4 was overlapped with ENTREP-EGFP. CXCR4 expression on the cell surface still remained (arrows). Scale bar 10 mm. The images are representative of at least three independet experiments.
Figure 6A. Motif analysis in functional KDM6A-bound regions, showing top ten de novo motifs ranked by P-value determined by HOMER software. B. Co-binding analysis in functional KDM6A-bound enhancer and promoter regions revealed that HNF1A was the most enriched co-bound TF amongst three other acinar cell TFs. Binding regions of TAL1 in a non-pancreatic cell type and random binding sites were used as negative controls. P-values was determined by Fisher's exact test for peak comparisons using all enhancer and promoter regions as background. C. The most downregulated genes in Kdm6apKO pancreas are shown ranked by q-value, and are almost invariably bound by HNF1A and down regulated in Hnf1aaKO pancreas, or known to be direct HNF1A-dependent target genes from other studies (red and purple, respectively). D,E. GSEA analysis on the Hnf1aaKO and Kdm6apKO ranked-ordered gene lists vs. their reciprocal up- or downregulated gene sets, demonstrated that KDM6A and HNF1A regulate similar genes. F. Expression changes in Hnf1aaKO and Kdm6apKO pancreas, showing that genes bound by KDM6A and downregulated in Kdm6apKO pancreas (red dots) were generally downregulated in Hnf1aaKO pancreas. G. HNF1A and KDM6A co-occupy the same regions in Pah, which is downregulated in Hnf1a and Kdm6a knock-out pancreas. H. Genes that were co-bound by KDM6A and HNF1A showed greatest downregulation in Kdm6apKO pancreas, compared with KDM6A-bound genes that were not bound by HNF1A. Box plots show median and IQR of Log2 TPM fold-changes and whiskers extend to highest and lowest data points within 1.5 x IQR outside box limits. P-values were determined by two-tailed Student's t tests and n=4 replicates per condition.
SECexplorer tool for customized interrogation of SEC-SWATH elution profiles.Figure 7SECexplorer web-interface for querying custom protein sets for co-elution behavior in the SEC-SWATH-MS data, viewing chromatograms for interpretation and with algorithmic assistance. Zoom in to high MW peak shoulder of holo-COP9 Signalosome (compare Figure 5), where defined co-elution signals of CSN substrate components CUL4A and DDB2 suggest the partial resolution of substrate-bound and free pools of CSN holo-complex. Estimation of the fraction of holo-CSN in the likely substrate-bound pool versus the free pool, with eight measurements along the eight subunits and based on Gaussian deconvolution of two signals underlying the observed peak and shoulder (also see Appendix Figure S7).
Figure 5A, Representative images of scaf1-/- and scaf1+/+ fish fed with the indicated diets. B, C, Changes in (B) length and (C) weight over time (Δ1 +/+ n=10, Δ1 -/- n=10, Δ2 +/+ n=10, Δ2 -/- n=12-13. D-F, Adipose tissue measurements on hematoxylin-eosin (H&E)-stained adult zebrafish sagittal sections. (D) Adipose tissue area per total section area (average of three sections/biological replicate) and (E) adipocyte size (average of 20-30 adipocytes of ventral adipose tissue per biological replicate) (Δ1 +/+ n=3, Δ2 n=8). scaf1Δ1 and scaf1Δ2 are represented with circles and squares, respectively. Adipose tissue measurements on hematoxylin-eosin (H&E)-stained adult zebrafish sagittal sections. (F) Representative images of ventral fat deposits (dotted lines). G, Number of eggs per clutch (standard diet Δ1 +/+ n=8, Δ1 -/- n=8, Δ2 +/+ n=7, Δ2 -/- n=12, double diet Δ1 +/+ n=7, Δ1 -/- n=8, Δ2 +/+ n=8, Δ2 -/- n=9). H, Representative images of H&E-stained ovaries. Black dotted lines outline the ovaries, blue dotted line indicates a mature follicle. I, Quantification of mature ovary follicles per ovary section (average of three sections/biological sample) (Δ1 n=2-3, Δ2 n=6).
Figure 6(A) CRISPR/Cas9 gene editing was used to generate peroxisome-null cell lines by knocking out PEX19. In wild-type HCT116 cells, catalase localized to peroxisomes (dot-like structures), whereas in PEX19 -/- cells catalase was diffusely localized throughout the cytosol, indicating loss of peroxisomes. Scale bars, 10 µm. Wild-type and PEX19 -/- HCT116 cells stably expressing HA-Parkin and 3Flag-MITOL were treated with 10 µM valinomycin for 3 hours, and then subjected to immunocytochemistry with anti-Flag, anti-catalase, and anti-Hsp60 (B) antibodies. In wild-type HCT116 cells, MITOL translocated to peroxisomes, whereas MITOL remained associated with mitochondria in PEX19 -/- cells. Arrowheads indicate representative examples of 3Flag-MITOL co-localization with catalase rather than Hsp60 in wild-type HCT116 cells. Higher magnification images of the boxed regions are shown in the small panel. Scale bars, 10 µm. Wild-type and PEX19 -/- HCT116 cells stably expressing HA-Parkin and 3Flag-MITOL were treated with 10 µM valinomycin for 3 hours, and then subjected to immunocytochemistry with anti-Flag anti-Tom20 (C) antibodies. In wild-type HCT116 cells, MITOL translocated to peroxisomes, whereas MITOL remained associated with mitochondria in PEX19 -/- cells. Higher magnification images of the boxed regions are shown in the small panel. Scale bars, 10 µm.
AosR regulates non-canonical cysteine biosynthesis pathway.Figure 4Non-canonical pathway for cysteine biosynthesis in Mtb. MoeZ transfers sulfur from a yet unknown source to the C-terminus of CysO to form thiocarboxylated CysO (CysO-SH). CysM, a non-canonical cysteine synthase, utilizes O-phospho-L-serine (OPS) and CysO-SH as substrates to generate CysO-cysteine adduct, which is cleaved by a protease Mec, to release cysteine and regenerate CysO(Burns et al., 2005). Hypothetical model for transcriptional regulation mediated by AosR. Upon oxidative stress, AosR binds to its promoter regulating the entire operon through a positive feedback loop, resulting in higher L-cysteine biosynthesis. Early log-phase cultures of indicated strains were subjected to 50 µM of CHP for 24 h, in the presence or absence of 1 mM L-cysteine, and CFUs were enumerated (mean ±SEM; n=3). Single-cell suspensions of indicated strains were used to infect pΦ from Balb/C at 1:10 MOI. Cells were either supplemented with 0.2 mM L-cysteine or left untreated and the CFUs were enumerated 96 h p.i. (mean ± SEM; n=3). pΦ from Balb/C were infected with indicated strains and bacterial viability was monitored 96 h p.i (mean ± SEM; n=3). 35 µg whole cell lysates (WCL) of Rv, Rv∆aosR and Rv∆aosR::aosR subjected to 0 or 50 µM of CHP for 24 h were resolved on 15% SDS-PAGE, transferred to nitrocellulose membrane and probed with α-AosR and α-SigA antibodies. Relative expression of indicated genes in Rvtreated with 50 µM CHP for 24 h as compared with Rv as measured by qRT-PCR. Data were normalized with respect to 16s rRNA and results are expressed as mean log2 fold change ± SD of three independent replicates and is representative of one of two biological replicates. Statistical significance was analysed using student's t-test (two-tailed, unpaired). WCLs of Rv, Rv∆aosR, and Rv∆aosR::aosR subjected to 0 or 50 µM of CHP for 6 h were resolved on 15% SDS-PAGE, transferred to nitrocellulose membrane, and probed with indicated antibodies. Relative expression of indicated genes in Rvtreated with 50 µM CHP for 24 h as compared with Rv as measured by qRT-PCR. Data were normalized with respect to 16s rRNA and results are expressed as mean log2 fold change ± SD of three independent replicates and is representative of one of two biological replicates. Statistical significance was analysed using student's t-test (two-tailed, unpaired). Early log-phase cultures of indicated strains were subjected to 50 µM of CHP for 24 h and the CFUs (mean ±SEM; n=3) were enumerated. pΦ from B6 were infected with indicated strains and intracellular bacterial viability was monitored 96 h p.i (mean ± SEM; n=3).
Figure 7(A) Illustration of the arena used for the open field test (OFT). (B) In the OFT, Cox6a2-/- mice travelled a longer distance and moved faster than WT mice. Cox6a2-/- mice also moved more often from the outer zone to the inner zone of the arena. Two-tailed unpaired Student's t-tests. (C) Illustration of the arena used for the social interaction test (SIT). (D) In the SIT, Cox6a2-/- mice also travelled a longer distance with higher mean velocity than WT mice. Additionally, both Cox6a2-/- and WT mice spent more time exploring the container with the stranger mouse than the empty container. Total distance moved, mean velocity, and f [number of crossings] outer to inner zone: two-tailed unpaired Student's t-test. Social interaction: two-way ANOVA followed by Tukey's multiple comparisons test. (E) Sensory gating was tested by the pre-pulse inhibition (PPI) of the acoustic startle response (ASR) test. (F) The startle responses were significantly lower in Cox6a2-/- mice when the loud stimulus (120 dB) was presented alone the first 5 times. Two-way ANOVA followed by Tukey's multiple comparisons tests. (G) The PPI of the acoustic startle response was similar at all pre-pulses for Cox6a2-/- and WT mice. Two-way ANOVA followed by Sidak's multiple comparisons tests.
KCC4 expression correlates with SIRT7 expression, and KCC4 activity is modulated by acetylation.Figure 1A In silico analysis of Sirt7 mRNA expression in different tissues of the BXD genetic reference population (www.genenetwork.org). Adr: Adrenal Gland mRNA, Bla: Bladder mRNA, Bone: Bone Tibia mRNA, CNS: CNS mRNA, Cec: Cecum mRNA, Epi: Epididymis mRNA, Eso: Esophagus mRNA, Eye: Eye mRNA, Fat: Peritoneal Fat mRNA, GI: GI Track mRNA, Hea: Heart mRNA, Kid: Kidney mRNA, Liv: Liver mRNA, Lung: Lung mRNA, Mus: Gastrocnemius Muscle mRNA, Ova: Ovary mRNA, Pro: Prostate mRNA, Sal: Salivary Gland mRNA, Skin: Skin Back mRNA, Sn: Sciatic Nerve mRNA, Spl: Spleen mRNA, Tes: Testis mRNA, Thy: Thymus mRNA, Ton: Tongue mRNA, Tra: Trachea mRNA, and Ute: Uterus mRNA. B Correlation plot for kidney mRNA expression of Sirt7 and Slc12a7 that codifies KCC4 in the mouse BxD genetic reference population (www.genenetwork.org). C Oocytes injected with cRNA encoding KCC4 were incubated with NAD+ 200 μM and NAM 10 mM for 4 h. Lysates were immunoprecipitated with an anti-KCC4 antibody and then analyzed by SDS-PAGE/immunoblotting using an anti-acetylated-lysine (AcK) or anti-KCC4 antibody. Non-injected oocytes (UI) were used as a control. D Oocytes expressing KCC4 were incubated with NAM for the indicated times, and KCC4 protein levels (upper panel), and activity (lower panel) were analyzed by SDS-PAGE/immunoblotting or Cl--dependent 86Rb+ uptake under hypotonic conditions. E Oocytes injected with H20 or KCC4 were incubated for the indicated times, and KCC4 protein levels were analyzed by SDS-PAGE/immunoblotting. F Oocytes were injected with either WT, K114R or K114Q KCC4 and incubated with vehicle (V), NAD+ or NAM for 48 h, and KCC4 protein levels (upper pannel), and activity (lower pannel) were analyzed as in D.
A schematic representation of the pcMNP-based viral RNA extraction method.Scheme2 A schematic representation of the pcMNP-based viral RNA extraction method.
The degree of polyploidy differs between etiologies and low tumor ploidy correlates with reduced disease-free survival.Figure 7(A) The ploidy index is inferred as 1/cell density of tumorous (T) and non-tumorous (NT) tissue of HCC patients with different liver diseases. ALD - alcoholic liver disease (n=65); HBV - hepatitis B virus infection (n=24); HCV - hepatitis C virus infection (n=85); NASH - non-alcoholic steatohepatitis (n=33). Statistical significance was determined by one-way ANOVA with multiplicity correction (Sidak-Holm); * p≤0.05, ** p≤0.01, *** p≤0.001. n-numbers refer to the number of patient samples analyzed. (B) All patients were grouped according to HCC tumor cell density as surrogate for tumor ploidy. Data were divided at the median and exact patient numbers are shown in the graphs. Median recurrence-free survival (RFS) for a period of 10 years for patients with low tumor ploidy was 4.8 years. For patients with high tumor ploidy the median RFS could not be defined as more than 50% of the patients survived without relapse. Patient characteristics are shown in Appendix table S1. A Log-rank test was used to determine statistical significance.
The CMA receptor LAMP2A is downregulated in CTNS-deficient cells.Figure 4The expression levels of the endogenous LAMP2A and LAMP1 proteins were analyzed in wild-type (WT) and Ctns−/−mousefibroblasts (A), kidney (B) and liverlysosomes (C) by Western blot. Quantitative densitometry analysis of immunoblots obtained from different experiments was performed by calculating the ratio between LAMP2A and actin signals in each lane. Results are mean ± SEM of four independent experiments with mousefibroblasts, 5 WT and 6 Ctns−/−mousekidneys, 3 WT and 3 Ctns−/−mouselivers (lysosomal extracts). Comparative analysis of the amount of LAMP2A in wild-type or Ctns−/−liverlysosomes was performed using two different anti-LAMP2A antibodies. 15 μg of lysosomal proteins were loaded in each lane. Ab1, anti-LAMP2A antibody raised against the twelve amino acids of the cytosolic region of ratLAMP2A largely validated to recognize mouseLAMP2A but not other LAMP2 isoforms (Cuervo Dice, 1996). Ab2, anti-LAMP2A Abcam Ab18528. Decreased lysosomalLAMP2A was evident using either antibody. LAMP1 was used as a loading control. Representative of two independent experiments with similar results.
Figure 5A. Immunofluorescence images of MAD1 and MAD1-pT716 kinetochore levels in nocodazole-arrested RPE1 cells treated with or without AZ-3146. Scale bars = 5µM. B. Electron micrographs of rotary-shadowed MAD193-718:MAD2 (top row), MBP-MAD1:MAD2 (middle row), and MBP-MAD1:MAD2 in complex with GST-CDK1:Cyclin B1 (bottom row). Scale bar = 50 nm. C. Quantifications (top) and corresponding immunofluorescence images (underneath) of relative kinetochore MAD1 and MAD1-pT716 levels in nocodazole-arrested of MAD1-WT-C14 and MAD1-3EK-C12 cells treated with different doses of AZ-3146 for 30 min. MG132 was included at the time of AZ-3146 addition to prevent mitotic exit. Each dot represents a cell, horizontal lines indicate the median and error bars show the 95% confidence interval. Note, when these vertical bars do not overlap the difference is considered statistically significant at a level of at least p<0.05 (see methods). 30 cells from 3 experiments. Both kinetochore intensity graphs display data that is relative to the WT-C14 untreated controls, which are normalised to 1. The mean level of the normalised controls is indicated by the dotted lines. D. Duration of mitotic arrest in MAD1-WT C14 or MAD1-3EK C12 cells arrested in nocodazole and then treated with indicated concentrations of AZ-3146. Note, the 1.25 µM AZ-3146 data is displayed in Figure 4D, but also included here to allow comparison with other drug doses. Graph shows cumulative mean (±SEM) of 3 experiments, 50 cells per condition per experiment.
Figure 3Representative false colour images of photosystem II (PSII) operating efficiency (ΦPSII) captured by chlorophyll fluorescence imaging from WT, stp1-1, stp4-1 and stp1stp4 plants. ΦPSII was measured at a photosynthetically active radiation (PAR) of 440 µmol m-2 s-1.    ΦPSII quantified over the phenotyping period. Data shown are means ± SEM; n = 10 per genotype and time point.   Whole-plant recordings of changes in CO2 assimilation (A) from WT, stp1-1, stp4-1 and stp1stp4 plants. Data shown are means ± SEM; n ≥ 3 per genotype. Whole-plant recordings of changes in CO2 assimilation (A) from self-grafted donor lines (WT/WT, stp1stp4/stp1stp4) and reciprocal grafting of shoot/root (WT/stp1stp4, stp1stp4/WT) plants. Data shown are means ± SEM; n = 3 per genotype. Quantification of leaf glucose (Glc). Data shown are means ± SEM; n = 8 per genotype and time point.   Quantification of leaf fructose (Fru). Data shown are means ± SEM; n = 8 per genotype and time point.   Quantification of leaf sucrose (Suc). Data shown are means ± SEM; n = 8 per genotype and time point.   Quantification of leaf starch. Data shown are means ± SEM; n = 8 per genotype and time point. Representative Red Green Blue (RGB) images of 3-(day 0) and 4-week-old (day 7) WT, stp1-1, stp4-1 and stp1stp4 plants. Projected rosette area over the phenotyping period. Data shown are means ± SEM; n = 10 per genotype and time point.
targeted arginines on p14ARF protein stability.Figure 4A, U2OS, HeLa and HEK293 cells were transfected with Flag-tagged wild type (wt) or mutant (RG, RF, RK) p14ARF-containing plasmids. Overexpression was analyzed by immunoblotting using α-Flag (exogenous p14ARF proteins) and α-β-TUBULIN (loading control) antibodies (A). As visualized here, p14ARF with the Flag-epitope tagged reproducibly showed a clear migration difference between wt and mutant p14ARF proteins, with the mutants (especially the RK mutant) migrating slowlier than wt protein in the SDS-PAGE. The staining signals of the p14ARF bands were densitometrically quantified and normalized to the respective β-TUBULIN signal, as specified by the numbers below the blots, with the protein signals of p14ARF wt set to 1. Transcript levels of wt and mutant p14ARF were determined by RT-qPCR in U2OS cells (B) as well as in HeLa and HEK293 cells (Appendix Fig. S3B, S3C). Values were normalized to GAPDH expression and presented relative to wt p14ARF, mean ± SD of triplicates. C: HeLa cells were transfected with the indicated siRNAs (five control/non-targeting siRNAs and four PRMT1-specific siRNAs). PRMT1 depletion and endogenous p14ARF protein levels were analyzed by immunoblotting using α-PRMT1, α-p14ARF and α-β-TUBULIN (loading control) antibodies. The p14ARF signals were densitometrically quantified and normalized to the respective β-TUBULIN signal, as specified by the numbers below the blot, with the siControl_2 condition set to 1. D: HeLa cells were infected with recombinant adenovirus encoding GFP (control) or wild type PRMT1. PRMT1 overexpression and endogenous p14ARF protein levels were analyzed by immunoblotting using α-PRMT1, α-p14ARF and α-CDK2 (loading control) antibodies. The p14ARF signals were densitometrically quantified and normalized to the respective CDK2 signal, as specified by the numbers below the blot, with the control condition set to 1. : HeLa cells expressing a doxycycline-inducible shRNA targeting PRMT1 were treated or not with doxycycline (Dox) for 6 days. PRMT1 depletion was monitored by immunoblotting using the indicated antibodies (E). The p14ARF signals were densitometrically quantified and normalized to the respective β-TUBULIN signal, as specified by the numbers below the blot, with the -Dox condition set to 1. PRMT1-proficient (-Dox) as well as PRMT1-depleted cells (+ Dox) were further treated with the protein synthesis inhibitor cycloheximide (CHX) and harvested after 0, 0.5, 1, 2, 3, 4, and 6 hours. Cell lysates were subjected to immunoblotting using α-p14ARF and α-β-TUBULIN (loading control) antibodies and conventional ECL detection (F). For precise quantification of the p14ARF protein stability in the -/+ Dox conditions during the CHX time course, immunoblotting was performed with independent cell lysates and primary antibodies, as in (F), which were then detected using fluorescence dye-coupled secondary antibodies and the LI-COR Odyssey system. The quantitative p14ARF signals were normalized to the corresponding quantitative β-TUBULIN signals and displayed in (G). The CHX-untreated samples (0 hour) were set to 100% for each condition.
Expression of active NADase plays a central role in the intracellular survival of GAS in oropharyngeal keratinocytes.Figure 3A. Intracellular survival of GAS strains 188, 188NADase-, and 188NADase(G330D). Deletion of the gene encoding NADase or inactivation of NADase by the amino acid substitution G330D resulted in a 20- to 40-fold reduction in intracellular survival compared to parent strain 188. *, P<0.001. NADase western blot (B) and activity measurements (C) of culture supernatants from GAS strains 188, 188NADase-, and 188NADase(G330D).
Figure 2Micronuclei (indicated by arrow head) in GFP-NLS- or GFP-hcGAS expressing HEK293 cells before (0 h) or 24 h after γ-irradiation (IR; 10 Gy). Scale bar: 10 μm (A). Micronuclei (indicated by arrow head) in GFP-NLS- or GFP-hcGAS expressing HEK293 cells before (0 h) or 24 h after γ-irradiation (IR; 10 Gy). The average MNs/cell. Graphs show Mean ± SEM (n=3 independent experiments) representing six different microscopic fields with over 200 cells. IFNB1 response in HEK293 cells stimulated with transfected plasmid DNA. Mean ± SEM of n=3 independent experiments. Experimental outline for micronuclei generation and cell death after γ-irradiation. Micronuclei (indicated by arrow head) and cGAS staining in WT and cGAS-/- BMDMos exposed to γ-irradiation (10 Gy). Scale bar: 10 μm. Average MNs/cell in BMDMos. MN graphs show Mean ± SEM (n=3 independent experiments) representing eight different microscopic fields with over 200 cells. Cell death in WT and cGAS-/- BMDMos that were first synchronized at G2/M, then γ-irradiated (10 Gy) followed by release and analysis at indicated time points. Mean ±SD, x biological triplicates (n=3) per treatment group are shown.
Figure 7(A) Quantification of spheroids' area in MDA-MB-468 cell line (NTC vs. shDVL3). The data are presented as the mean ± SD, n = 3 showing statistical difference after 32h *p < 0.05. (B) Quantification of the spheroids' area in the MCF7 cell line (NTC, shA DVL3 and shB DVL3). The data are presented as the mean ± SD, n = 3 showing statistical difference after 100h *p < 0.05. (C) IVIS images showing representative tumors generated by injecting luciferase-expressing MDA-MB-468 cells (NTC, shA DVL3 or shB DVL3) in the mammary fat pad of SCID mice. Color scale (luminescent signal intensity): blue, least intense signal; red, most intense signal. (D) Quantification of luminescence changes in tumors generated by injecting luciferase-expressing MDA-MB-468 cells (NTC, shA DVL3 or shB DVL3) in the mammary fat pad of SCID mice, Average luminescence was used to measure volume of tumor; n ≥ 9. ( (E) Tumor pictures of tumors resulting from the injections of MDA-MB-468 (NTC, shA DVL3 or shB DVL3) in the mammary fat pad of SCID mice. Scale shown in cm. (F) Final weight of tumors resulting from the injections of MDA-MB-468 (NTC, shA DVL3 or shB DVL3) in the mammary fat pad of SCID mice, One-way ANOVA was used for statistical comparison, n ≥ 9. (G) IVIS images showing representative tumors generated by injecting luciferase-expressing MCF7 cells (NTC, shA DVL3) in the mammary fat pad of SCID mice. Color scale (luminescent signal intensity): blue, least intense signal; red, most intense signal. (H) Quantification of luminescence changes in tumors generated by injecting luciferase-expressing MCF7 cells (NTC, shA DVL3) in the mammary fat pad of SCID mice. Average luminescence was used to measure volume of tumor. n=7.