EMBO/sd-character-level-ner huggingface dataset:
Phage infection dynamics in BREX‐containing cellsCulture dynamics over an extended period (> 30 h) for SPO1‐infected wild‐type (black) versus BREX‐containing (red) strains of B.Figure 2A Culture dynamics over an extended period (> 30 h) for SPO1‐infected wild‐type (black) versus BREX‐containing (red) strains of B. subtilis BEST7003. Each curve represents a single technical replicate grown in a single well on a 96‐well plate. Culture decline is temporally reproducible for the BREX‐lacking strain but occurs later, at apparently stochastic time points, for the BREX‐containing strain. Re‐growth following culture crash represents phage‐resistant mutants. B Phage production during a one‐step phage growth curve experiment with wild‐type (black) and BREX‐containing (red) strains of B. subtilis BEST7003 infected with SPO1. Error bars represent SD. Y‐axis represents absolute phage concentrations. Black and red arrows point to the time point of maximal burst for BREX‐lacking and BREX‐containing strains, respectively. C Phage production during a one‐step phage growth curve experiment with wild‐type (black) and BREX‐containing (red) strains of B. subtilis BEST7003 infected with Φ3T. Error bars represent SD. Y‐axis represents relative phage concentrations normalized to the value at the beginning of the infection. D Multiplex PCR assay showing lysogeny during a phage infection time course in the strain lacking BREX (black), but not in BREX‐containing strain (red), or uninfected (U) strains. Amplicons for the bacterial DNA, phage DNA, and lysogen‐specific DNA are 293, 485, and 1,218 bp, respectively.
Figure 3 (A)Venn diagram showing the overlap between four published YAP signatures [39-42] (B) Venn diagram showing the overlap between the YAP signatures obtained in Oral SCC and Breast (YAP Signature 1) and CRC and HCC (YAP Signature 2) .(C .(C) Venn diagram showing the overlap between YAP Signature 1 and the genes upregulated by more than 1.5 fold in BCC compared to IFE[28 (D) Venn diagram showing the overlap between YAP Signature 1 and the genes upregulated by more than 1.5 fold in well-differentiated SCC versus IFE and in EMT containing SCC versus HF[33] (E) Venn diagram showing the genes commonly upregulated in YAP signature 1 and in BCCs, well-differentiated and EMT-containing SCCs (F) Venn diagram showing the overlap between YAP signature 2 and the genes upregulated by more than 1.5 fold in BCC compared to IFE [28] (G) Venn diagram showing the overlap between YAP signature 2 and the genes upregulated by more than 1.5 fold in well-differentiated SCC versus IFE and in EMT containing SCC versus HF [33 (H) Venn diagram showing the genes commonly upregulated the YAP Signature 2 and in BCCs, well-differentiated and EMT-containing SCCs. HCC: hepatocellular carcinoma; CRC: colorectal cancer; Well-diff SCC: well-differentiated SCC; EMT SCC: EMT containing SCC
IGF2R blockade results in CD20 phosphorylation.Figure 1(a) RT-PCR expression and quantification of IGF1, IGF1Rβ and IGF2 levels in untreated C2C12 and shCD20 C2C12 myoblasts and 10 nM IGF1-treated C2C12 and over-expressing CD20 C2C12 myoblasts. Each experiment was replicated independently four times. (b) Immunofluorescence for IGF2 (in green) in untreated and 10 nM IGF1-treated C2C12 myoblasts. Scale bars = 75 μm. Representative CD20 immunoprecipitation using anti-pSer+pThr in (c) C2C12 cells treated with IGF1 for two hours (2 h) or overnight (ON) as indicated. Densitometric analysis of data are expressed as the ratio of CD20/vinculin or pSer+pThr/CD20 and are shown normalized to vinculin in arbitrary units in the lower panels shown Two-way ANOVA. *p<0.05; **p<0.01; ****p<0.0001. Each experiment was performed in triplicate wells. All values are expressed as the mean ± SEM. Representative CD20 immunoprecipitation using anti-pSer+pThr in (d) sh-empty (shCTR)- and shCD20-treated C2C12 cells treated with anti-Flag and 10 nM anti-IGF2R, as indicated. Densitometric analysis of data are expressed as the ratio of CD20/vinculin or pSer+pThr/CD20 and are shown normalized to vinculin in arbitrary units in the lower panels shown Two-way ANOVA. *p<0.05; **p<0.01; ****p<0.0001. Each experiment was performed in triplicate wells. All values are expressed as the mean ± SEM. Representative CD20 immunoprecipitation using anti-pSer+pThr in (e) C2C12 cells treated with IGF1 and IGF2, as indicated. Densitometric analysis of data are expressed as the ratio of CD20/vinculin or pSer+pThr/CD20 and are shown normalized to vinculin in arbitrary units in the lower panels shown Two-way ANOVA. *p<0.05; **p<0.01; ****p<0.0001. Each experiment was performed in triplicate wells. All values are expressed as the mean ± SEM. (f) Representative WB of IGF2R and phosphorylated IGF2R (pIGF2R) in untreated, shCD20-treated and anti-IGF2R-treated C2C12 cells. Densitometric analysis of data are expressed as the ratio of IGF2R/actin or pIGF2R/IGF2R in arbitrary units in the lower panels. One-way ANOVA. *p<0.05; Each experiment was performed in triplicate wells. All values are expressed as the mean ± SEM. (g) IGF2R immunoprecipitation products were immunoblotted for Gαi2 and WB expression of IGF2R in untreated, IGF2-treated, anti-IGF2R-treated and IGF2+anti-IGF2R treated C2C12 cells. Densitometric analysis of data are expressed as the ratio of Gαi2/IGF2R in arbitrary units in the lower panel. (h) Representative WB of IGF1Rβ and IGF1Rβ immunoprecipitation products immunoblotted for p-Tyr in untreated, IGF2-treated and IGF2+anti-IGF2R-treated (1:500, 1:1000 and 1:2000 dilutions of anti-IGF2R) C2C12 cells (cells were treated for 2 and 24 h). Densitometric analysis of data are expressed as the ratio of pTYR/IGF1Rβ in arbitrary units in the lower panel. Each experiment was performed in triplicate wells. All values are expressed as the mean±SEM.
The C-terminal region of ALFY interacts with GABARAP.Figure 1A Transfected GFP−Atg8 homologs were immunoprecipitated with μMACS™ from total U2OS cell extracts followed by immunoblot analysis with anti−GFP and anti−ALFY antibodies. Data are representative of two independent experiments. B An overview of the deletion mutant constructs of ALFY used for GST pull‐down experiments in (C). C The indicated 35S−labelled in vitro−translated GFP−tagged constructs were incubated with GST, GST−LC3B or GST−GABARAP conjugated to glutathione Sepharose, and their binding was evaluated by autoradiography (ARG). 5% of the in vitro−translated protein (arrow head) used was loaded. Equal amounts of GST proteins were used as shown by Coomassie Brilliant Blue (CBB) staining. Data are representative of three independent experiments. D Recombinant ALFY (aa 2981-3526) was incubated with GST‐Atg8 proteins conjugated to glutathione Sepharose. The pulled‐down complexes were subjected to SDS-PAGE and anti‐ALFY and anti‐GST immunoblotting. 5% of the recombinant ALFY protein used was loaded as input. Data are representative of three independent experiments.
p62 forms a shell around huntingtin protein aggregates.Figure 7.(A) HeLa cells transiently expressing a Flag-tagged NH2-terminal fragment (amino acids 1-171) of huntingtin containing a 68-polyglutamine expansion, Flag-N-HttQ68. After 48 h, the cells were fixed, and Flag-N-HttQ68 was stained green using a Flag mAb. Endogenous p62 was stained red using the p62 pAb. (B) Endogenous p62 forms a shell around aggregated GFP-N-HttQ68. Transiently transfected HeLa cells were fixed 48 h after transfection, and endogenous p62 was detected using a p62 mAb (red). (C and D) The p62 shell surrounding GFP-N-HttQ68 was detected independently of the use of antibodies. (C) GFP-N-HttQ68 and DsRed-tagged p62 were cotransfected into HeLa cells, and images of live cells were obtained after 48 h of expression. The left panel shows one confocal plane of a representative GFP-N-HttQ68 and DsRed-p62 double-positive structure, and the right panel shows the Z-stack of planes with side views of the aggregate at the side and at the top. N, nucleus. (A-C) Nuclei were detected using Draq5. (D) GFP-N-HttQ68 and tdTomato-tagged p62 were cotransfected into HeLa cells, and images of live cells were obtained after 24 h of expression. (E) Transiently expressed Flag-N-HttQ68 and myc-LC3 colocalize in GFP-p62-positive structures. S-GFP-p62 cells were fixed 48 h after transfection, Flag-N-HttQ68 was stained red using a Flag tag mAb, and myc-LC3 was stained far red (blue) using a chicken myc tag pAb (top). The bottom panel shows a live cell image of a GFP-N-HttQ68 aggregate surrounded by a shell containing tdTomato-LC3. Bars, 5 μm.
Figure 3A, PTPN3 and PTPN3 (D811A) stabilize TβRI. TβRI and PTPN3 were detected by Western blotting with appropriate antibodies as indicated in HaCaT cells stably expressing PTPN3 or PTPN3 (D811A). B, shRNA PTPN3-mediated reduction of endogenous TβRI level is restored by MG132. HaCaT cells expressing shRNA PTPN3-1, shRNA PTPN3-2 or control shRNA were treated with MG132 (20 μM) for 4 h. Cell lysates were analyzed by Western blotting with anti-TβRI, anti-PTPN3 and anti-GAPDH antibodies. C, PTPN3 rescues the degradation of TβRI induced by Smurf2. HEK293T cells were transfected with the indicated plasmids. Cells were harvested after 24 h post-transfection. Western blotting was done with the indicated antibodies. D, PTPN3 and PTPN3 (D811A) attenuate ubiquitination of TβRI. HEK293T cells were transfected with the indicated plasmids. 24 h after transfection, IP and immunoblotting analysis were performed with the anti-FLAG antibody. Levels of TβRI ubiquitination and other proteins were examined by Western blotting with the indicated antibodies. E, PTPN3 blocks Smurf2-mediated ubiquitination of TβRI. HEK293T cells were transfected with the indicated plasmids, and treated with MG132 (20 μM, 4 h) before harvest. TβRI IP was performed with the anti-HA antibody, and TβRI ubiquitination was determined by Western blotting with the anti-Myc antibody. Protein levels were examined by Western blotting with the indicated antibodies. F, Depletion of Smurf2 reverses the shPTPN3-induced degradation of TβRI. HEK293T cells were transfected with expression plasmids for shPTPN3, shSmurf2 and HA-TβRI. 48 h post-transfection, cells were harvested for immunoblotting analysis. TβRI level was determined by Western blotting with the anti-HA antibody. Levels of other proteins were done by Western blotting with the indicated antibodies. G, Dominant negative Smurf2 mutant C716A prevents shPTPN3-induced TβRI degradation. HaCaT cells stably expressing shPTPN3 were transfected with FLAG-Smurf2 (C716A). Cells were harvested for Western blotting with anti-TβRI, anti-FLAG, anti-PTPN3 and anti-GAPDH antibodies.
a) IL-8 enzyme-linked immunosorbent assay (ELISA) on U2OS cells starved of glutamine or treated with BPTES (10 μM) for 24 h.f7(a) IL-8 enzyme-linked immunosorbent assay (ELISA) on U2OS cells starved of glutamine or treated with BPTES (10 μM) for 24 h. (b) RT-PCR analysis of IL-8 and CHOP mRNA expression in U2OS cells starved of glutamine for 24 h and co-treated with DMαKG. (c) Metabolite analysis of U2OS cells starved of glutamine, treated with BPTES or glutamine-starved co-treated with DMαKG. (d) IL-8 ELISA of U2OS cells starved of glutamine or treated with BPTES for 24 h and co-treated with DMαKG. (e) Immunoblot analysis of pS6K and pJNK in U2OS cells starved of glutamine for 24 h and co-treated with DMαKG. Error bars in all figures represent s.d. of three biological replicates. OD, optical density.
Rab8A is not required for the activation of Parkin E3 ligase activity at mitochondria in response to PINK1 activation by CCCP.Figure 6Rab8A is not required for the activation of Parkin E3 ligase activity at mitochondria in response to PINK1 activation by CCCPWild‐type (WT) or Rab8A knockout (KO) HeLa cells were transfected with WT or Cys431Phe (C341F)‐mutant Parkin. After transfection for 24 h, cells were treated with DMSO as a vehicle control or 10 μM CCCP for 6 h. Mitochondrial enriched extracts (mitochondrial lysate) were incubated with ubiquitin‐binding resins derived from his‐halo‐ubiquilin1 UBA domain tetramer (UBAUBQLN1). Captured ubiquitylated proteins were subject to immunoblotting with anti‐CISD1 and anti‐mitofusin 2 antibodies. Mitochondrial lysate and total lysate were also subjected to immunoblotting with indicated antibodies for loading and protein expression controls. Phospho‐Ser111 Rab8A was detected after Rab8A immunoprecipitation from 200 μg of mitochondrial lysate with anti‐Rab8A antibody.
Figure 9A Schematic depicting ATP-generating pathways in eukaryotic cells: glycolysis, the Pentose Phosphate Pathway (PPP), fatty acid oxidation (FAO), the TCA cycle, and the mitochondrial respiratory electron transport chain (ETC). Blue stars mark pths targets. Green indicates individual metabolites with statistically significant upregulation in atosPBG compared to the control. Cellular metabolites were measured by LC-MS-based untargeted metabolomics from extracts of Stage 11 embryos; biological replicates for control n=5, for atosPBG n=7. (B) Normalized ATP/ADP ratio values are decreased in atosPBG compared to control embryos (p=0.028). Data information: Mean±SEM, ns=p>0.05, *p<0.05, **p<0.01, ****p<0.0001. unpaired t-test Cellular metabolites were measured by LC-MS-based untargeted metabolomics from extracts of Stage 11 embryos; biological replicates for control n=5, for atosPBG n=7. Heatmap of non-targeted metabolites in atosPBG compared to wild-type embryos shown with average log2fold change (FC) in the non-targeted LC-MS/MS analysis reveals (C) an increase in atosPBG in substrates of the dGR/HPR enzyme, including 4-hydroxy α-ketoglutarate and hydroxyproline (HLP) and a smaller decrease in its products, glycolate and glycerate, (D) a significant increase in some dipeptides including those containing hydroxyproline. Data information: Values in heat maps are obtained from untargeted metabolomic analysis, unpaired t-test Cellular metabolites were measured by LC-MS-based untargeted metabolomics from extracts of Stage 11 embryos; biological replicates for control n=5, for atosPBG n=7. (E) Quantification shows an increase in atosPBG in the pyruvate/glucose ratio (p=0.035), but none for the lactate/glucose ratio (p=0.65). Data information: Mean±SEM, ns=p>0.05, *p<0.05, **p<0.01, ****p<0.0001. unpaired t-test Cellular metabolites were measured by LC-MS-based untargeted metabolomics from extracts of Stage 11 embryos; biological replicates for control n=5, for atosPBG n=7. Heatmap of non-targeted metabolites in atosPBG compared to wild-type embryos shown with average log2fold change (FC) in the non-targeted LC-MS/MS analysis (F) We observe increases in atosPBG in intermediates of mitochondrial fatty acid β-oxidation (FAO), including different carnitine-conjugated lipids, and (F-G) a significant increase in ketone body substituents compared to the control. Data information: Values in heat maps are obtained from untargeted metabolomic analysis, unpaired t-test
Figure 4A. Schematic diagram of the social interaction device indicating the social and the empty chambers.B, C. Mice were simultaneously exposed to an empty container and a caged unfamiliar juvenile mouse (social enclosure, stranger 1). Lrig1-mutant mice exhibit social interaction defects as determined by the time spent interacting (sniffing) with the stranger enclosure (B) and the percentage of total interaction time with stranger in the three-chamber social interaction test (C). Dashed line in (C) represents chance-level performance (i.e. 50%) when mice equally explore the social enclosure and the empty container. Data represent means ± SEM of independent determinations performed in n=8-9 mice of each genotype, and the statistical significance between wt and knockout mice are *p<0.05 by Students t test. D. Schematic diagram of the social novelty test. In the test for social novelty, a second stranger (stranger 2) mouse was introduced in the empty container.E. In the preference for social novelty task, wt mice showed a preference for social novelty, while mutant mice showed no significant preference for novel target (stranger 2). Mutants also spent significantly less time interacting with the novel mice compared to controls. Data represent means ± SEM of independent determinations performed in n=8-9 mice of each genotype, and the statistical significance are as follow: *p<0.05 and **p<0.005 by ANOVA followed by Student-Neuman Keuls multiple comparison test.
Figure 2A. Western analysis of nuclear (N) or cytoplasmic (C) fractions from whole cell lysates (W) from A549 cells (RL WT), RNase L knock out A549 cells (RL KO), or A549 cells with either wild-type RNase L (NLS-RL-WT) or RNase L-R667A (NLS-RL-CM) fused to a nuclear localization signal sequence. Short and long exposure with anti-RL antibody. GAPDH protein used as cytoplasmic marker. Histone H3 protein used as nuclear marker. B. Graph depicting the fraction of RNase L found in the nucleus or cytoplasm. Mean and SEM of two experiments with individual experiment values plotted. C. FISH with probes to GAPDH mRNA or oligo(dT) to detect poly(A)+ RNA in A549 cells without or with either NLS-RL-WT or NLS-RL-CM either mock transfected or transfected with poly(I:C) for 4 hours. Scale bar 5 micron. D. Graph depicting median and the value of the intensity of oligo(dT) fluorescence in individual nuclei divided by nuclear volume in the indicated cell types either mock transfected or transfected with poly(I:C) for 4 hours. At least 43 nuclei analyzed per condition.
Structure of dynactin's shoulder A.Figure 2Gaussian surface rendering of dynactin showing the arrangement of the upper subdomain (greens) and the lower subdomain (blues) of the shoulder on the filament (gray). Different features are shown in different color shades, and the dimerization domain is colored magenta. Upper subdomain of the shoulder, shown in ribbon (main) or surface representation (inset), with the arm, hook and paddle colored in shades of greens. Lower subdomain of the shoulder, shown in ribbon (main) or surface representation (inset), with the arm, hook and paddle colored in shades of blue.
Figure 6Schematic of SCAP targeting to ER exit sites under different conditions. The red puncta represent the Sec12-positive ER exit sites, and the yellow puncta represent the SCAP-positive ER exit sites. Cideb enriches SCAP to ERESs via interacting with Sec12. CHO-K1 cells transfected with GFP-SCAP, HA-Cideb or HA-Cideb-K128A were arrested with 5 µM Nocodazole for 30 min, then fixed with methanol and stained with antibodies against Sec12 and HA. Scale bars represent 10 µm (left), and 2.5 µm (Inlay). Number of SCAP-positive ER exit sites per cell.   Normalized fluorescent intensity of SCAP at each individual ER exit site.   Total fluorescent intensity of SCAP at ER exit sites per cell.   Number of Sec12-positive ER exit sites per   cell. 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. In panel C-F, quantification of images from 3 independent experiments. Model depicting the role of Cideb in enhancing SREBP processing. When cellular sterol level is high, SCAP binds to Insig, resulting in the retention of SREBP/SCAP on ER and low SREBP activation. The absence of sterol frees SCAP from Insig, and induces the interaction between Cideb and SCAP, leading to the targeting of SREBP/SCAP to the ERESs via the interaction between Cideb and Sec12 and the enrichment of SREBP/SCAP in Golgi. Cleaved SREBP enters nucleus and activates the expression of fatty acids/cholesterol synthesis genes.
| Identification of macrophages in the lung of SARS-CoV-2-infected hamsters with and without ivermectin treatment and characterization of their transcriptomic profile related to M1/M2 polarization.Figure 3a, representative histopathology photomicrographies of lungs according to the different groups: mock_saline, CoV_saline and CoV_ivermectin. Top panels: whole lung sections. Bottom panels: high magnification. CoV_saline section exhibits important congestion (*), edema associated with few mononuclear cells (white arrowheads). Note the thickening of the alveolar walls. CoV_ivermectin section exhibits important amounts of mononuclear cells (black arrowheads) and less marked signs of congestion or edema. Hematoxylin and Eosin. Scale bars = 1 mm (top panels) and 20 μm (bottom panels). b, representative immunofluorescence photomicrographies of neutrophils (Ly-6G), monocytes/macrophages (Iba1), M2 macrophages (Arg1) and SARS-CoV-2 (NP) in the lung. Scale bars = 50 μm. c, quantification of Iba1+ cells, Arg1+ cells and Iba1+Arg1+ cells in the lungs. mock_saline n=3 (males), mock_ivermectin n=4 (males), CoV_saline n=9 (6 males and 3 females), CoV_ivermectin n=6 (4 males and 2 females). Data information: M: male hamsters; F: female hamsters. Horizontal lines indicate medians. The p value is indicated in bold when significant at a 0.05 threshold. Mann-Whitney test d, heatmaps showing the differentially-expressed genes related to the M1/M2 polarization in comparison to mock-infected hamsters. * indicates Benjamini-Hochberg adjusted p-value<0.05 in the comparison between saline and ivermectin within the same sex. Color gradient represents the transcription log2 fold change comparing infected and mock-infected. e, lung weight-to-body weight ratio in the different groups (n=4/sex/group). Data information: M: male hamsters; F: female hamsters. Horizontal lines indicate medians. The p value is indicated in bold when significant at a 0.05 threshold. Mann-Whitney test
Figure 6Western blots showing DgcM-3xFLAG translated in vitro in the presence or absence of OmrA or OmrB. A - The inclusion of purified wild-type or mutant Hfq proteins is indicated, and the translation assay was conducted The different Hfq interaction site mutant proteins are indicated. Data information: Quantification of DgcM translation rates, relative to that of the unaffected OmpA control, is shown below the gels. The ratio of DgcM/ OmpA signal was set to unity for the lane lacking sRNA in each set of three combinations (-sRNA, +OmrA, +OmrB), indicated by "1.00*". Effects due to OmrA/ OmrB addition can be seen by comparing lanes 2 and 3 to lane 1 in each set. Western blots showing DgcM-3xFLAG translated in vitro in the presence or absence of OmrA or OmrB. B - An ssDNA-oligo (Hfq_BS that base-pairs specifically to the Hfq binding site on dgcM mRNA (nt 42-66) was preannealed to the mRNA prior to OmrA/B addition and translation. OmpA-3xFLAG was included as internal control. Data information: Quantification of DgcM translation rates, relative to that of the unaffected OmpA control, is shown below the gels. The ratio of DgcM/ OmpA signal was set to unity for the lane lacking sRNA in each set of three combinations (-sRNA, +OmrA, +OmrB), indicated by "1.00*". Effects due to OmrA/ OmrB addition can be seen by comparing lanes 2 and 3 to lane 1 in each set.