EMBO/sd-character-level-ner huggingface dataset:
Figure 6(A) XPO1 and STK38 are required for Beclin1 cytoplasmic accumulation upon nutrient starvation-induced autophagy. HeLa cells were transiently transfected with the indicated siRNA (control and KPT conditions = siNT). 72 hours later, cells were incubated with XPO1 inhibitors KPT-185 or KPT-330 as indicated (final concentration = 1 µM) or with DMSO for all other conditions for 2 hours prior incubation with DMEM or EBSS supplemented (or not) with inhibitors for 2 hours. Cells were fixed and stained for endogenous Beclin1. Graphical representation of endogenous Beclin1 nuclear staining/cytoplasmic staining (n > 30 cells from 3 independent experiments, mean ± SEM; *p<0.05, ***p<0.001, ns, not significant, Mann-Whitney test). XPO1 activity inhibition by both KPT-185 and KPT-330 as well as STK38 silencing failed to induce Beclin1 cytoplasmic accumulation upon nutrient starvation (B) XPO1_S1055 phosphorylation is required and sufficient for Beclin1 nuclear exit upon nutrient starvation. Genome edited XPO1 mutant HAP1 cells were incubated with IMDM (complete) or EBSS (starvation) for 2 hours. Cells were then fixed and stained for endogenous Beclin1. Graphical representation of endogenous Beclin1 nuclear staining/cytoplasmic staining (n > 30 cells from 3 independent experiments, mean ± SEM; ***p<0.001, ns, not significant, Mann-Whitney test). Here, phosphonegative XPO1 failed to induce Beclin1 cytoplasmic accumulation upon nutrient starvation whereas phosphomimetics variant (S1055D & S1055E) potentiated Beclin1 cytoplasmic accumulation (C) Genome edited XPO1 mutant HAP1 cells were transiently transfected with siRNA targeting endogenous STK38 (siSTK38#206 or with non-targeting siRNA (siNT)). 72 hours later, cells were incubated with IMDM (complete) or EBSS (starvation) for 2 hours. Cells were then fixed and stained for endogenous Beclin1. Graphical representation of endogenous Beclin1 nuclear staining/cytoplasmic staining (n = 30 cells from 2 independent experiments). Here, phosphomimetic XPO1 (S1055D) potentiated Beclin1 cytoplasmic localization in both complete and starvation condition even in the absence of STK38 as compared to cells carrying wt XPO1
Figure 6A) Low magnification merged and split channels confocal images of striatal sections showing immunolabelling for ChAT (cyano), DRD2 (red), and the nuclear stain DAPI (white). Bar = 200 μm. B1,B2) Top. Representative ChAT-positive (cyano) cholinergic interneurons, labelled for DRD2 (red), from a Tor1a+/+ (B1) or Tor1a+/- (B2) striatal section. The colocalization mask of the two signals is shown in white (right). Bar = 10 μm. Bottom. Higher magnification images (bar = 5 μm) of representative Tor1a+/+ (B1) and Tor1a+/- (B2) ChIs showing the lack of colocalization of DRD2 with the ER marker PDI (green). A detail at higher magnification is shown in the insets
Figure 5(A) Diagram showing DNA double strand break (DSB) induced first phase NF-κB activation through an ATM/TRAF6 and PARP1/PIASy dependent IKK activation mechanism Hinz et al, 2010Stilmann et al, 2009Wu et al, 2006(; ; ). (B) SDS-PAGE western blot of whole cell lysates of U2-OS cells irradiated (20 Gy) at time points indicated prior to harvest. ut, untreated. LDHA. Representative gel shown from n = 4 biological replicates. (C) U2-OS cells transfected with TRAF6 siRNA or scrambled control siRNA and TRAF6 and IKKβ levels analyzed by western blotting (top panels). NF-κB activity was determined by EMSA in untreated cells (ut) and after IR at indicated time points (lower panel). ns, unspecific band. Representative gels are shown from n = 2 biological replicates. (D) U2-OS cells were exposed to IR at indicated time points prior to harvest and analyzed by western blotting for levels of IκBα, NIK, IL-1α and PARP1. Treatment with proteasomal inhibitor MG132 (10 μM) started 4 hours prior to harvest. NIK serves as positive control for efficient proteasome inhibition. Quantitation from n = 4 biological replicates and SD is shown for IκBα (right panel). Brown-Forsynthe ANOVA test: p = 0.0005. (E) U2-OS CRISPR IKBKB knockout and control cell lines were irradiated (20 Gy) and harvested at indicated time points. NF-κB activity was analyzed by EMSA (lower panel). ns indicates an non-specific band. Upper panel, western blot analysis of actin and IKKβ. Representative gels shown from n = 3 replicates. (F) Irradiated U2-O2 control and IKBKB knockout cells (as above) were analyzed by RT-qPCR at the time points indicated for expression of NF-κB target genes identified by RNA-Seq (Dataset EV1). Significantly regulated targets, cutoff set at p = 0.05 (as determined by ANOVA with Tukey multiple comparison), are shown.
Figure 5(A) 293T cells were transfected S proteins with each variant-associated mutation for 24h and stained with biotinylated ACE2 and fluorescent streptavidin before analysis by flow cytometry. (B) Left Panel: EC50 values (concentration of ACE2 needed for 50% binding) for Alpha and associated mutations. Colour code corresponds to location on S protein functional domains and lower EC50 values signifies higher affinity to ACE2 binding. Right Panel: EC50 values for Beta and associated mutations. Data set for N501Y and D614G reference mutations are duplicated between left and right panels as mutations are common to both variants. (C) S protein transfected 293T cells were stained with human monoclonal antibodies targeting the S2 (mAb10), RBD (mAb48 and mAb98), and the NTD (mAb71). Cells were analyzed by flow cytometry. The percentage of positive cells is indicated.
Shp2 ablation inhibits mammary gland tumor formation in PyMT mice Kaplan-Meier analyses of tumor formation for control (PyMT;Shp2fl/fl, blue, n = 33), heterozygous Shp2 mutant (PyMT;MMTV‐Cre;Shp2+/fl, green, n = 15), and homozygous Shp2 mutant (PyMT;MMTV‐Cre;Shp2fl/fl, also called PyMT;coShp2, orange, n = 34) mice.Figure 6Kaplan-Meier analyses of tumor formation for control (MLN4924;Shp2fl/fl, blue, n = 33), heterozygous Shp2 mutant (MLN4924;MMTV‐Cre;Shp2+/fl, green, n = 15), and homozygous Shp2 mutant (MLN4924;MMTV‐Cre;Shp2fl/fl, also called MLN4924;coShp2, orange, n = 34) mice. ***P 0.001 (log‐rank test). Mammary gland whole‐mounts from control MLN4924 and MLN4924;MMTV‐Cre;Shp2fl/flmice at indicated ages. The characteristic lymph node of the mammary glandfat pad is marked by white arrowheads; premalignant lesions are marked by red arrowheads. Scale bar, 4 mm.Quantification of premalignant lesions in mammary glands of control MLN4924 and MLN4924;MMTV‐Cre;Shp2fl/flmice, as shown in (B). Error bars represent SEM (n = 6). *P 0.05 (Student's unpaired t‐test).
PAT1 is phosphorylated in planta and in vitroTandem MS spectrum of the phosphopeptide SSFVSYPPPGSISPDQR in which the underlined serine is phosphorylated.Figure 2Tandem MS spectrum of the phosphopeptide SSFVSYPPPGSISPDQR in which the underlined serine is phosphorylated. Y‐ and b‐ions are indicated that localize phosphorylation to the SP site. MPK4 and MPK6 were immunoprecipitated from extracts of Col‐0 seedlings treated with 200 nM flg22 for 10 min. For the negative control (−), extracts were incubated with agarose beads without antibodies. IPs were incubated with His6‐PAT1, His6‐PAT1 S208A or MBP for 60 min at 37°C before boiling and SDS‐PAGE. Autoradiogram (top panel), Coomassie‐stained gel for loading control (bottom).
Rab7 activity influences organization and motility of the Arl8b compartment.Figure 1A/B. Hybrid Rab7/Arl8b compartment. A. Representative confocal images of fixed HeLa cells expressing Arl8b-GFP (green), immunolabeled against endogenous Rab7 (eRab7, magenta). Zoom insets (3x) highlight select regions of colocalization (white), white arrowheads point to vesicles positive for both GTPases. B. Colocalization (Mander's overlap) of endogenous Rab7 with Arl8b-GFP versus free GFP (EV), n EV=4, n Arl8b=9 images (3≥ cells per image) analysed from 2 independent experiments. Significance: 2-tailed student's t-test, * p<0.05. C-F. Analysis of Arl8b compartment organization and dynamics as a function of Rab7 activity status. C. Left and middle panels: representative confocal images of live HeLa cells expressing mCherry-Rab7 or its mutants Q67L and T22N (white), together with Arl8b-GFP (green), taken at the start of time-lapse (t0). Right panels: tracks followed by Arl8b-positive null during the time-lapse lasting 50 s recorded at 0.5 s per frame, with highest displacement rates for each track depicted on a rainbow colour scale (blue: immobile; red: maximum mobility per time interval). Zoom insets (2.8x) highlight select peripheral (PP) and perinuclear (PN) cell regions D. Plot of Arl8b-positive pixel distribution expressed as fractional distance along a straight line from center of nucleus (0) to the plasma membrane (1.0), numbers of (pixels) plotted given above each scatter, n=7 cells analysed per condition from 2 independent experiments. E/F. Quantification of mean Arl8b vesicle displacement and maximum speed, respectively, n Rab7=21, n QL=25, n TN=24 images (3≥ cells per image) analysed from 2 independent experiments.
Figure 8Topical rapamycin treatment twice daily for 4 weeks showed an obvious therapeutic effect in rosacea patients. (A, B) Photos of patients topically applied with rapamycin (n=10) or placebo (n=8) ointment at 0 week and 4th week. Topical rapamycin treatment twice daily for 4 weeks showed an obvious therapeutic effect in rosacea patients. (C) The change of patients' CEA score showed in scatter histogram at 0 week and 4th week. (D) The change of patients' IGA score showed in scatter histogram at 0 week and 4th week. "The Change value < 0" indicates that skin lesions show positive improvement after treatment; "The Change value > 0" indicates skin lesions show exacerbation after treatment; "The Change value = 0" indicates that skin lesions show no change after treatment; Data represent the mean ± SEM, and two-tailed unpaired Student's t-test was used (C, D).
Figure 1Representative micrographs of a time-lapse microscopy of K and KM non-regressed tumour cells (H2B-GFP green). Top: Mitotic cell with a chromatin bridge (yellow arrow). Bottom: Mitotic cell with misalignment and cytokinesis failure resulting in binucleation (yellow arrow). Percentage of mitotic errors in K and KM primary tumours and in K and KM non-regressed tumours. Percentage of cells in K and KM primary tumours and in K and KM non-regressed tumours with the indicated mitotic errors. Scale bar 20 µm.
Quantitative dissection of cell-to-cell-communication in the developing brain.Figure 5Automated microinjection was performed on organotypic slices of mouse E14.5 - E15.5 dorsal telencephalon using a solution containing Dextran-A555 (not shown) and Alexa488 (green). Slices were fixed after microinjection (n = 71 cells total) and were stained for Tbr2 (white, H-J). (A-C) Representative examples of a non-coupled cell (A, cartoon on the right), a 2-cell cluster (B, cartoon on the right) and a 5-cell cluster (C, cartoon on the right). The asterisks indicate the Dx-A555-positive, microinjected cell. Percentage of microinjected cells found in a coupled cluster (coupled, green). Cluster size, expressed as % of total. Distribution of coupled cells expressed as the distance from the ventricular surface (0 µm = ventricle surface. Distribution of coupled cells divided into 11 bins and expressed as % of total. the images on the left are maximum intensity projections (MIP) of 18, 17, 23, 12 and 30-focal planes, respectively A representative picture of a 2-cell cluster (cartoon in the middle, nuclei are numbered from 1 to 2. Microinjected cells are stained for Tbr2. A representative picture of (H) a 2-cell cluster (cartoon in the middle, nuclei are numbered from 1 to 2) and (I) a 7-cell cluster (cartoon in the middle, nuclei are numbered from 1 to 7), the latter containing one Tbr2 positive cell. Microinjected cells are stained for Tbr2. The asterisks indicate the Dx-A555-positive, microinjected cell. comparison of Tbr2-positive cells (expressed as % of total) among all the cells in the VZ (VZ; n=1003 from 4 different confocal images from 3 independent experiments; error bar represents standard deviation) and in coupled clusters in the VZ (coupled clusters, VZ; n= 104 from two independent experiments). Coupled cells were scored based one presence/absence of apical and/or basal polarity cues, and results are expressed as % of total. the images on the right are single optical sections corresponding to the nucleus (white dotted line); scale bars are 20 µm for MIP and 10 µm for single focal planes. VZ, ventricular zone; SVZ, subventricular zone; CP, cortical plate.
Figure 3(A, B) Control and GRASP55 KO clones were transfected with HA-tagged GSL biosynthetic enzymes, treated with nocodazole (33 µM) for 3 hours, and processed for immunofluorescence with anti-HA (green), anti-GM130 (red), and anti-TGN46 (blue) antibodies. The relative position of HA-tagged enzymes with respect to GM130 and TGN46 was measured by line scanning and expressed as normalized positions of the peak intensity with the start of GM130 peak indicated as Cis and the end of TGN46 peak indicated as trans in the graph. The double headed arrows show the distance between the peak localization of GM130 and the enzymes. Scale bar 1μm. (C) Control and GRASP55 KO cells were transfected for 16 hours with GCS-HA or LCS-HA and processed for cryoimmunolabeling with anti-HA antibody (10-nm gold particles) and anti-GM130 antibody (15-nm gold particles) in case of GCS-HA and anti-HA antibody (15-nm gold particles) and anti-GM130 antibody (10-nm gold particles) in case of LCS-HA. Representative images of the distribution of GCS-HA and LCS-HA are shown. Scale bar 200nm. Red asterisk marks C4 cisterna and blue circles indicate GM130 labelling. (D, E) Distribution of indicated enzymes across the Golgi stack was quantified and represented as fraction of Gold particles in each cisternae for GCS-HA (D) and LCS- HA (E) (n indicated in the graph; data are Mean ± SEM).
Figure 4The ∆tagA mutant displays long and bent but still dynamic structures. Scale bar: 2 µm. Frames from time lapse movies showing sheath dynamics in the ∆tagA mutant. Sheath polymerization does not stop when the distal end reaches opposing membrane (white arrow). Continued polymerization causes the sheath to tilt and distal end to move towards the bacterial cell pole, ultimately effecting bending of the whole structure. C. Sheath detaches from its membrane anchor (orange arrow), keeps extending to maximum cell length and then contracts. Scale bars: 1 µm. Time lapse series of T6SS activity in tagged strain (VipA-mCherry2 TagAVC-mNeonGreen). Orange arrow indicates sheath distal end, white arrow indicates initiation site of another T6SS sheath. Scale bar: 1 µm. Kymographs of dynamics observed for TagAVC. E. TagAVC localizes at the distal end when sheath reaches the cell periphery. Signal of TagAVC-mNeonGreen disappeared and reappeared on this site repeatedly. F. TagAVC localizes at the distal end when sheath reaches the cell periphery and stays associated to this site until contraction. G. TagAVC briefly localizes at T6SS initiation site. H. TagAVC briefly localizes at T6SS initiation site and at distal end, where it stays attached after contraction.
Figure 1List of human methyltransferases and their orthologs in Drosophila melanogaster. Empty cells indicate that no ortholog could be identified (see Methods for details). LC-MS/MS measurements of m6A levels in total RNA (B) or in poly(A)+ RNA (C) upon KD of predicted methyltransferases in Drosophila S2R+ cells. m6A abundance in total RNA is significantly reduced when Mettl5 is depleted, while its depletion has no effect on m6A level in mRNA. As expected, the KD of Mettl3 and Mettl14 reduce m6A levels in mRNA. Bar chart represents the mean ± standard deviation of three technical measurements from three biological replicates. *P < 0.05, **P < 0.01, ***P < 0.001 (two-tailed t-test).
Figure 4Significantly differentially expressed hallmarks in ArCK cells compared to euploid control cells are shown in the first column in the heatmap (FDR q value0.05). Normalized enrichment scores are plotted. The corresponding NES for these hallmarks in doxorubicin (Doxo), palbociclib (Palbo), nutlin3 (Nutlin) and torin1 (Torin) treated cells are also plotted. Hallmarks did not reach statistical significance (FDR q value0.05) are shown in grey. Enrichment plots for "Interferon alpha response", "TNFalpha signaling via NFKB" and "Interferon gamma response" hallmark signatures in ArCK cells compared to the euploid control cells. Significantly differentially expressed RELA and RELB target genes in ArCK cells compared to euploid control cells were identified by ingenuity pathway analysis based on RNA sequencing data (Log2 fold change, p value0.05). RT-qPCR quantifying NF-κB downstream target gene expression in ArCK and euploid control (Ctrl) cells. n=6 biological replicates; mean± SEM. IL-1b, *p= 0.029; IL-6, *p= 0.032; IL-8, **p= 0.008; unpaired t-test. Measurement of NF-κB activity with NF-κB alkaline phosphatase (SEAP) reporter assay. The reporter was expressed in RPE1-hTERT cells by transient transfection. 10 hours after transfection, cells were treated with either DMSO (Ctrl) or reversine (500 nM; Aneuploid senescent) for 96 hours and the secretion of alkaline phosphatase in the culture supernatant was measured. The secretion level was normalized to cell number for each condition. n=3 biological replicates; mean± SEM; unpaired t-test, ****p<0.0001.
a, Heat map of HCIPs (represented by APSMs) for HA–Flag–PARKIN and mutants in response to depolarization in 293T cells, with or without Btz or BafA.f3a, Heat map of HCIPs (represented by APSMs) for HA-Flag-PARKIN and mutants in response to depolarization in 293T cells, with or without Btz or BafA. Proteins indicated had weighted and normalized D-scores ≥ 1.0, Z-score ≥ 5, and APSMs ≥ 2 unless otherwise noted (see Methods). b, Summary of PARKIN-interacting proteins in 293T and HeLa and integration with diGly sites.
MM primary tumors are highly susceptible to mycolactone toxicity and synergy with BZ.Figure 6Mononuclear cells from bone marrow aspirates of newly diagnosed (#2, #5, #9 and #13) or relapsed (#7 and #12) MM patients were treated with mycolactone and/or BZ at the indicated concentrations, or vehicle as control, for 18 h. Then, MM cells were identified by staining with anti-CD38 and anti-CD138 antibodies A Following treatment, induction of apoptosis/necrosis was measured by exposure of annexin V and PI incorporation. Data are Mean % of live cells from technical duplicates, relative to controls. Mononuclear cells from bone marrow aspirates of newly diagnosed (#2, #5, #9 and #13) or relapsed (#7 and #12) MM patients were treated with mycolactone and/or BZ at the indicated concentrations, or vehicle as control, for 18 h. B Synergy between mycolactone and BZ in treated tumors. Data are Mean Loewe scores from technical duplicates, shown as heatmaps.
Figure 2(A) Quantification of iFRAP experiments using HeLa cells expressing SMC3-LAP in the wild-type form (wt) or with lysines 105-106 mutated to glutamines (QQ) or arginines (RR) and synchronized in G1-phase. Error bars denote s.e.m., n > 25 cells per condition.(B) Quantification of chromatin residence time of SMC3-LAP alleles in G1-phase. (C) Quantification of iFRAP experiments using cells synchronized in G2-phase. Error bars denote s.e.m., n > 26 cells per condition.(D) Quantification of stably bound SMC3-LAP alleles in G2-phase. (E) Western blot of mouse fibroblasts expressing SMC3-LAP in the presence or absence of endogenous Smc3 after gene deletion. Extracts were prepared after subculturing two times. Cells without SMC3-LAP do not proliferate after deletion of Smc3. (F) Western blot showing HeLa cell extracts after treatment with siRNA as indicated and synchronized in G2-phase. Note that ESCO1 immunoblot signals were also reduced in sororin depleted cells. We currently do not know if this is an effect of sororin depletion, an off-target effect or an artefact of unequal sample loading. (G) iFRAP quantification of stably chromatin bound SMC3-LAP after siRNA treatment using cells synchronized in G2-phase. Error bars denote s.e.m., n > 11 cells per condition.
Figure 3(A) Western blot analysis of ATXN1 after knockdown of RSK3 in Daoy cells. Bottom left panel: qRT-PCR results of RSK3 mRNA. Bottom right panel: densitometry of the western blot image in the top panel (n = 3 except for shATXN1 where n = 1). Data information: Mean ± SD, *P<0.05, **P<0.01, ***P<0.001 ****P<0.0001, one-way ANOVA. (B) Western blot analysis of ATXN1 after overexpression of RSK3, ZBTB7B, or eGFP in Daoy cells (n = 3). Data information: RSK3 antibody used in is only suitable for detecting overexpressed RSK3 but not for endogenous RSK3. Mean ± SD, *P<0.05, **P<0.01, ***P<0.001 ****P<0.0001, one-way ANOVA. (C) Western blot analysis of ATXN1(82Q) after overexpression of either wild-type or inactive RSK3 in Daoy cells expressing ATXN1(82Q) (n = 3). Data information: RSK3 antibody used in is only suitable for detecting overexpressed RSK3 but not for endogenous RSK3. Mean ± SD, *P<0.05, **P<0.01, ***P<0.001 ****P<0.0001, one-way ANOVA. (D) In vitro kinase assay of RSK3 with ATXN1. MSK1 was used as a positive control. Asterisk: auto-phosphorylated RSK3. (E) In vitro kinase assay of four different RSK members with ATXN1. (F) In vitro kinase assay of RSK3 with N-terminal (F1), middle (F2), and C-terminal fragment (F3) of ATXN1. (G) Mass spectrometry analysis of phosphorylated residues in ATXN1 F3 after in vitro kinase assay with RSK3. (H) Western blot analysis of ATXN1 after a treatment with RSK3 inhibitor (BI-D1870) on SCA1 patient iPSC-derived neurons (n = 3). Data information: Mean ± SD, *P<0.05, **P<0.01, ***P<0.001 ****P<0.0001, one-way ANOVA. (I) Western blot analysis of total and phospho-S776 ATXN1 after a co-treatment with MSK1 (SB-747651A) and RSK3 inhibitor (BI-D1870) on SCA1 patient iPSC-derived neurons (n = 3). Data information: Mean ± SD, *P<0.05, **P<0.01, ***P<0.001 ****P<0.0001, one-way ANOVA.
STAMP2 promotes PCa growth in vitro and in vivo.Figure 2A LNCaP cells were transfected with either control siRNA or two independent STAMP2 siRNAs, and membrane fractions of the cells were prepared followed by Western blotting analysis. STAMP1 is used as a loading control. B LNCaP cells were cultured in RPMI 1640 medium containing 10% CT-FBS and treated with or without 1 nM R1881 for 24 h before being transfected with the indicated siRNAs. The cells were then cultured for the indicated times, and cell growth was measured by Cell Counting Kit-8. n = 3, *P < 0.0001. Error bars indicate SD. C LNCaP cells were cultured and transfected with the indicated siRNAs as described in (B). After transfection, the cells were cultured for 10 days. The colonies formed were stained and photographed. D LNCaP cells stably expressing shRNA against STAMP2 or control shRNA were treated with or without 1 nM R1881. The cells were then collected, and membrane fractions were prepared followed by Western blot analysis. E LNCaP cells stably expressing shRNA against STAMP2 or control shRNA were cultured for the indicated times, and the cell numbers were determined using a hemocytometer. n = 3, *P = 0.047; **P = 0.0046. Error bars indicate SD. F LNCaP cells stably expressing shRNA against STAMP2 or control shRNA were cultured in soft agar for 2 weeks as described in Materials and Methods. The plates were then stained and photographed.G Quantification of data from (F). n = 3, *P = 0.022. Error bars indicate SD. F LNCaP cells stably expressing shRNA against STAMP2 or control shRNA were cultured in soft agar for 2 weeks as described in Materials and Methods. The plates were then stained and photographed.G Quantification of data from (F). n = 3, *P = 0.022. Error bars indicate SD. H LNCaP cells stably expressing shRNA against STAMP2 or control shRNA were subcutaneously implanted into male SCID mice. Tumor size was measured after 8 weeks. n = 9, *P = 0.037. Error bars indicate SEM. I VCaP cells were transfected with the indicated siRNA as described in (B). After transfection, VCaP cells were cultured for 2 weeks. The colonies formed were stained and photographed.J Quantification of the data from (I). n = 3, *P < 0.0001. Error bars indicate SD.