EMBO/sd-character-level-ner huggingface dataset:
Filamentation in mutant DHFR strains is completely reversible and does not represent TLD.Figure 4Mutant W133V was grown in amino acid supplemented M9 medium (M9+AA) for 4 hours at 42°C, and subsequently placed on M9 agar pads and their growth was monitored at room temperature. Shown are phase contrast images taken from different time points throughout the time-lapse experiment. Unlike cells experiencing TLD, an irreversible phenomenon, W133V DHFR cells recover and resume growth at low temperature. WT cells were treated with different concentrations of Tmp at 42°C for varying amounts of time in amino acid supplemented M9 medium (panel B) or in M9 media supplemented with both amino acids and GMP (panel C), following which they were spotted on M9+AA plates and allowed to grow at 30°C. Colonies were counted next day. In the presence of only amino acids, there was no loss in viability for any concentration of Tmp (panel B), despite extensive filamentation (Figure EV1E). In contrast, in the presence of amino acids and GMP, the cells showed sharp loss in viability when grown at high Tmp concentrations. In both panels, error bars represent SD of two biological replicates. The dashed line represents cfu at one-hour timepoint. WT (with and without 5μg/ml Tmp) and mutants were grown for 6 hours at 42°C in M9+AA medium without or with GMP, and subsequently diluted serially and spotted on M9+AA agar plates and allowed to grow at 30°C till visible colonies were formed. While WT treated with high Tmp shows loss in cfu, indicating TLD, no loss in viability was observed for WT or mutants. Expression of SOS response genes recA, recN and sulA when mutant strains and Tmp treated WT cells are grown in M9 medium supplemented with amino acids (black bars) or in the presence of both amino acids and GMP (gray bars) at 42°C (I91L+W133V was grown at 40°C). Error bars represent SD of 2-3 biological replicates.
Beclin‐1‐dependent autophagy stimulated by ABT737.Figure 3(A, B) Detection of autophagic vacuoles by LC3‐GFP and their modulation by ABT737 and by Beclin‐1‐specific siRNAs. HeLa cells were transfected with control or Beclin‐1‐specific siRNAs, 24 h later re‐transfected with LC3‐GFP, cultured in complete medium (CM) for 24 h, and finally kept 12 h either in CM or in nutrient‐free (NF) conditions, in the presence or absence of 1 μM ABT737. Representative microphotographs of cells cultured in NF medium are shown in (A) and the percentage (means±s.d., n=3 separate experiments) of LC3‐GFP‐transfected cells bearing LC3‐GFP aggregates in the cytoplasm (LC3‐GFPvac) are quantified in (B). The insert in (B) demonstrates the efficiency of the Beclin‐1‐specific siRNAs, as quantified by immunoblot. (C, D) Detection of cytoplasmicvacuoles using chloromethylfluorescein diacetate (CMFDA). Cells were transfected with control or Beclin‐1‐specific siRNAs, cultured for 48 h in CM, washed, cultured in CM (D) or NF (C, D) for 12 h, stained with CMFDA, and either photographed (C) or subjected to the quantification of the cells that bear at least one discernible cytoplasmicvacuole (arrow head) (means±s.d., n=3 separate experiments). (E) Ultrastructure of autophagic vacuoles induced by ABT737. Transmission electron microphotographs are shown. (F) Detection of dead and dying cells in the cultures. Cells treated as in (A) were stained with the ΔΨm‐sensitive dye DiOC6(3) and the vital dye propidium iodide (PI). The black portions of the columns refer to the DiOC6(3) low PI+ population (dead) and the remaining part of the column corresponds to the DiOC6(3) low PI− (dying) population. Results are means±s.d. of three independent experiments.
Figure 1A549 cells stably expressing lentivirus vector containing random shRNA or Atg7 specific shRNA were generated. (A) Cells were incubated for 2 hours in the presence or absence of 50 nM of lysosomal inhibitor chloroquine (CQ). Cell lysates were evaluated by immunoblotting. A549 cells stably expressing lentivirus vector containing random shRNA or Atg7 specific shRNA were generated. (B) Cells were infected, for 1 and 4 hours, with K. pneumonia at MOI of 10 followed by 1 hour of gentamycin treatment. Cell lysates were plated in LB agar for 18 hours and CFU were determined. Data information: the triangles and dots represent the individual test (three technical replicates per individual test) on control and Atg7 knock down cells. The bars represent the mean CFU of each group (three biological replicates each group), error bars represent standard deviation. Statistical significance between assays were calculated using two-tailed student's t test with Welch's correction. NS: not significant; *p ≤ 0.05; **p ≤ 0.01. (C) Schematic of WT P. aeruginosa and ΔpscD P. aeruginosa mutant. A549 cells stably expressing lentivirus vector containing random shRNA or Atg7 specific shRNA were generated. (D-E) Cells were infected, for 1-4 hours, with WT (D) or ΔpscD P. aeruginosa (E) at MOI of 10. All infections were followed by 1 hour of gentamycin treatment. Cell lysates were plated in LB agar for 18 hours and CFU were determined. Data information: , the triangles and dots represent the individual test (three technical replicates per individual test) on control and Atg7 knock down cells. The bars represent the mean CFU of each group (three biological replicates each group), error bars represent standard deviation. Statistical significance between assays were calculated using two-tailed student's t test with Welch's correction. NS: not significant; *p ≤ 0.05; **p ≤ 0.01.
Figure 2(A) Representative immunoblotting of Lrp4, MuSK, Transferrin Receptor (TfR), CTGF, and β-actin in whole-cell lysates and the plasma membrane fraction of C2C12 myotubes treated with 1 pM (1 ng/ml) agrin and/or 250 pM (100 ng/ml) CTGF for 24 h. CTGF increases Lrp4 on the plasma membrane in the presence of agrin. (B) C2C12 myotubes were treated with 1 pM agrin and/or 250 pM CTGF for 6 h. Lrp4 was immunoprecipitated (IP) with an anti-Lrp4 antibody, and phosphorylated Lrp4 was immunoblotted with an anti-phosphotyrosine (p-Tyr) antibody. (C) Representative immunoblotting of Lrp4, MuSK, CTGF, and TfR in whole tissue lysates and the plasma membrane fraction of lower limb muscles at E18.5 of wild-type (WT) and Ctgf-/- mice. (D) Representative immunoblotting of Y755-phosphorylated MuSK, total MuSK, and β-actin in the diaphragm at E18.5 of wild-type (WT), Ctgf-/-, and Ctgf+/- mice.
Gata6 expression is conserved in human normal skin and found in tumors harboring a sebaceous differentiation.Figure 6(A) Normal human scalp skin sections stained with Gata6 and PanKeratin antibodies. Endogenous Gata6 expression is found in the JZ, upper SG and SD. As in mouse skin, Gata6 is not expressed in sweat glands (SwG). (B) Representative sections of human skin tumors stained with Gata6 and PanKeratin antibodies. Quantification of the percentage of Gata6-positive tumors within each histopathological group (middle left panel) and quantification of the percentage of Gata6-positive cells within each positive sample (middle right panel). Note that the least positive sample showed about 3% of Gata6-positive cells. The analysis was performed on 73 different tumor samples. SSC : squamous cell carcinoma, SCAP: syringocystadenoma papilliferum, EMPD: extramammary Paget's disease, nod./superf./scler. BCC: nodular/ superficial/ sclerodermiform basal cell carcinoma, Steatocystoma M.: steatocystoma multiplex, FSCH: folliculosebaceous cystic hamartoma. Heat-map displaying the -log(p-value) of χ2 test computed between the different tumor types (bottom left panel). (C) Gata6 expression (measured in Transcripts Per Million, TPM) in human SCC (n=6), BCC (n=6) and sebaceous carcinoma (n=14) samples from (North et al, 2018). Data are means ± SD. (**) p-value ≤ 0.005, Student's t-test. Data information: (A and B) Scale bars: 100 μm.
Figure 4(A) RFP-labeled MDA-MB-231 cells stably overexpressing LIMT or eGFP were injected into the tail vein of 5-week old female SCID mice (150,000 cells/mouse). Eighteen days after injection, lungs were excised and imaged. The number of metastatic nodules in each lung was quantified and presented in a dot plot. The horizontal lines represent median number of nodules per animal of each group. Each dot of the left panel represents one animal. The experiment was repeated twice (N = 18 and 14 for LIMT and eGFP overexpression, respectively). Shown are representative fluorescence images of lungs from the LIMT overexpression group and the control group. Scale bar, 0.5 cm. A two-way ANOVA was applied to evaluate differences between groups. (B) The experiment described above was carried out with RFP-labeled MDA-MB-231 cells stably expressing shRNAs against LIMT (N = 7) or control shRNAs (N = 8). Shown are representative fluorescence images of lungs from the LIMT knockdown group and the control group. Scale bar, 0.5 cm. A student's t-test was applied to evaluate differences between groups.
A cell-autonomous activity of HST enables movement of amiRSUL, but not of free GFP(A) Representative vein-chlorosis phenotype caused by the genetic mosaic rescue of pSUC2::HST:GFP co-expressed in pSUC2::amiRSULhst-1 plants (see also Appendix Fig.Figure 5Representative vein-chlorosis phenotype caused by the genetic mosaic rescue of pSUC2::HST:GFP co-expressed in pSUC2::amiRSULhst-1 plants (see also Appendix Fig.S11A). Bright field and GFP fluorescence images of pSUC2::GFP and pSUC2::tmGFP9 seedlings. Localization of HST:GFP and, as a reference, of tmGFP9 expressed under the pSUC2 promoter in Arabidopsis primary leaves (C) or roots (D). CW: calcofluor white staining. Scale bars: 20 µm. Confocal fluorescence images of the differentiation and division zones of Arabidopsis root tips expressing pSUC2::HST:GFP, pSUC2::GFP or pSUC2::tmGFP9. Scale bars: 100 µm. Confocal fluorescence images of the differentiation and division zones of pSUC2::GFPhst-1 or pSUC2::tmGFP9hst-1 root tips. Scale bars: 100 µm. Bright field and GFP fluorescence images of pSUC2::GFPhst-1 and pSUC2::tmGFP9hst-1 seedlings.
Depletion of lamin A/C increases the formation of F-actin, which has an adverse effect on ciliogenesis in RPE cells.Figure 4A-C The RPE cells as described in Fig 3 were stained for paxillin (as a marker for focal adhesion; green), F-actin (red) and DNA (blue). The representative images are shown (A). Scale bars, 20 μm or 5 μm (insets). The cell spreading area of the cells (B, n ≥ 108) and the number of focal adhesions per cell (C, n ≥ 30) were measured. D-G The cells were serum-starved for 32 h and then treated with (+) or without (-) Cyto D (250 nM) for another 16 h. The cells were stained for acetylated tubulin (white), pericentrin (green), F-actin (red) and DNA (blue). The representative images are shown (D). The left insets show the merged signals of acetylated tubulin and pericentrin. Scale bars, 20 μm or 2 μm (magnified images). The numbers of actin filaments within 60 μm2 around the basal body (as illustrated by the circle) from the cells under serum-starvation without Cyto D treatment were measured (E, n ≥ 132). The percentage of the cells with cilia in the total counted cells (F, n ≥ 357) and the length of cilia (G, n ≥ 126) were measured.
XIAP BIR1- and -BIR2 support melanoma growth via TAB1- and RIPK2-dependent signalling A B16F1 WT, B16F1-XIAP∆BIR1 and B16F1-XIAP∆BIR2 subcutaneous melanoma tumour growth in BL/6 WT mice (n=3 each genotype).Figure 3A B16F1 WT, B16F1-XIAP∆BIR1 and B16F1-XIAP∆BIR2 subcutaneous melanoma tumour growth in BL/6 WT mice (n=3 each genotype). B B16F1 WT, B16F1-XIAPKO (clone 1 and 2), B16F1-TAB1KO and B16F1-RIPK2KO (clone 1 and 2) subcutaneous melanoma tumour growth in BL/6 WT mice (n=5 each genotype). C qRT-PCR analysis of different cytokines/chemokines in the indicated melanoma tumours in mice. Dots represent individual mice (n=3). Data are presented as mean ± SD. One-way analysis of variance (ANOVA) followed by Dunnett's post-analysis.
a) Δ133p53α protein is polyubiquitinated.f5(a) Δ133p53α protein is polyubiquitinated. FLAG-tagged versions of wild-type Δ133p53α (wt) and mutant Δ133p53α (mut; from c below), along with vector control (−), were retrovirally transduced into MRC-5 fibroblasts. The wild-type Δ133p53α-expressing cells had a pair of STUB1 siRNA-transfected (+, siRNA no. 1 for 4 days) and untransfected (−) counterparts. These cells were maintained for 8 h under amino acid- and serum-starved conditions with bafilomycin A1 (100 nM). Protein lysates were used either in IP with anti-ubiquitin (Ub) antibody, followed by IB with anti-FLAG antibody (top), or directly in IB for FLAG-Δ133p53α, STUB1 and β-actin (lower three panels). Smear signals indicate polyubiquitinated FLAG-Δ133p53α protein (Poly-Ub). Asterisk indicates a nonspecific band in negative control. These experimental conditions resulted in similar amounts of FLAG-Δ133p53α in the presence and absence of STUB1 knockdown, allowing quantitative analysis of ubiquitination. The relative densitometric values of wild-type FLAG-Δ133p53α polyubiquitination in the presence and absence of STUB1 knockdown (normalized with total FLAG-Δ133p53α) are shown. ( (b) Δ133p53α is ubiquitinated at the C-terminal lysine residues. MRC-5 fibroblasts expressing wild-type FLAG-Δ133p53α in the absence of STUB1 knockdown were used in IP with anti-FLAG antibody, and the resulting immunoprecipitates were analysed using mass spectrometry. Representative MS/MS spectrum of an [M+3H]3+ peptide from FLAG-Δ133p53α shows sites of ubiquitin modification. The peptide sequence for the spectrum is shown, with the sites of modification marked as asterisks: amino-acid positions 248 and 249, which correspond to positions 381 and 382 in full-length p53. The same peptide was also observed in the unmodified state. (c) Lysine (K)-to-arginine (R) substitutions at amino-acid residues 248 and 249. (d) Mutation of the ubiquitination sites renders Δ133p53α resistant to degradation. The same three cells as in a were cultured under amino-acid- and serum-starved conditions for 8 h (without bafilomycin A1), transfected with STUB1 siRNA (no. 1 for 4 days) or untreated, and examined in IBs for FLAG-Δ133p53α, STUB1, p62/SQSTM1 and β-actin. The effect of starvation was confirmed by decreased p62/SQSTM1 in each cell. The upregulation of p62/SQSTM1 by wild-type and mutant FLAG-Δ133p53α is likely associated with enhanced cell proliferation by Δ133p53α overexpression.
The NFκB-A20-RIPK3 incoherent feedforward loop discriminates TNF stimulus dynamics (A) Modeling schematics depict TNF-induced activation of complex I (C1) and IKK to induce transcriptional activity of NFκB.Figure 3(A) Modeling schematics depict TNF-induced activation of complex I (C1) and IKK to induce transcriptional activity of NFκB. C1 can also initiate activation of RIPK1 and RIPK3 to induce phosphorylation of necroptosis executor MLKL (pMLKL). TNF-induced expression of IκB attenuates NFκB, whereas A20 inhibits IKK and RIPK3. (B Computational simulations and microscopy analysis of death time distributions (left, representative data of three independent experiments) and death rates (right, mean of three independent experiments ± standard deviation) in TNF-treated L929 (B) wildtype (wt) cells. C) Computational simulations and microscopy analysis of death time distributions (left, representative data of three independent experiments) and death rates (right, mean of three independent experiments ± standard deviation) in TNF-treated L929 (C) RelA-knockout (KO) cells. (D) Simulated death time distributions with 2- or 4-fold increased constitutive A20 expression in the absence of inducible transcription (300 simulated cells per condition). (E) Death time distributions in TNF-treated parental RelA KO cells (-) or RelA KO cells expressing A20 from a constitutive transgene (pBabe-A20, +, representative data of three independent experiments). (F) Average death rates (<12 hours) in TNF-treated cells (mean of three independent experiments ± standard deviation; two-tailed Student's t-test *P<0.05, or no statistically significant difference, n.s., P>0.05). (G,H) (G) Distribution of death times (representative data of three independent experiments), or (H) death rates in TNF-treated wt, parental A20 KO cells, or A20 KO cells reconstituted with an NFκB-inducible transgene (fIL8-A20; mean of three independent experiments ± standard deviation). (I) Simulations of 24-hour fractional survival after varying durations of transient or sustained (24h) TNF stimulation (mean of three independent experiments ± standard deviation; two-tailed Student's t-test ***P<0.001, or no statistically significant difference, n.s., P>0.05). (J) experimental measurements of 24-hour fractional survival after varying durations of transient or sustained (24h) TNF stimulation (mean of three independent experiments ± standard deviation; two-tailed Student's t-test ***P<0.001, or no statistically significant difference, n.s., P>0.05).
Figure 4Scatter plots of percent distal polyA site usage indices (PDUIs) in control cells (siCont), Srsf3-silenced cells (siSrsf3; left panel), and both Srsf3- and Hnrnpk-silenced cells (siSrsf3 + siHnrnpk; right panel). PDUIs of all 3′ UTRs are plotted. The 3′ UTRs shortened and extended by siSrsf3 exceeding the thresholds of |ΔPDUI| > 0.2 and p < 0.01 are indicated in red and blue, respectively. 3′ UTRs that were shortened by Srsf3 silencing were first selected. PDUIs of these 3′ UTRs in C2C12 cells treated with control siRNA (C), Srsf3 siRNA (S), Hnrnpk siRNA (K), or both Srsf3 and Hnrnpk siRNAs (S+K) are plotted. ****p < 0.0001 by Steel-Dwass test. The box plot shows the interquartile range (boxes), the median (central band) and the minimum and maximum except for the outliers at the ends of whiskers (n = 965). mRNA expression levels of seven CPSF factors in C2C12 cells treated with the indicated siRNA normalized for those treated with the control siRNA. **p < 0.01 by CuffDiff (Trapnell et all., 2010). Western blots showing protein expression levels of CPSF6 and β-actin (ACTB). Whole cell lysates were harvested 42 h after siRNA transfection. Distributions of interactions between RNA and CPSF6 (red), hnRNP K (black), or SRSF3 (blue) around the distal polyA sites that are downregulated in Srsf3-silenced cells. The standard error of the average density of tRIP reads is shown as a semi-transparent shade around the average curve.
YAP enriches in F-actin rich areas in the apical compartment of hepatocytes during regeneration A EM image of immunogold-labelled YAP on liver tissue sections at 1.Figure 4EM image of immunogold-labelled YAP on liver tissue sections at 1.8 d post PH. Image (A) shows a BC in between two hepatocytes. Indicated regions (black squares) of the apical (a') and the basolateral (a'') membrane of the hepatocyte are shown as magnification. Arrows indicate gold particles. Correlative light and electron microcopy of F-actin (B) and YAP (C) on a liver tissue section at 1.8 d post PH. YAP was detected by immunogold-labelling, F-actin by fluorescence staining with phalloidin. Indicated BC regions with high (c', c'') or low (c''') F-actin levels are shown as magnifications. Arrows indicate gold particles.
Figure 4(A) SEC chromatograms of given loading concentrations of SPOP28-359 are shown. The concentrations were normalized to the monomer molecular weight, i.e., identical concentrations of dimeric SPOP ΔBACK (see Fig EV2) and oligomeric SPOP28-359 contain identical numbers of protomers. (B) SEC chromatograms for SPOP constructs defective in self-association in one or both oligomerization domains are shown. Proteins were injected at the same loading concentration (533 µM) and the elution volume of globular molecular weight standards is noted above the graph. (C) SEC chromatograms of SPOP28-359 (200 µM), SPOP mutBACK (718 µM), and mixtures of the two (ratios given in the Figure, SPOP at 200 µM) are shown. The elution volume of globular molecular weight standards is noted above the graph. (D) Constructs for expressing full-length HA-SPOP or HA-SPOP mutants capable of oligomerization through only one (or neither) of the self-association domains were transfected into NIH 3T3 cells. DAPI was used to stain the nucleus and SPOP localization was identified using an anti-HA antibody. Experiments were performed at least twice on four biological samples. Multiple cells were examined and representative cells are shown. For additional images see Appendix Fig S3. Scale bar represents 5 µm.
Figure 3A-D Proximity between IgM-BCR on the surface of 3046SM (A, C) or 3046M (B, D) cells before and after a 1-min stimulation with the indicated reagents, assayed by Fab-PLA. Results are presented as representative microscopy images (A, B) and box plots after quantification (C, D). Scale bar: 5 μm. In the box plots, the median values are highlighted as thick lines and the whiskers represent the minimum and maximum value. PLA signals (dots/cells) were counted from at least 100 cells for each sample and p-values were calculated by Kruskal-Wallis one-way ANOVA. E-H Calcium flux measured by FACScan for 3046SM (E, F) and 3046M (G, H) cells after stimulation with NIP15-BSA (30 pM), 1NIP-pep (80 nM), Ac146Fab (25 nM) or Ac38Fab (25 nM). Arrows indicate the addition of the stimuli to the cells.
Figure 7A Transmission electron microscopy (TEM) cross sections of WT, dhhc2kd and ∆isp1/3 parasites showing the arrangement of SPM underneath IMC. Approximately 60 recognizable hollow microtubules (green) are associated with IMC (red), distributing evenly around circumference with an interval of 100-120 nm in WT ookinetes. Microtubules lost association with IMC in the ookinetes of dhhc2kd and ∆isp1/3 parasites. Left: one representative cell in each parasite is shown; Right: two areas are magnified to show the details. Lower panel is the quantification of distance between adjacent microtubules associated with IMC. Values are mean ± SD for n=204 (WT), 189 (dhhc2kd), and 211 (∆isp1/3) measurements in 20 cells each group from two independent experiments; Kolmogorov-Smirnov test, ****, P<0.0001. B Apical polar ring and the emanating SPM in WT, dhhc2kd and ∆isp1/3 parasites examined by negative staining and TEM. A dome-like SPM structure radiating from the apical polar ring was observed in early (3 hours) or mature (12 hours) WT ookinetes, but impaired in mutant parasites. n is the number of cells analyzed from three independent experiments. More images in the Appendix Figure S5. Scale bar = 1μm. C SiR-tubulin staining show the SPM structures of WT, dhhc2kd and ∆isp1/3 parasites. Arrowheads point to cell apical. Scale bar = 5 μm. D Proposed role of DHHC2/ISP1/ISP3 in maintaining the dome-like SPM structure consisting of 60 microtubules in ookinete elongation. Microtubules lost association with IMC in the ookinetes of either dhhc2kd or ∆isp1/3 parasites.
Electrophysiological characterization of AAV-dCAS induced neurons.Figure 6A, Firing pattern of a neuron reprogrammed by the endogenous activation of Ascl1, Lmx1a, NeuroD1 and expression of miRNA218 (ALNe-218). All analyzed cells (n=10) showed electrophysiological properties of immature neuron/glia-like cells (i.e. lack of APs and a relatively low Rin). B, Firing pattern of a neuron reprogrammed by the endogenous activation of Ascl1, Lmx1a, Nr4a2 (ALN). The majority, 14 out of 15 cells, exhibit action potentials, while one showed electrophysiological properties of immature neuron/glia-like cells (i.e. lack of APs and a relatively low Rin). Bottom right: example of spontaneous synaptic events recorded from a ALN reprogrammed neuron.