EMBO/sd-character-level-ner huggingface dataset:
Dynamic interaction of the CIP2A, PP2A, and mTORC1 complex regulates mTORC1 activity.Figure 5.(A and B) In vitro translated full-length CIP2A was incubated with purified human PP2A complex and HA-Raptor (A) or HA-S6K1 (B) purified from transiently transfected MCF7 cells. Protein complexes were immunoprecipitated with anti-HA antibody and analyzed by immunoblotting as indicated. Nonspecific IgG served as a control. Note that human reticulocyte (RC) lysate used for in vitro translation of CIP2A contained detectable amounts of PP2Ac. (C) Endogenous CIP2A protein complexes immunoprecipitated from MCF7 cells were analyzed by immunoblotting and PP2A activity assay. Immunoblots of corresponding cell lysates are shown on the right. (D) Endogenous raptor protein complexes immunoprecipitated from MCF7 cells stably infected with control (Ctr) or CIP2A shRNA lentiviruses (Fig. 3 A) and starved for amino acids for 20 min when indicated were analyzed by immunoblotting and PP2A activity assay. Immunoblots of corresponding cell lysates are shown on the right. (E) Raptor protein complexes immunoprecipitated from MCF7 cells treated with control (C) or PPP2R1A (R1A) siRNAs for 56 h were analyzed by immunoblotting with the indicated antibodies. Immunoblots of corresponding cell lysates are shown on the left. (F) HA-S6K1 protein complexes immunoprecipitated from MCF7 cells stably infected with control or CIP2A shRNA lentiviruses (Fig. 3 A), transiently transfected with HA-RPS6K1 cDNA and starved for amino acids for 20 min when indicated, were analyzed by immunoblotting. Immunoblots of corresponding cell lysates are shown on the right. (G) Enzymatic activity of a purified PP2A holoenzyme incubated with human reticulocyte lysate with or without in vitro translated full-length CIP2A. Error bars are SDs for four (D) independent experiments or a representative triplicate experiment (C and G). (H) Representative confocal microscopy images of untreated and amino acid-starved (4 h) MCF7 cells stained for endogenous CIP2A and raptor. Black lines indicate that intervening lanes have been spliced out. −AA, amino acid starvation; IP, immunoprecipitation; a.u., arbitrary unit. Bar, 10 µm.
Figure 2A. Representative pictures of immunostaining for F4/80 (green) in Control and CriptoMy-LOF TA sections at day 2 (top panel) and 5 (bottom panel) after injury. B. Quantification of F4/80 staining/damaged area (μm2) at day 2 (top graph) and 5 (bottom graph) after injury. Data information: Nuclei were counterstained with DAPI (blue). Scale bar: 50 μm. Magnification of the boxes is 3.5 x. Data are expressed as box plots displaying minimum, first quartile, median, third quartile and maximum (n≥5 biological replicates; **P<0.01, Student's t-test). C. Representative pictures of immunostaining for CD206 in Control and CriptoMy-LOF TA sections at day 2 (top panel) and 5 (bottom panel) after injury, respectively. D-E. Quantification of CD206+ MPs per area (mm2) at day 2 (D) and 5 (E) after injury. Data information: Nuclei were counterstained with DAPI (blue). Scale bar: 50 μm. Magnification of the boxes is 3.5 x. Data are expressed as box plots displaying minimum, first quartile, median, third quartile and maximum (n≥5 biological replicates; **P<0.01, Student's t-test).
Figure 1Potentially phosphorylated residues in the MICU1 (NM_144822) sequence were detected using the Scansite 3 software (http://scansite3.mit.edu). The different values refer to the surface accessibility scores (Scansite) or the phosphorylation scores, which were obtained with both NetPhos 3.1 (http://www.cbs.dtu.dk/services/NetPhos/) and NetPhorest (http://www.netphorest.info) software HeLa cells overexpressing the HA-tagged MICU1 WT, MICU1 S124D or MICU1 S124A mutants were stained for HA or HSP60 (mitochondrial marker). Merged images are indicated (merge). Scale bar 10 μm Representative images of the 2mt-GCaMP6m 474/410 ratio of ShMICU1 HeLa stable cells expressing an empty vector (ctrl) or the MICU1 WT, MICU1 SD, and MICU1 SA. Scale bar 10 μm Resting mitochondrial calcium levels, evaluated through ratiometric imaging of the mitochondrial targeted GCaMP6m, in ShRNA control (plko) or ShRNA MICU1 HeLa stable clone cells transfected with the indicated constructs (n=5 independent experiments; 55-67cells) Representative kinetics (E) and analysis (F) of aequorin-based [Ca2+]m measurements in ShRNA MICU1 HeLa stable clone cells transfected with the indicated constructs and challenged with 20 μM 2,5-di-tert-butylhydroquinone (TBHQ) in the absence of extracellular Ca2+ (n=3 independent experiments) Representative kinetics (G) and analysis (H) of aequorin-based [Ca2+]m measurements in intact ShRNA MICU1 HeLa stable clone cells transfected with the indicated constructs and challenged with 10 μM cyclopiazonic acid (CPA) in the presence of 100 μM EGTA (n=3 independent experiments) Western blot analysis for the presence of MICU1 in clones arising from single cells generated by CRISPR/Cas9-mediated genome editing. The results for 12 of the 36 clones that were examined are shown Resting mitochondrial calcium levels, evaluated through ratiometric imaging of the mitochondrial-targeted GCaMP6m, in MICU1-KO cells generated using the CRISPR/Cas9 technique and transfected with the indicated constructs (n=3 independent experiments; 30-56 cells) Representative kinetics (K) and analysis (L) of aequorin-based [Ca2+]m measurements in intact MICU1-KO cells generated using the CRISPR/Cas9 technique, transfected with the indicated constructs and challenged with 20 μM TBHQ in the absence of extracellular Ca2+ (n=3 independent experiments.) Representative kinetics (M) and analysis (N) of aequorin-based [Ca2+]m measurements in intact MICU1-KO cells generated using the CRISPR/Cas9 technique, transfected with the indicated constructs and challenged with 10 μM CPA in the presence of 100 μM EGTA (n=3 independent experiments).
Figure 3Average DamID values plotted against average expression levels of eight genes activated by CRISPRa (red; n = 2) or in control cells that were treated either without sgRNA or with one of various sgRNAs targeting promoters on different chromosomes (blue; n ranging from 19 to 27).
ATP8A1 knockdown causes an increase in RE luminal PS levels A His‐tagged 2xPH was mixed with liposomes that were composed of 75% (mol/mol) PE, 20% (mol/mol) PC, and 5% (mol/mol) of PS, PA, PI, PIPs, or sulfatide.Figure 6A His‐tagged 2xPH was mixed with liposomes that were composed of 75% (mol/mol) PE, 20% (mol/mol) PC, and 5% (mol/mol) of PS, PA, PI, PIPs, or sulfatide. In the case of PE/PC, liposomes of 80% (mol/mol) PE and 20% (mol/mol) PC were used. After 15 min, the mixture was spun at 100,000 g for 30 min, and the resultant supernatant (S) and pellet (P) were subjected to SDS-PAGE. The gels were stained with Coomassie blue. B COS‐1 cells were incubated in medium containing Alexa 488‐Tfn for 40 min, washed, fixed, and stained with recombinant 2xPH. Magnified image is shown in the right panel. Scale bar, 10 μm. C COS‐1 cells were transiently transfected with GFP‐lact‐C2 and stained for Rab11. Scale bar, 10 μm. D, E COS‐1 cells were transiently transfected with GFP‐lact‐C2 and stained with recombinant 2xPH and anti‐GM130 antibody. The asterisk indicates a cell expressing GFP‐lact‐C2, and the arrowhead indicates a non‐expressing cell. The graph in (E) shows fluorescence intensity per pixel of recombinant 2xPH stain in RE area, which was delimited by GM130. Data are normalized to the 2xPH intensity in GFP‐lact‐C2 (−) cells (mean ± SD, n = 13 cells for each condition). Scale bar, 10 μm. F-H COS‐1 cells stably expressing GFP‐lact‐C2 were treated with ATP8A1 siRNA1 or EHD1 siRNA1 for 72 h and stained with recombinant 2xPH and anti‐GM130 antibody. The intensities of 2xPH staining and GFP‐lact‐C2 in RE area were measured. The intensity of 2xPH (G) and the ratio of 2xPH to GFP‐lact‐C2 intensities (H) are shown. In (H), data are normalized to that of control cells. Data represent mean ± SD and were analyzed with two‐tailed Student's t‐test (n > 26 cells from three independent experiments). **P 0.01; ***P 0.001; NS, not significant. Scale bars, 10 μm.
Bax and Bak are regulated by the same mechanism.Figure 6A FLIP of BaxTBakSS without (○, broken line) or with (●, straight line) overexpressed Bcl-xL. Fluorescence of a neighboring cell is shown as control (, dotted line). Data represent averages ± SEM from 20 (-Bcl-xL) and 16 (+Bcl-xL) ROI measurements. B Western blot analysis of PARP cleavage in HCT116 Bax/Bak DKO cells overexpressing Bax, Bax S184V, BaxTBak or BaxTBak V197/198S. The experiment was carried out in the absence of apoptotic stimuli. Cells transfected with pcDNA3.1 vector serve as control for PARP cleavage. Similar sample loading is controlled by actin. n = 4. C Colony formation of Bax/Bak DKO cells transfected with pcDNA, BaxTBak or BaxTBakSS with Bcl-xL overexpression in the absence of apoptotic stimuli. n = 3. D Caspase 3/7 activity was measured in HCT116 Bax/Bak DKO cells overexpressing Bax, Bax S184V, BaxTBak or BaxTBak V197/198S. Caspase activity is displayed in relative fluorescence units (RFU). The experiment was carried out in the absence of apoptotic stimuli and pcDNA3.1-transfected cells served as control. Data represent averages ± SEM. n = 4. p-values according to One Way ANOVA using the Holm-Sidak method are displayed. E Comparison of Bax and Bak localization and activity dependent on the MA (Bax MA, blue rectangle) using wild-type Bax (i, blue), Bax S184V (ii), BakTBax (iv) and BakTBax S184V (iii). Direct comparison of Bax S184V (ii) and BakTBax S184V (iii) reveals MA-independent influences of Bax and Bak on protein localization and activity (I). The impact of the S184V substitution (*) on translocation, retrotranslocation and activity is shown by differences between BakTBax and BakTBax S184V (II). The additional effect of Bax MA binding to the hydrophobic groove of Bax (III) is indicated by the comparison of both Bax variants (i+ii) versus both Bak variants (iii+iv). F STS-induced caspase 3/7 activity of HCT116 Bax/Bak DKO cells expressing Bax, Bax S184V, BakTBax or BakTBax S184V is displayed normalized to Bax activity. Data represent averages ± SEM. n ≥ 3. G Western blot analysis of PARP cleavage after STS treatment in HCT116 Bax/Bak DKO cells overexpressing Bax, Bax S184V, BakTBax or BakTBax S184V. Empty vector-transfected cells serve as control for PARP cleavage and actin is the loading control. n = 3. H Plot of Bax (●) and Bak (○) retrotranslocation rates vs. hydrophobicity of the MA of the constructs. R2 = 0.95.
Figure 2(A-B) IHC labeling of Sirt7 and Ki67 expression in the skin at different HF phases in the second hair cycle and in the depilation-induced hair cycle in WT mice. P56, P69, P74 and P84 indicate the postnatal day in the second hair cycle. Pd, post depilation. Dashed line indicates bulge (Bu) of a hair follicle and solid line indicates the Ki-67 positive staining region. (C) The hair coats of Sirt7f/f and Sirt7f/f;K15-Cre mice were clipped at P60 and images were captured on P60, P125 and P150. (D) The postnatal day of hair coat recovery by 25%, 50% 75% and 100% in shaved Sirt7f/f and Sirt7f/f;K15-Cre mice. n = 6 mice per genotype. Box-and-whisker plots: mid-line, median; box, 25th and 75th percentiles; whiskers, minimum and maximum. (E) H&E staining of the skin in Sirt7f/f and Sirt7f/f;K15-Cre mice at P100. (F) Representative immunofluorescence images showing Ki67/Pcad expression (upper) and Cd34/Ki67 expression (lower) in Sirt7fl/fl and Sirt7f/f;K15-Cre HFs on P56, P74 and P85. The white dashed line indicates bulge of hair follicle. (G) The percentage of Ki67-positive cells per HG; n = 3 mice and 10 HGs were counted in each mouse. (H) Ki67-positive cells per bulge; n = 3 mice and 10 bulges were counted in each mouse. (I) Schematic illustrating the HF cycle in Sirt7f/f and Sirt7f/f;K15-Cre mice after mifepristone-induced Cre activation. The arrow indicates the time of mifepristone treatment (details in methods); the red star indicates the end of the telogen phase in Sirt7f/f;K15-Cre HFs.
Figure 3A The experiment was performed as described in Fig 2C. Instead of IFNB, ISG54 and IL6 inductions were measured by qPCR. B HEK293 cells were transfected with si-TRAF3IP3 oligos and then treated with various stimuli. Following subcellular fractionation, S5 fractions were obtained and subjected to native PAGE for detecting IRF3 dimer by immunoblotting. The whole cell lysates were also subjected to SDS-PAGE for anti- IRF3 and tubulin immunoblotting. C The experiment was performed as described in Fig 2E. ISG54 and IL6 induction was measured by qPCR. D Wild type and Traf3ip3-/- HEK293T cells were infected with or without VSV. At indicated time points, Cells were collected and analyzed by immunoblotting. Following subcellular fractionation, S5 fractions were subjected to native PAGE to examine IRF3 dimer, and P5 fractions contained mitochondria were subjected to SDD-AGE to examine MAVS aggregation. The mitochondrial outer membrane protein Prohibitin was immunoblotted as an internal control.
Figure 1(A-C) The effect of hypoxia was estimated in C57BL/6J mice (n=3) by an HRE-luciferase (HRE-luc) reporter plasmid at indicated time points. All mice were injected according to body weight. (A) Experimental set-up. (B) Imaging of luciferase activity (purple signal) in the liver of control mice and mice under normoxic and hypoxic conditions at indicated time points. Red circles indicate the region of interest selected for the measurement of the luciferase activity. (C) Bioluminescent photon counts normalized to the PBS control group in liver of mice in normoxia and hypoxia at indicated time points. P-values were calculated using one-way ANOVA followed by post-hoc Tukey's test to correct for multiple testing during the pairwise multiple comparisons. Data information: All bars represent mean ± SEM. Each individual data point represent individual mice. P-values were calculated using two-way ANOVA followed by post-hoc Šídák's multiple comparisons test to correct for multiple testing during the pairwise multiple comparisons, except if otherwise stated. *** P<0.001; ** P≤ 0.01; * P≤ 0.05. N = normoxia, H = hypoxia. Signal around the perimeter of the Petri dishes represent aspecific luciferase signal. (D, E) Imaging of luciferase activity in the liver of HIF1aAlbKO (D) and HIF2aAlbKO (E) mice after a hydrodynamic tail vein injection with a HRE-luc reporter plasmid or control (PBS) according to body weight followed by 6h normoxia or hypoxia. (F, G) Bioluminescent photon counts normalized to the PBS control group in liver of (F) HIF1aAlbKO and (G) HIF2aALbKO mice and their wild-type littermates 6h after normoxia of hypoxia. Data information: All bars represent mean ± SEM. Each individual data point represent individual mice. P-values were calculated using two-way ANOVA followed by post-hoc Šídák's multiple comparisons test to correct for multiple testing during the pairwise multiple comparisons, except if otherwise stated. *** P<0.001; ** P≤ 0.01; * P≤ 0.05. N = normoxia, H = hypoxia. Signal around the perimeter of the Petri dishes represent aspecific luciferase signal.
Figure 2A minimal model of antibiotic action. Antibiotic is transported between the intracellular and extracellular environments ( and ) by influx and efflux rates ( and ). reversibly binds to target ribosomes to form the complex CA, with binding and dissociation rates and , respectively. This complex can then be degraded through the intermediate CA′. These intracellular dynamics influence the overall population recovery rate; the maximum growth rate is dependent on the ribosome concentration . Thus, recovery can be quantified both on the individual level, as a function of , or on the population level, in terms of cell density. Concurrent antibiotic transport and ribosome inhibition dictate recovery dynamics. During treatment, , and the intracellular antibiotic concentration can be linearly approximated by , where is assumed to be constant and represents the treatment duration (leftmost schematic and panel 1). Antibiotic influx is greater than efflux; binds to target ribosomes and strongly inhibits the upregulation of ribosome synthesis. When extracellular antibiotic is removed, decreases (middle and rightmost schematics, green shading and panel 2), and ribosome synthesis resumes when is sufficiently small (panel 3). Ain accumulates linearly with treatment duration for sufficiently short treatment durations (panel i). After the removal of Aout, efflux and inhibition dynamics combine to delay the synthesis of ribosomes in a concentration-dependent manner (panel ii). Colors indicate increasing antibiotic concentration, as shown in panel ii. Antibiotic turnover timescale sets intracellular recovery RTcell. Regardless of the antibiotic treatment history, the relationship between intracellelular antibiotic and ribosome concentration approaches the same asymptote, indicating that the timescale of individual detoxification sets the timescale of ribosome synthesis. Colors indicate increasing antibiotic concentration, as in Figure 2C-D; for these representative trajectories, treatment duration was set to 120 minutes. Population recovery time is dictated by total antibiotic exposure. Lower right inset shows that RTpop is an increasing function of dose duration; these data collapse onto a single relationship as a function of total antibiotic exposure (main figure). Individual (RTcell) and population recovery time (RTpop) are strongly correlated, suggesting that intracellular dynamics lead to population level recovery (R2 = 0.86).
M45 induces the formation of autophagosomes and aggregates.Figure 8(A) NIH-3T3 cells stably expressing GFP-LC3 were transduced with retroviral vectors expressing M45 or Ct3. Three days later, cells were analyzed by confocal laser scanning microscopy. GFP-LC3 dots were counted in 50 cells per sample. Results are shown as box and whisker plots. The bottom and top of the box represent the first and third quartile, respectively. The horizontal line within the box represents the median. The minima and maxima within 1.5-fold interquartile range are shown as whiskers. Dots represent outliers. Significance was determined using ANOVA (***, p<0.001). M45 and Ct3 were detected by immunoblot (B) Fluorescence images of cells described above. Large GFP-LC3 aggregates are indicated by arrow heads. (C) Electron micrograph of a double-membrane autophagosome in an M45-expressing cell. (D) Autophagosomes were counted in 10 randomly selected M45- and Ct3-expressing cells (2-3 TEM sections per cell). Results are shown in box and whisker plots as described for panel A. Significance was determined using the student's t-test (***, p<0.001). (E) Epifluorescence image of an M45-expressing cell with large GFP-LC3 structures (F) TEM image of the same cell reveals large aggregates marked with 'A'. (G, H) Successive enlargement of the region marked in panel F shows an example of an aggregate-associated autophagosome.
Mutation of the human IFITM3 endocytosis motif converts IFITM3 into an enhancer of SARS-CoV-2 infections.Figure 3HEK293T-ACE2-GFP stable cells were transiently transfected with human IFITM plasmids or vector control for 24 h. A) Flow cytometry gating strategy for analyzing cells with high ACE2-GFP expression and IFITM expression. HEK293T-ACE2-GFP stable cells were transiently transfected with human IFITM plasmids or vector control for 24 h. B) Flow cytometry histograms showing expression of each of the IFITM constructs, and the gating as indicated in (A) and by the dotted line, which was used for examining percent infections in IFITM-expressing cells. HEK293T-ACE2-GFP stable cells were transiently transfected with human IFITM plasmids or vector control for 24 h. C) Transfected HEK293T-ACE2-GFP cells were infected with SARS-CoV-2 (MOI 1) for 24h and analyzed for percent infection by flow cytometry. Representative example plots are shown for each transfection condition. HEK293T-ACE2-GFP stable cells were transiently transfected with human IFITM plasmids or vector control for 24 h. D) Graph depicts normalized infection percentage measurements from 2-3 independent experiments, each performed in triplicate, from cells infected Bars represent averages with individual data points shown as circles. Error bars represent SD. #p<0.05 compared to vector control by ANOVA followed by Tukey's multiple comparisons test. HEK293T-ACE2-GFP stable cells were transiently transfected with human IFITM plasmids or vector control for 24 h. E) Representative example flow cytometry histograms of ACE2-GFP from non-infected samples. HEK293T-ACE2-GFP stable cells were transiently transfected with human IFITM plasmids or vector control for 24 h. F) Normalized measurements of ACE2-GFP mean fluorescence intensity (MFI) of non-infected samples from three separate experiments. Bars represent averages with individual data points shown as circles. Error bars represent SD. Statistical significance (p<0.5 by ANOVA followed by Tukey's multiple comparisons test) was not observed for comparisons between any samples. HEK293T-ACE2-GFP stable cells were transiently transfected with human IFITM plasmids or vector control for 24 h. G) Confocal microscopy of HEK293T cells transfected with empty vector, hIFITM3, hIFITM3-Y20A, or hIFITM3-L23Q. Green, anti-IFITM3; Blue, DAPI. White arrows highlight plasma membrane localization of IFITM3-Y20A and IFITM3-L23Q. Scale bars represent 10 µm.
a) Lysates extracted from neurospheres of Ctrl, FIP200GFAP cKO, Trp53GFAP cKO and 2cKO mice and analyzed by western blot using antibodies to p53 and actin.figf4(a) Lysates extracted from neurospheres of Ctrl, FIP200GFAP cKO, Trp53GFAP cKO and 2cKO mice and analyzed by western blot using antibodies to p53 and actin. (b) Mean ± s.e.m. of relative mRNA level (normalized to Ctrl mice as 1) of Cdkn1a, Bbc3, Fas, Pax6 and Stat3 in neurospheres of Ctrl, FIP200GFAP cKO, 2cKO and Trp53GFAP cKO mice are shown (n = 3 mice for each). (c,d) Mean ± s.e.m. of the number (c) and size (d) of primary and secondary neurospheres from Ctrl, FIP200GFAP cKO, 2cKO and Trp53GFAP cKO mice at P28 (n = 3 mice for each). (e-h) Analysis of immunofluorescence or staining on frozen-sectioned neurospheres for nestin, Ki67, TUNEL, PAX6, phosphorylated STAT3 (pSTAT3) and DAPI, as indicated. Mean ± s.e.m. of the percentage of Ki67+ (e), BrdU+ (f) and TUNEL+ (g) cells in neurospheres from Ctrl, FIP200GFAP cKO, 2cKO and Trp53GFAP cKO mice are shown (n = 3 mice for each, counting of >500 cells for each mouse). Representative images are shown in h. *P 0.01. Full-length blots are presented in Supplementary Figure 11.
Figure 4A) Transient expression analysis in Arabidopsis mesophyll protoplasts shows enhanced defense gene expression in mkkk7 protoplasts after flg22 treatment. Protoplasts were isolated from 4 week old plants and transfected with pWRKY29:fLUC (WRKY29) or pFRK1:fLUC(FRK1) constructs together with 35S:rLUC, as indicated in the graph. Protoplasts were treated for 4 hrs with 10 µM flg22 or mock treated as indicated. The horizontal axis indicates the treatment while the vertical axis represents expression levels relative to the mock treated control sample, as fold induction. All measurements were normalized to the rLUC activity. Bars represent means ± STDEV (n=2). Experiment was repeated 6 times with similar results. B) WRKY29 transcripts measured by qRT-PCR in flg22 treated leaf material. Leaf strips of Col-0 and mkkk7 were treated with 1 µM flg22 for t=0, 1, 2, and 4h. WRKY29 transcripts were normalized against Ubiquitin transcript as described before [62]. Bars represent mean value and error bars show SE (n=3). (* p<0.05, ** p<0.01, student t-test). C) FRK1 transcripts measured by qRT-PCR in flg22 treated leaf material. Leaf strips of Col-0 and mkkk7 were treated with 1 µM flg22 fort=0, 1, 2, and 4h. FRK1 transcripts were normalized against Ubiquitin transcript as described before [62]. Bars represent mean value and error bars show SE (n=3). (* p<0.05, ** p<0.01, student t-test).