EMBO/sd-character-level-ner huggingface dataset:
(I) HeLa CCL2 cells infected with either sh-control luciferase (shGL) or shβ-catenin lentiviruses were treated with nocodazole (200 ng/mL) for 18 h (metaphase) and released by incubation in fresh medium for 2 h (telophase). Cell lysates were analyzed by pull-down assays using rhotekin RBD beads, and pulled-down proteins were separated by SDS-PAGE followed by immunoblot analysis using the indicated antibodies. The membrane was then stained with coomassie (CBB) to confirm the amount of RBD precipitated.
(F, G). The percentages of pre-spike foot (F) and stand-alone foot (G) for wild-type MUNC18-1 or different mutants were calculated. Data information: indicates MUNC18-1, # indicates MUNC18-1 overexpression group compared with the control group. P<0.05; **, P<0.01; *** P<0.001 N.S. means no significant difference. Data were from 6 independent experiments. All the data were analyzed using one-way ANOVA, and showed as the mean ± s.e.m.
B eEF2K WT and KO MEF treated for 6h with indicated concentration of NFR, Rapamycin (Rapa), AICAR or starved for indicated time in PBS (Starv.), were analyzed by immunoblot using indicated antibodies. Tubulin is used as loading control.
Genome browser view of FUS gene, centered on altFUS. The 'Transcript' track contains the beginning of the canonical FUS transcript (ENST00000254108) in blue. The 'Protein' track contains the beginning of the FUS protein (green) and the whole altFUS protein (red). In F, the 'Peptide' track contains all the peptides identified by the OpenProt resource using the classical spectrum-centric approach. The peptides sequences are indicated and are unique to altFUS or its isoform
HeLa cells stably expressing HA-E2F1 WT and HA-E2F1 ∆RxL under the control of a retroviral promoter were treated with Cycloheximide (CHX) at 50 μg/ml for the indicated minutes (min) Quantification of E2F1 half-life
(C) MG activity upon knockdown of host genes. HEK293T cells were transfected with scrambled siRNA or siRNA targeting RBBP6, hnRNP L, hnRNPUL1 or PEG10. Twenty-four hours post-transfection, cells were transfected with MG assay plasmids including VP30 at 12.5 and 25ng doses. Data information: , the data represent the mean ± S.D. from one representative experiment in which each transfection condition was performed in triplicate (n=3). The experiment was reproduced in two additional, independent experiments Statistical significance was calculated using ANOVA with Tukey's multiple comparisons test. ****p<0.00005; ***p<0.0005, **p<0.005, *p<0.05.
C. Premature activation of a mutant APC/C (1-E562A) in Xenopus egg extracts. The purified recombinant WT APC/C or Apc-loop500-mutant APC/C (1-E562A) was incubated with its substrates (35S-labelled cyclin B and a version of cyclin B lacking the N-terminal 67 residues, ∆67) in APC/C-depleted (ΔAPC) interphase extracts supplemented with non-degradable cyclin B at 23˚C. Samples taken at indicated time points after addition of substrates were analysed by SDS-PAGE and autoradiography (Fig S7A). The relative cyclin levels are shown, normalized with reference to the intensities found at time 0 for each time point. Error bars, SEM from three independent experiments. A representative result is shown in Fig S7A.
C: Solid phase binding assay to test for binding of different Dam1 complexes to Bim1. Recombinant GST-Bim1185-344 was immobilized on glutathione sepharose beads and incubated with either recombinant Dam1WTc or Dam1∆SxIPc. Input and pull down samples were analyzed by SDS-PAGE.
The indicated constructs were transfected into MCF-10A cells for 24h, then the GFP fluorescence was detected using fluorescence microscope (F) and fusion protein levels were determined by western blot with anti-GFP and anti-CIP2A-BP antibodies respectively
Healthy control (filled circles) or BLM-induced mice were treated with vehicle (filled squares), 1 nmol (empty circles), 3 nmol (empty squares), or 10 nmol (filled triangles) FA-TLR7-54 and then sacrificed on day 21 for analysis. (C) Quantitation of total hydroxyproline content per right lung (Healthy control, n=5; others, n=7-9).
Quantification of HDAC1 and TDP-43 expression levels in urea-soluble fractions. N = 5 per group. Data information: All data represent mean ± SEM, **** p < 0.0001 by t-test.
K. Mean mitochondrial section area in Ifnb+/+ and Ifnb-/- MEFs transfected with Drp1 or phosphomimetic Drp1. Error bars are SEM from 10 transfectants.
Mutation strategy of Hdac6-binding site on Prom1. The lysine (K) 138 in the first cytoplasmic loop of Prom1 was mutated to glutamine residue (Q). TM, transmembrane domain.
C, Representative examples of HLH-30::3xFLAG::eGFP subcellular localization: Cytoplasmic, diffuse GFP distribution along the whole body of the worm; Weak nuclear, some cells in the posterior part of the worm display nuclear GFP labeling but no nuclear labeling is found in the head cells; Strong nuclear, distinct nuclear GFP labeling in cells along the worm body, including neurons in the head area. Arrowheads denote nuclear localization of HLH-30::3xFLAG::eGFP. Scale bar 200 µm and 50 µm. D, Quantification of the subcellular distribution of HLH-30::3xFLAG::eGFP and HLH-30(C284A)::3xFLAG::eGFP upon treatment with different stimuli. Data are from three independent experiments with at least 50 animals per experiment.
A, B Immunohistochemistry analysis of CD8a, CD4 (A), PCNA, and Foxp3 (B) in tumors from WT and Tet2-/- (KO) mice subcutaneously injected with Hepa1-6 hepatoma cells. Corresponding bar charts display positive staining, quantified using Image Pro Plus 6.0 software (n=3). Pictures were captured at 400X magnification. Scale bar = 50 μm.
I. U2OS cells transfected with empty vector (EV) or indicated RFC subunit expression plasmids were subjected to FLAG IP and immunoblotted with indicated antibodies.
(H) Endogenous WASH colocalizes with Beclin 1 via confocal microscopy. HeLa cells cultured in CM or EBSS for 1 h were stained with antibodies against Beclin 1 and WASH. The colocalization rate (Pearson's correlation coefficient) between Beclin 1 and WASH was calculated and shown as means±s.d. in the lower panel. Scale bar, 10 μm.
B Quantification of transwell migration assays performed for PC3-LN4 EDC3 wild type, EDC3 S161A, EDC3 KO, and EDC3 S161D cell lines. The number of migrated cells (40 h) was counted as outlined in Materials and Methods. Data information: All invasion and migration assays were performed in triplicates and 6 different fields at 10x magnification were counted in 3 separate inserts. The data are presented as the mean +/- S.D. p values<0.05, calculated using unpaired student's t-test.
E. left, western blot analysis of transient overexpression of wild type ABL1 (WT) and ABL1-R351W, compared to empty vector control (EV) in HEK293T cells. Right, quantification of p-CRKL signal based on densitometry analysis of blots from 6 independent experiments. Error bars represent ±S.D., n=6, *P=0.0004 by two-tailed unpaired Student's t-test.
Through the synthesis/secretion of soluble chemoprotective factors including IL-6, CAFs induce pancreatic cancer cell chemoresistance. High protein synthesis rate in CAFs is dependent on the robust intrinsic activation of the mTOR pathway resulting in inhibition of the protein translation inhibitor 4E-BP1. Secreted IL-6 participates in an autocrine feed-forward loop in the intrinsic activation of mTOR and subsequent high protein synthesis.CAFs express the somatostatin receptor sst1. Upon activation with the novel SOM230 somatostatin analogue (Pasireotide® Novartis) presenting a high affinity for sst1, the mTOR pathway is inhibited, resulting in reduced synthesis/secretion of chemoprotective factors including IL-6 through inhibition of mRNA translation. As consequences, the autocrine feed-forward IL-6/mTOR/protein synthesis/IL-6 loop is abrogated, and pancreatic cancer cells are re-sensitized to the apoptotic action of chemotherapies.
(C) The interaction of TRB1 with HAM1 and HAM2. TRB1-Flag transgenic plants were crossed to HAM1-Myc and HAM2-Myc transgenic plants and their progeny were used for co-IP.
(B) 2D 1H-15N TROSY-HSQC spectra of DMPC/DHPC bicelle-bound Bax (α2-α5) (blue) and soluble Bax (α2-α5) (red) at 600 MHz. The magnified regions highlight the spectral changes for some residues in the bicelle-bound and soluble proteins.
(F-H) Proportion or numbers of ILC2s in the spleens, kidneys and islet graft of mice with or without IL-33 treatment. Data shown are the mean ± SEM (n=4-6 per group) and a one-way ANOVA was performed; **P<0.01, ***P<0.001.
G, H. Control or OPA1 KO HCT116 cells were maintained in normoxia or hypoxia (1% O2) for 24 hours. The glucose uptake (G) and lactate production (H) were measured, and the values were normalized to the protein concentration. Statistical significance was assessed by a two-way ANOVA; error bars are presented as mean ± SD of three independent experiments; N.S., not significant, *p < 0.05, **p < 0.01.
Histogram showing the distribution of the fluorescence peak time expressed as a percent of generation time. Bars shaded in plum indicate moderately pulsing cells (n = 185) and magenta bars indicate highly pulsing cells (n = 57). Significance by Mann-Whitney test. The data shown are from 3 independent experiments. Peaks mainly occur between 80% and 100% of the generation time.
(b) DLD1 parental or NCOA4−/− cell extracts were resolved using SEC and each fraction was analysed for the presence of NCOA4, FTH1 and FTL by western blot.
The effects of individually mutating the first two residues of Ams1 on its Nbr1-binding ability. Nbr1-Ams1 interactions were examined by the Pil1 co-tethering assay. Scale bar, 3 μm.
E Time-lapse imaging of condensates formed by cGAS, G3BP1 and dsRNA. Data information: Representative images are shown Scale bars, 10 μm 45 μM cGAS-mCherry, 10 μM G3BP1-mEGFP 2 μM dsRNA were used in the assays.
Angle change curve of cell, nucleus and Golgi. Red-dashed line: laser angle; Blue line: nucleus angle; Orange line: cell angle; Green line: Golgi angle. H: control cell; I: CAMSAP2 KD cell; J: CLASPs&GCC185 KD cell. Box-whisker plot shows the total time required for the nucleus, Golgi and cell body reaching the given laser angle. Blue: time required for nucleus; Green: time required for Golgi; Orange: time required for cell body. (1 representative of 3 independent experiments and n= 14 cells). The ends of the whiskers are set at 10% and 90% of the entire population, ***p < 0.001, ns, no significant difference, unpaired t-test.
(E) In a first series of experiments, animals [WT (N=13) and HET (N=18)] were trained to press a lever to obtain a pellet of food in an illuminated Skinner box with a fixed-ratio (1:1) schedule. After ten days of training, they were transferred to a light on/light off protocol where lever presses were only rewarded when a small light bulb located over the lever was switched on. Lever presses while the bulb was off were punished with a time penalty of ≤ 10 s during which the bulb would not turn on. (F,G) Mean days to criterion (F) and mean number of lever presses during the last two training sessions (G) for the fixed-ratio (1:1) paradigm. No significant differences were observed.
(e) Decreased ERK phosphorylation in Atg5−/− MEFs occurs independently of changes in ERK phosphatases. Immunoblots for the indicated proteins in 2 h serum-deprived WT MEFs transfected with scrambled siRNA (scr), and Atg5−/− MEFs transfected with scr or siRNAs against MKP3 or PP2A in the presence or absence of EGF. The bars represent mean±s.e.m., n=3.
TFEB silencing inhibits VEGFR2 internalization. Bar graphs of VEGFR2 internalization expressed as the percent of internalized VEGFR2 versus PM VEGFR2 after VEGF-A stimulation
(E) Cumulative distribution plot of the fold-difference in expression after 8 days of 4-OHT treatment for mRNAs, relative to day 0 of treatment (tpm≥1) with (n=6034) and without (n=7887) evidence of AGO2-binding by HEAP (Li et al., 2020).
A. Western blot illustrating abundant Bcl11b and Gata3protein expression in Myc/Bcl2+DN2- but not LSK- or GMP-derived blasts. Thymus (Thy) and bone marrow (BM) extracts from healthy WT mice served as controls. Detection of α-tubulin or valosin-containing protein (VCP) ensured comparable loading.
(D) SNARE protein staining was compared for control and SFT2D2 KD HeLa cells and quantified. The graph shows the change in cellular intensity measured for each post-Golgi SNARE investigated. Images illustrate the increased cellular intensity of VAMP3 in SFT2D2 KD cells.
d) Bulk degradation activity of Rubicon knockdown cells. The bulk degradation activity in control and Rubicon knockdown cells in nutrient-rich medium was measured. Wortmannin (W, 100 nM) was added as a negative control. The data are mean ± s.d. of three independent experiments.
I. Transepithelial electric resistance of the Caco2 cells shscramble or shPkd2 (sequence 1). n=6.
(E) Oxygen consumption rates in intact heart mitochondria in the presence of pyruvate-glutamate-malate (C I) and pyruvate-glutamate-malate + succinate (C I + C II) as substrates. State 3 (substrates+ADP); State 4 (+oligomycin); Uncoupled (+FCCP). (n = 3). Bars represent mean ± S.E.M. (Student's t test, *p < 0.05, **p < 0.01, ***p < 0.001).
(c) Immunoblot analysis of LC3b and p62 at various stages of P14 differentiation. Data are representative of two independent experiments with samples pooled from 5-20 mice for each time point.
Retrotransposon transcription in WT (Hu303), fft2Δ (Hu1955), fft3Δ (Hu1867), and fft2Δfft3Δ (Hu2000). F) The sequence of a Tf2 retrotransposon transcript from the 'R' sequence of the upstream LTR to the start codon of the coding region. The long and short RACE products (E) were sequenced and the transcript start sites are highlighted (yellow). Orange text, R. Light blue, U5. Dark blue, self-primer and primer binding site. Green, start codon.
(A) qRT-PCR analysis for Islr, Chd1 and Vim in total intestinal tissues, intestinal epithelium and lamina propria. n = 4.
(a) Endogenous TR3 is localized in the MIM in response to THPN stimulation. Left, isolated mitochondria from cells treated with THPN (20 μM, 6 h) were incubated with proteinase K (20 μM and 40 μM) for 30 min. TR3 expression was determined. Tom20 in MOM and Tim23 in MIM were used as controls. Right, the mitochondria were further subjected to submitochondrial fractionation. VDAC1, Tim23 and Hsp60 were used to represent MOM, MIM and mitochondrial matrix protein, respectively. '-' represents vehicle treatment only.
(E) Graph represents the levels of Spindly phS499 at unattached KTs for cells Spindly phS499 levels were determined relative to CID and all values were normalized to the control mean fluorescence intensity, which was set to 100% (n≥1246 KTs from at least 63 cells for each condition, n=2 independent experiments). Statistical analysis was calculated using a Kruskal-Wallis test for multiple comparisons. p values: ****, <0.0001 Data information: Data are shown as mean ± SD. Scale bar: 5 μm.
Comparison of protein synthesis rate of endogenous E. coli genes measured using Ribo-seq from this study (in molecules/s units) and from that by Li et al. (Li et al, 2014) (in molecules/generation units). Each point corresponds to a single gene and color denotes the ratio of transcription initiation rate to translation initiation rate (giving RNAP/ribosome) capturing whether transcription (light yellow) or translation (dark blue) is more dominant.
E. HEK 293T cells were co-transfected with pCS2-Endouc and phuORFchop-luc for 24 hr and then subjected to DMSO or TH treatment for 2 hr. The lysates of transfected cells were then applied to PPA. Total RNAs were isolated from heavy polysome fractions followed by performing 5`RACE to characterize the nucleotide sequences of truncated (E) endogenous huORFchop-CHOP transcripts. The first sequence at N-terminus of RNA samples from the truncated forms derived from exogenous huORFchop-luc (n=14) and endogenous huORFchop-CHOP transcripts (n=11) was determined; the percentages of positive colonies containing various 5'UTR lengths (truncated forms) of luc and CHOP transcripts among total colonies examined were calculated.
(E) The Sch9-2D3E mutant did not suppress the rapamycin sensitivity of bur1-∆C cells. bur1-∆C which expresses pRS426 (mock), pRS426 BUR1, pVT102-Ura (mock), pVT102-Ura SCH9, pVT102-Ura sch9-2D3E, pVT102-Ura sch9-3A-2D3E, pVT102-Ura sch9-3D/E, or pVT102-Ura sch9-3D/E-2D3E were cultured in liquid SC-Ura medium and serial dilutions spotted onto SC-Ura plates with 0 or 8 ng/ml of rapamycin added. Plates were incubated at 24 ̊C for 3 days.
(E and F) Western blots for acetyl-histone H3 (K9) (Ace-H3-K9), histone H3 in WT (E) or MZsinhcaf -/- (F) PG, PV, EV, MV and FG follicles. α-tubulin was used as a loading control. Data information: EV, early vitellogenic stage (early stage III); FG, full-grown stage (late stage III); MV, mid vitellogenic stage (mid stage III); PG, primary growth (stage I); PV, previtellogenic stage (stage II).
Representative immunoblots (C) and quantification (D) of UCP1 protein expression in sWAT of male mice at 20wks of age (WT n=4, TG n=6, TGxKO n=4). Data are from male wild type (WT), Ucp1-TG (TG), and Ucp1-TGxGdf15-KO (TGxKO) mice.
K,L Quantified fluorescence of UbG76V-GFP normalized to mRFP in the hypodermis of animals of the indicated genotype at L4+48 hours. *P<0.001 using ANOVA with Dunnett's posthoc comparison to the wild-type control equivalent time point, with N=20 animals per genotype and time point. Data has been normalized to wild type. Error bars indicate SEM.
(G) The fold change in mean YFP fluorescence increases with increasing stress levels, shown for two biological repeats (adjacent boxplots). Each day's fold change distribution consisted of >40 manually corrected single-cell traces. On each box, the central mark indicates the median, and the bottom and top edges of the box indicate the 25th and 75th percentiles, respectively. The whiskers extend to the most extreme data points not considered outliers.
B. The EBV infection efficiency in HK1 cells transfected with the indicated siRNAs was analyzed by FACS. The cells were infected with EBV-GFP virus at 24 h after siRNA transfection. The ratio of GFP-positive cells was quantified by FACS at 24 hpi. The mean value of the GFP-positive rates in the siNC transfected HK1 cells was normalized to 1.
(G and H) ChIP-qPCR showing enrichment (percent input) of H3K9me2 (G) and H3K27me3 (H) around the SELE locus. Primer pairs targeting distinct regions are listed on the X axis. Graphs are representative of three independent experiments. Data are shown as means ± SD. *P < 0.05 compared to TNF-α (-). Statistical differences were analyzed by the Student's t test.
E Quantization of the autophagy markers LC3-I, LC3-II (pCMV vs pCMV+Leupeptin P = 0.032, TFEB vs TFEB+Leupeptin P = 0.014), and p62 (pCMV vs pCMV+Leupeptin P = 0.019, TFEB vs TFEB+Leupeptin P = 0.024), and Tau species markers Tau1, PHF1 (pCMV vs pCMV+Leupeptin P = 0.00082, TFEB vs TFEB+Leupeptin P = 0.022), and total Tau normalized for loading to y-tubulin from (D). P-values determined by t-test, n = 4. (*P < 0.05) Each bar represents average ± s.e.m.
M-P Immunofluorescence analysis on adjacent sections revealing laminin organization (green) and myosin heavy chain (MyHC) expression (red) at 4 weeks (M) and 8 weeks (N) after grafting (M, N displayed items are obtained from a multi-photo collage); DAPI staining (blue) shows centrally located nuclei in developing fibres (O) and peripheral nuclei in mature myofibres (P). Scale bar: (M, N) 200 μm, (O, P) 50 μm.
B) Illustration of H/M ratio meaning. For proteins whose degradation is retarded by proteasomal inhibitors, H-labeled copies are degraded at slower rates than M-labeled copies, resulting in H/M ratios greater than 1. For proteins whose degradation is not affected by proteasomal inhibitors, H-labeled and M-labeled proteins are degraded at similar rates, resulting in H/M ratios of ~1.
Distribution of endogenous MITOL in the mitochondria‐rich or peroxisome-rich fraction following cellular fractionation. The distribution of endogenous MITOL was examined using MITOL-3Flag knock-in HCT116 cells. Graphic data represent results of two biological replicates. In scatter plot, dots indicate individual data points. Black dots indicate the ratio of MITOL-3Flag collected in mitochondria-enriched fractions, and red dots are the ratio of MITOL-3Flag collected in the peroxisome-enriched fractions. Mean values are also shown.
I-J. The roles of ubiquitin contact residues in the activity of MvcA. I. The indicated amounts of MvcA or its mutants were incubated with 2 μg UBE2N-Ub for 5 min in 25 μl reactions. The ratio of cleavage was analyzed by SDS-PAGE and then densitometry. Each bar represents the ratio of cleavage obtained from three independent experiments. Bars and error bars: means and standard error of the mean (SEM). J. The activity of the MvcA mutant harboring three mutations. 0.05 μg, 0.25 μg and 1.25 μg of MvcA or the mutant was incubated with 2 μg UBE2N-Ub in 25 μl reactions. After 30 min incubation, the products resolved by SDS-PAGE were detected by CBB staining. Note that the mutant did not detectable cleave UBE2N-Ub even in the reaction received the highest amount of protein.
B. Measurement of the viability of normal human endothelial cells (HuAR2T), normal human astrocytes (NHA), and embryonic kidney (HEK293T) cells at the indicated clemastine concentrations and time points (n = 12). A dashed line marks the 50 % cell viability.
(E) Immunoblot showing the levels of the MAVS protein in HEK293T cells expressing Flag-MAVS with increasing amounts of HA-RNF34 or its H342A mutant. α-Tubulin was used as a loading control. Cell-based studies were performed independently at least three times with comparable results.
D) Graph depicts normalized infection percentage measurements from two experiments with cells infected as in (C) and one experiment with MOI 0.1. 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.
Quantification of ciliary length. Results are mean ± SE of longitudinal length of acetylated-tubulin (blue), a marker of primary cilia, in EGFP-INVS (green) transfected in hTERT-RPE1 cells (n=58 for EGFP vector and for EGFP-INVS WT, and n=52 for EGFP-INVS 3A mutant, respectively). Three independent experiments showed similar results. Statistical significance was determined by student's t test.
A-D. Wild type embryos injected with control MO or with cyp26c1 MO. (A) Lateral views of the embryos at 55 hours post fertilization (hpf). (B) Dorsal view and magnification on the lateral view of the embryos. Dotted line, pectoral fins; *, otic vesicle. cyp26c1 morphants show smaller fins compared to controls (n = 30 embryos).
C, Quantification of FAK protein in cytosolic and membrane fractions in degron ORP2 cells with or without IAA. Cells were starved overnight in LPDS and loaded with 50 µg/mL LDL +/- IAA for 2 h. The proportion of FAK signal in the membrane fraction (of total FAK in cytosol+membranes) is presented ± SD. For no IAA n=13 and for IAA n= 11, 2 independent experiments. Student's t-test.
(A) Schematic representation of human ribosomal precursors with rRNAs sequences displayed as grey (18S; small ribosomal subunit) or white boxes (5.8S and 28S rRNAs; large ribosomal subunit). These three rRNAs are flanked by external (5'ETS, 3'ETS) and internal transcribed spacers (ITS1, ITS2). The position of endonucleolytic cleavages is represented by vertical lines along these transcribed spacers, or corresponds to 5' and 3' ends of rRNA sequences. (B) Graphical representation of DNA probes used for Northen blot hybridization displayed along the 47S primary rRNA transcript.
Quantiﬁcation of chlorophyll content of WT and osprr73 mutants Data are presented as mean ± SD. n = 3, biological replicates, and asterisks represent significant difference among means by student's t-test with (*) P ≤0.05, (**) P ≤ 0.01, (***) P ≤ 0.001.
WT and LRRK2 KO RAW264.7 macrophages were infected with Lm WT or Lm Δhly. Growth measured for 2 h. Data show the mean ± SEM of three independent biological experiments. ns=non-significant, *p≤0.05, **p≤0.01 by One-way ANOVA followed by Sidak's multiple comparisons test.