EMBO/sd-character-level-ner huggingface dataset:
WT cells and the indicated mutants were left untreated (0 min) or were treated with cycloheximide (CHX) to block protein synthesis for 90 min and 180 min at 26°C or at non-permissive temperature (37°C) where indicated. Total cell lysates were analyzed by SDS-PAGE and Western blotting with the indicated antibodies. (A) Graphs display the FLAG-Orm2 protein levels determined by densitometric quantification of Western blots from cell lysates of WT cells and the indicated mutants 0, 90 and 180 min after the addition of CHX. Each experiment was repeated at least three times, FLAG-Orm2 levels were normalized to Pgk1 loading controls, and each time-point was related to t=0 min (set to 1). Data are presented as mean ±standard deviation.
Densities (cells/mm2) of BrdU+ cells in the OB GCL 16 months after TAM treatment. n=4, n.s. not significant. Scale bar: 50 μm.
PCA analysis plot (top) of control (black) and HMGB1-knockdown replicates (blue). Scatter plot (bottom) showing significantly up- (>0.6 log2-fold change; orange) or downregulated genes (<-0.6 log2-fold change; green) upon HMGB1 knockdown.   Bar plots showing GO terms associated with the up/downregulated genes from panel B and their enrichment P-values (-log; right).
(A) Human embryonic kidney 293A (HEK293A) cells were transfected with HA-tagged ArfGAP1 or HA-tagged RFP1 as a control for 24 hours. Cell lysates were immunoprecipitated with anti-HA beads. Immunoprecipitates (IP) or whole cell lysate (WCL) samples were probed for HA and the mTORC1 components (mTOR, Raptor and mLST8). Vinculin was probed as a control.
enzymes activity (n=9-10) of (J) HK, (K) LDH, and (L) SDH in vector or siSUNCR1 transfected C2C12 cells treated with 0 or 2 mM SUC (n=5-6).
Representative cell; top and side view. 3D reconstruction of the plasma membrane based on CellMask Green staining. Z-stack 5 µm above the coverslip with intracellular peripheral mask. Quantification of mitochondria in peripheral mask based on MitoTracker DeepRed staining.
B. Schematic representation of prazosin treatment in vivo of glioblastoma-bearing immunocompetent mice.
(j) Growth of trypanosomes in the absence (control, black lines) or presence (TbMCU-OE, red lines) of 1 μg ml−1 tetracycline (means±s.d., n=3). (k) Dead cells in representative fields of control (−Tet) and TbMCU-OE (+Tet) PCF are indicated (white arrows). Scale bar, 10 μm.
(D) Quantification of cell index measurements plotted as percentage of contraction following different concentrations of U46619. Data shown as mean ± SD from three independent experiments. P value <0.05 (Student's t test) was considered significant. The difference between wt and FLNAΔECS was significant at every concentration of U46619.
F: Control, ELDR overexpressed and ELDR-depleted Cal27 cells were probed with an antibody against ILF3 (red) and DAPI (blue). Representative confocal microscopic images show nuclear and cytoplasmic expression of ILF3. Arrows indicate cytoplasmic expression. Magnifications 60X, scale bar 20 μ.
C) Fractionation of sEVs by Sucrose Density Gradient: Ptc partially co-fractioned with Synt1A, Lbm and Hsp70. Note that while unprocessed form of co-receptor Ihog does not co-fraction with Ptc, the processed form does in the same subpopulation.
E) Frequency of new firing origins (relative to total structures counted) from Mybl2+/+ and Mybl2Δ/Δ ESCs treated or not with Mirin. At least 450 replication structures were counted per treatment from 3 independent repeats. Error bars represent SEM. Statistical analysis using two- tailed unpaired t-test.
(f) Western blot analysis of VAMP2, actin and LC3-II in HeLa cells stably expressing VAMP2-HA wild type or VAMP2-HA mutated in the CALM-binding site at different levels (wild-type clone 6, wt Cl6: low level; wild-type clone 11, wt Cl11: high level; mutant clone 11, AA Cl11: low level; mutant clone 12, AA Cl12: high level). The cells were treated with Baf A1 as indicated. (SE, short exposure; LE, longer exposure). Quantification of LC3-II/actin ratio is shown. Data are representative of three independent experiments and shown as mean ±s.d. (*P0.05; two-tailed t-test).
Schematic showing replacement of wild-type SecA in a translocation intermediate by Walker B (WB) SecA mutants (green).
Immunoblot in lysates and media collected from HEK293DAX cells following 16 h of activation of XBP1s [by 1 g/mL dox] and/or ATF6 [by 10 M TMP], as indicated (Shoulders et al, 2013). Immunoblots of the ATF6 target protein BiP (reactive with the KDEL antibody) and the XBP1s target Sec24D confirm the small molecule activation of these transcription factors.
(F) Cell fractionation of the two indicated strains (seperated by the horizontal broken line). Postnuclear supernatants (PNS) were subjected to differential centrifugation resulting in a 30,000 g organelle pellet (P) and supernatant fraction (S).
D) Schematic of Miro1 truncation mutants used in E-G. TM denotes the transmembrane domain.
(J) LC3-LC3 and mCherry-Parkin stably expressing TBC1D15−/− cells in the presence of HA-tagged TBC1D15 WT or F280A mutant were treated with valinomycin for 3 hr. Cells were subjected to immunofluorescence microscopy with anti-HA antibody. Scale bars, 10 μm. (K) The YFP-LC3 morphology of cells in (J) was quantified. The error bars represent ±SD from three independent replicates. Over 50 cells were counted in each well.
(e) H2O2 production with 0.5 mM NADH, complex I-linked substrate, was determined in the absence or presence of 2 μM complex I inhibitor rotenone using Amplex Red (n=5-8 per group).
(E) The robust metabolic transformation algorithm (rMTA, Valcárcel et al. 2019) was used to predict metabolic reactions whose knock-out can further transform the metabolic state of the remdesivir-treated SARS-CoV-2-infected cells back to the normal control state, using the Vero E6 cell samples (Methods). The significant metabolic pathways (FDR<0.1) enriched by the top 10% MTA-predicted targets are shown. Axes and the meanings of dot color and size are similar to (D).
(P) Proposed model of the mechanism by which Wip1 controls the transcriptional activity and stability of Smad4. See the discussion for details.
(G) Knockdown of IP3R1 prevents calcium mobilization from the ER to the cytosol. The measurements were similarly performed as in (E), and were from the same batch of cells. Data information: N = 20 cells for one replicate. Data are mean ± SEM (n = 3 technical replicates). ns, no significant difference; ***P < 0.001; two-tailed Student's t-test.
B Ribbon view of the asymmetric crystallographic unit containing a (yNap1c2-H2AΔ14-H2BΔ28)6 assembly. For clarity only layer 1 of the complex is shown. Layer 1 contains three protomers A-C that are related by two perpendicular non-crystallographic dyads ().C Side-view of the complex. The layer 2 omitted from panel B is shown in grey. The yNap1 dimer is shown in green and yellow, H2A in red and H2B in blue. See also Fig EV1.D View of the yNap1-H2A-H2B protomer complex with labeling of secondary structure elements.
(c) Midguts expressing mCherry-Atg8a in all cells, and with Atg7d4 loss-of-function clone cells (lacking GFP) at 2 h after puparium formation. Representative images are shown.
(D) Probability of HP2 closure calculated by integrating the densities (Mean ± SD from 1000 bootstrap samples). Statistical significance was tested by comparing bootstrapped confidence intervals (*, 95% confidence intervals do not overlap).
Relative gene expression of selected DEGs measured by qRT-PCR in Rv compared with Rv treated with CHP (g) and Rv as compared with Rv∆aosR under oxidative conditions (h). Data were normalized with respect to 16s rRNA and results are expressed as mean log2 fold change + SD of three independent replicates and is representative of one of two biological replicates.
(F) Graph reporting the volume of blood loss (µL) over 30 minutes following the tail clip. WT: wild-type littermates of F9-/- mice. The grey area represents the range of blood loss of F9-/- mice that received AAV-vectors expressing human FIX. Data are presented as mean±SD (n=3-4/group), and were analyzed in a 1-way ANOVA with Tukey's correction for multiple comparisons. **p<0.01 Boxes represent 25%-75% quartiles, with the line indicating the median. Whiskers span min to max.
E. HeLa cells expressing either lncRNA-MIF or control shRNA were transfected with miR-586 inhibitor or NC inhibitor as indicated. Twenty-four hours after transfection, levels of lactate in the culture medium were measured and normalized to cell number. Data shown are mean ± SD (n = 3; *P < 0.05, two-tailed t-test).
Immunofluorescence staining against Galectin3-GFP in BV2 microglial cells treated with or without SM (B) Scale bar, 10 μm.
m6A-dependent splicing of FAM214A intron 6. HeLa cells were transfected with control (siControl), WTAP (siWTAP) or YTHDC1 (siYTHDC1) siRNA and cultured for 48 h. PCR primers are the same as in A. Splicing efficiency was calculated using the method described in C and normalized to the control (siControl). Data are shown as the mean ± SD (n=3); p-values (Dunnett's multiple comparison test) are shown above.
F No polarization of DHHC2 in the dhhc2::6HA gametocytes with only either XA or 22°C stimulation. Three replicates performed, values are means ± SD.
(A) Effect of beclin 1 knockdown on EGFP-LC3 puncta formation in spns1-depleted zebrafish embryos. Injection of control (water) injection, spns1 MO (4 ng/embryo) or coinjection of spns1 MO (4 ng/embryo) and beclin 1 MO (12 ng/embryo) into Tg(CMV:EGFP-LC3) fish was performed to assess whether the beclin 1 knockdown reduces or eliminates aggregated LC3 puncta induced by Spns1 depletion at 84 hpf. Scale bar, 10 µm. Quantification of data presented in panel A (n = 12) is shown in the right graph; the number (n) of animals is for each morphant or water-injected control. Three independent areas (periderm or basal epidermal cells above the eye) were selected from individual animals.
B. Stacked bar plot of patients number diagnosed as various breast cancer subtype in TCGA group by the expression level of CDH1 or MMP1 or hTrmt13 (low as the first quartile, high as the fourth quartile).
C Immunofluorescence (IF) of PC3LN4 cells under normal culture condition with anti-phopsho-EDC3, anti-EDC3 Abs, and DAPI. Images captured under 40x magnification; scale bar: 10µm.
A Representative western blot analysis of pp53 in WT (n=6), DM (n=6) and PINK1-/-DM mice (n=3). GAPDH served as the loading control (DM ; **p=0.006 vs. WT group, PINK1-/-DM; **p=0.004 vs. DM group).
A - Relative Irs2 mRNA levels in HEK cells upon transfection with Gfp- or Irs2-encoding plasmids. Mean ± SEM; t-test, ****P < 0.0001. N = 6 replicates per group.
(H, I) Induction of Ctcflos and Ucp1 in vivo during cold-induced iWAT browning. Relative expression levels of (H) Ctcflos transcript 1 and (I) Ucp1 in iWAT of C57BL/6J mice held at 5°C for one week compared to age and weight matched mice held at 30°C or 23°C, assessed by quantitative PCR (qPCR). Mean and individual values, n=7 (biological replicates), one-way ANOVA (Šídák-test), **p< 0.01, ****p<0.0001.
A, PTPN3, PTPN3 (D811A) and PTPN3 (L232R) are similarly expressed in Huh7 stable cell lines. Expression levels of indicated proteins were detected with appropriate antibodies in Western blotting.
H. Proliferation curves of BJ-T cells transduced with a non-targeting or a PNRC1-specific shRNA construct. Results are shown as the average ± SD of 3 biological replicates with 3 technical replicates each. An unpaired t-test was performed for each time point (** p<0.01, *** p<0.005, **** p<0.001).
G) HEK293T cells were transfected with control (ev) or TMED2 plasmid and then used in the ERMIT assay with AGR2 WT (n=3).
The expression of FKBP5 and UBE2C determined by RT-qPCR analysis and normalized to GAPDH mRNA levels was examined in 22Rv1 cells stably expressing shRNA for each of the seven genes. Cells were cultured in 2% CSS. Data represent two independent experiments performed in duplicate or triplicate, showing the mean ± s.e.m., and normalized to their shGFP controls. Kruskal-Wallis test (p value < 0.0001, two-tailed) and Dunn's multiple comparisons test were performed. * Significant at a p value < 0.05, ** p value < 0.001.
(a) 2,3,5-Triphenyltetrazolium chloride (TTC)-stained brain sections from WT and HDC−/− mice showing the infarct area in those receiving saline, the H3R antagonist thioperamide (THIO), the H1R antagonist pyrilamine (PYRI; 10 mg kg−1, i.p., at reperfusion), the H2R antagonist cimetidine (CIME; 10 mg kg−1, i.p., at reperfusion) and α-FMH (50 mg kg−1, i.p., 2 h before ischemia) after 24 h reperfusion (n=6 per condition; scale bar, 5 mm).
(d-e) Average levels of (d) mRNA quantified by RNAseq (red) or qRT-PCR (grey) or (e) protein quantified by mass spectrometry (light green) or western blot (grey) for select p53 target genes under oscillatory p53. Data information: , n=2 biological replicates, error bars represent std dev.
(B) Tracking analysis of central MTOCs. In the left graph, the positions of central MTOCs along the spindle axis are shown over time. MTOC positions were used to calculate distance to the spindle equator. In the right graph, lines showing temporal changes in MTOC-equator distance are aligned based on the time when the MTOC reached a position closest to the spindle equator. Horizontal lines at 7 and at −7 μm denote the thresholds used for the definition of the middle and polar regions of the spindle.
A RT-qPCR analysis showing that BIC1 upregulates the transcriptional expression of PIF4. The Col-0, 35S:BIC1-YFP, 35S:BIC1-Flag and bic1bic2 plants were grown for 5 days under long-day conditions. The PP2A gene served as internal control. Data are means ± SD (n=3). Different letters indicate significant differences (Fisher's LSD, P< 0.05).
; representative FACS histograms and bar graphs show relative levels of pRb (Ser807/811) (I) Data shown in bar graphs are mean fluorescence intensity of indicated protein expression in PKCδ cKO HSPCs subsets relative to WT controls (n=4-6 mice per genotype per target). Data information: The statistical significance of difference was assessed using Two-tailed Student's unpaired t-test analysis for comparison of control and PKCδ cKO HSPCs. All data are presented as mean± SEM., *p<0.05, **p<0.001.
Representative images of P6 SCG neurons treated with IFNα (600 IU/ml) and stained for STAT1 at 18 hours and 8 days post-treatment. Scale bar, 100μm. Representative images of P6 SCG neurons treated with IFNα (600 IU/ml) and stained for Mx1 at 18 hours and 6 days post-treatment. Scale bar, 20μm.
(A) Plot from flow cytometry analysis of neutrophils in forepaw skin from 10-wk-old WT and RDEB mice. (B) Bar graph of flow cytometry analyses as in A of neutrophils in forepaw skin of newborn, 4-wk-old and 10-wk-old WT and RDEB mice. Plotted as fold increase compared to mean of age-matched WT paws; replicates per age = 5-6. ( Data information: Individual data points, mean ± S.E.M are shown. The data were analyzed by one-way ANOVA with Tukey's correction P values < 0.05 are considered significant.
C. Immunofluorescence of ULK1 and ATG13 puncta in wt and KO-OFD1 cells cultured in HBSS (90min). Green, ATG13 and ULK1; blue, Hoechst for nuclei. Scale bar=10μm. (Right) Graphs display quantification of puncta/cell, data are expressed as mean ± SEM; n=5 independent experiments, n≥200 cells/antibody.
H Proliferation of melanoma cells treated with LDHAi, m-TORC1 inhibitor (Rapamycin, 100 nM), or a combination of the two. Data information: Statistical analysis was performed by ordinary one-way Anova for the comparison of more than two groups. Unpaired t-test was used for the comparison of two groups, and two-way Anova was used for the proliferation. Data are shown as the mean ± SD, n = 3. ns: not significant, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001
(b) Binding between eIF4G and the Rag GTPase complex is mainly mediated by RagC, detected by expressing and immunoprecipitating either RagA or RagC alone. Representative of two biological replicates.
(A) Cartoon shows strategy for investigating conservation of uORF regulatory activity
Nitrocefin (2 mM) was added to the supernatant from 25°C overnight Yersinia cultures expressing the indicated CNFY derivatives to determine β-lactamase activity by measuring changes in absorbance at 390 nm (yellow) and 486 nm (red). The data represent the mean ± SD of three independent experiments, carried out in triplicates.
Macrophages transduced with non-specific scrambled shRNA (shNS) or CEBPB shRNA (shCEBPB) were treated for 24 h with CL097, ssRNA40, or ssRNA41. (A) Cells were lysed and immunoblot performed with antibodies to CEBPB and β-actin.
(A) Amino acid sequence of mouse Phf5a protein. The 12 Cys residues indicated in red are part of the PHD finger-like domain. Each CxxC motif is underlined and color coded according to their pairing with another CxxC motif to form a zinc finger (ZF). A pair of CxxC motifs hold the zinc atom in each ZF. Arrangement of 4 Cys residues in ZF1-3 are schematically shown below the Phf5a sequence.
(B) Immuno-EM verified that GFP-Lam6 overexpression indeed causes an expansion of contact site size. While vCLAMP was hardly visible in WT cells, it could be detected easily in the OE strain (albeit often had ER tubules invading it). The NVJ underwent expansion as well as evoked PMN in OE strains, and the ER-mitochondria contact became large and elongated instead of small and distinct. N, nucleus; M, mitochondria; V, vacuole. Scale bar represents 200 nm (see also Figure S3I).
(E) Increased DNA damage signaling upon CCT depletion and A3A expression. Depletion of CCT7 in K562-A3A cells was achieved by siRNA targeting. Expression of A3A was induced by dox treatment. Intracellular staining with anti-γH2AX antibody was analyzed by flow cytometry. Bar chart shows quantification of FACS results obtained over three biological replicates. Statistical analysis was performed using a two-tailed t-test, n=3; error bars, SEM. Immunoblot analysis of cell lysates showed decreased expression of CCT7 after siRNA targeting and A3A expression upon dox treatment. Ku-86 is a loading control. **p-value <0.01, ***p-value <0.01, ****p-value <0.0001, n.s. non-significant.
(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.
(E) Impact of BafA1 on LC3 lipidation. MEFs with the indicated genotypes were cultured in complete medium supplemented with BafA1 for 12 h and the proportion of LC3‐I/LC3‐II was determined by immunoblotting. GAPDH levels were monitored to ensure equal loading.
DICER-KO B cells were co-cultured for 24 hours with OTII-derived T cells in the presence or absence of OVA. Flow cytometry analysis of B lymphocytes co-cultured with OT-II isolated T cells in the presence or absence of OVA apoptosis (cleaved Caspase-3 expression) 24h, 48h and 72h after IS formation.
(a) Cytosolic ATP levels in WT and p53−/− HCT116 cells in the absence of glucose. Measurements were performed on luciferase-expressing HCT116, after perfusion of the cells with medium first containing then missing glucose.
(G) Total RNA isolated from the brain of mock and CHIKV infected (MOI 1x107 PFU/mouse, 6 dpi) irgm1+/+ and irgm1-/-mice and was subjected to qRT PCR for quantitation of viral load The total RNA used for qRT PCR with the brain is four times more than muscles.
HeLa cells with mock treatment (Mock) or depleted of Stx17 (Stx17 KD) were fixed and double immunostained for PGAM5 and Tom20. Scale bar, 5 μm.
Schematic model of the molecular events triggered by PNRC1 inside nucleoli. PNRC1 functionally interacts with DCP1α/DCP2 decapping complex acting as a scaffold to tether the decapping machinery inside nucleoli and promoting the m3G-cap hydrolysis from U3 and U8 snoRNAs, ultimately leading to rRNA processing block.
(E and F) Percentages of CD11b+ and B220+ in PB (E), and c-Kit positivity in myeloid fraction in PB (F left) and BM (F right) of p53 KO and lethargic/dead DKO mice (n = 8-9 mice per group).
(D) ELISA of IL-1β in the supernatants of two clones of NLRP3 KO THP-1 cells with or without ZIKV infection (MOI=1) at the indicated time points.
SCE events per metaphase on UQ.CreERT2/Nsmce2lox/lox MEFs grown in the presence of 4‐OHT for 72 h. ***P 0.001.
(d,e) USP33 interacts with both RALA and RALB. At 48 h post-transfection with tagged USP33 and RAL expression constructs, the indicated proteins were then immunoprecipitated with anti-Flag (M2) or anti-HA-agarose, followed by immunoblotting using anti-Flag or anti-HA antibodies. WCL, whole-cell lysate; IP, immunoprecipitates.
(A, B) Murine cardiac endothelial cells (MCEC) were treated with 2 mM D-β-hydroxybutyrate (β-OHB) or 2 mM acetoacetate (AcAc) for 24 hours. Quantification of concentrations of TCA cycle intermediates compared to control treatment. Data are presented as mean ± SD. n≥4; ns, not significant; *, p<0.05; **, p<0.01; ***, p<0.001 unpaired Student's t-test.
(E) CHIP analysis for hFOXM1,mFoxm1 and hFOXM1▲exon9 binding to the region of the Lin28a promoter in the N2A cells. (n=3 biological repeats).
d, FRAP experiment of SSTR3-GFP in RPE1 WT and SEPT2 KO cells. Cells were starved for 48h before the beginning of inspection and imaged every 30sec for 10mins after bleaching. Scale bar: 5µm. e, Percentage of normalised SSTR3-GFP signal recovery at whole cilia after photobleching from (d). n=20 cilia are quantified from three biological replicates Data include mean ±s.d. and P values are calculated by two-tailed unpaired student t-test.
A. Western blot of LC3B in lymphoblasts of OFD type I patients and controls, either untreated (-) or exposed to Baf-A1 (10nM, 2h)(+). Cells were cultured in complete medium (FM, top) or under serum starvation for 4h (STV, bottom). ACTIN and GAPDH were used as loading controls.
A Effect of mycolactone on MM.1S, JIM3 and KMS-11 cell viability after 48 and 72 h of treatment, as measured by exposure of Annexin V and incorporation of PI. Data are Mean % of live, apoptotic and dead cells from technical duplicates , relative to total cells.
(C) Quantification of the average number of ATMpS1981 foci per cell detected as in (B) in wild-type and SMCHD1 KO γ-irradiated HeLa cells. Data represent mean of 3 independent experiments ± SD (>200 cells/condition/experiment) with individual average values indicated.
(G) Distribution profile of Rab7 in WT and MSD lysosomal membranes. Values represent the mean±s.e.m. values of three independent experiments (B, C, E). *P0.05, Student's t‐test: (B): WT versus MSD, WT versus MPS‐IIIA, WT versus WT+cholesterol, MSD versus MSD+MβCD, and MPS‐IIIA versus MβCD for each analysed SNARE; (C, E): WT versus MSD, WT versus WT+cholesterol and MSD versus MSD+MβCD for each analysed protein. Scale bar: 0.3 μm (D).
CFP-FLIM of seeded HEK sensor cells expressing only CFP-TauRDP301L. The dense packing of CFP-TauRD in Tau accumulations induces fluorophore quenching that can be detected as decrease in CFP-lifetime. Fit-free analysis of lifetime phasor plots enables the assignment of distinct lifetime clusters (circles in phasor plot) to different Tau accumulation types. LT-2=NE Tau, LT-3=CYT and NUC Tau, and LT-1=soluble CFP-TauRD in the cytosol of cells without accumulations. Graphs show lifetimes of CFP-lifetime components assigned to soluble, NE, and CYT+NUC Tau in 6 different images (left graph, RM one-way ANOVA with Tukey post-test) and lifetime reduction relative to LT-1 in each respective image (right graph, one-way ANOVA with Tukey post-test.). N=2 experiments, 2-3 cells measured per experiments. Data shown as mean±SD. Scale bars=10 μm. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.
A. Western blot analysis of phosphorylation levels of tau and expression levels of indicated proteins in the cortical and hippocampal regions of the brain in APP/PS1mice after administration of Nec-1 (6.25 mg/kg) or vehicle (2.5% DMSO in PBS). The membranes analysed in (A) are identical to those in Fig 5F.
Action of EV20/MMAF on mitotic spindle formation. BT474 and BTRH cells were treated (10 nM, 48 hours), fixed, stained for β-tubulin (green) and DAPI (blue). Scale bar: 7.5 μm.