EMBO/sd-character-level-ner huggingface dataset:
(D) PARP2 and DDL interaction in vitro. GST-PARP2 protein immobilized on glutathione beads was used to pull-downHIS-SUMO-DDL-HA proteins (left panels). A PARylation reaction was performed prior to the pull-down assay (the third lane of left panels). The PARylated PARP2 and DDL were detected with α-PAR antibody (right panels).
(b) DamID domainograms after activation of ADAM22, ABCB1, or the genes ABCB4, ABCB1, ADAM22 and RUNC3B simultaneously. All data are from human RPE-1 cells. DamID data of activation of ADAM22 and ABCB1 alone are same as in Figure 4a, b.
C) Wnt activation in Apc5 homozygous mutant organoids drives a drastic shift in organoid morphology from budding structures to cystic spheres and increases the YAP protein level. Scale bar is 250µm. n > 10 organoids in each experiment. Quantification of YAP protein levels from crypt and villus regions of control organoids as well as from the regions bounded by rectangles in Apc5 organoids are shown on the right, with statistical significance in an unpaired 2-tailed t-test.
(A) Normal basal excitatory synaptic transmission at SC-CA1 synapses of PtenΔC/ΔC mice (4-5 weeks), indicated by the input-output curve of the amplitudes of field excitatory postsynaptic potentials (fEPSPs) plotted against stimulus intensities. (n = 10 slices from 4 mice for WT and 9 (4) for ΔC, genotype P=0.8378, two-way repeated measures ANOVA with Bonferroni's multiple comparison test). The error bars represent Standard error of the mean SEM (see Appendix table S1 "Statistics" for full details.
Representative topographic images of stiffness map of H1299control and H1299RASSF1A lung primary tumours provided by AFM.
C Expression of EGFP in primary rat neurons transfected with (CGG)100 (+1) EGFP and either anti-DDX3X LNAs or a control (one-way ANOVA with Tukey's multiple comparisons test; n=2408-5689 cells/condition). All graphs depict pooled data, normalized first within the replicate. Data Information: For all panels, **** P≤0.0001 for the specified statistical test (compiled from ≥3 replicates).
(A, B) Schematic representation (A) of the stalling situation on poly(A) tract mRNA. Cryo-EM density map of the poly(A) stalled 80S RNC filtered according to local resolution and used to build the molecular model (B).
(A) The expression of BCL9L or β-catenin in stiff or soft 4T1, MCF-7, B16 or MP-1 cells was determined by western blot. (B) The expression of β-catenin and BCL9L from SGGFP, BCL9L-SGs-4T1, MCF-7, B16 or MP-1 cells were determined by western blot. Data information n = 3 independent experiments in (A and B).
B - D In CDKL5 KOs (CDKL5 -/Y), comet distance (B) and lifetime (C) is increased when compared to WT (CDKL5 +/Y). Comet velocity (D) is unchanged. Student's t-test: n = 16/615 and 15/381 neurons/comets in WT and KO, respectively
Change in CD8 CD28+ T cells from baseline to post-therapy in G1 patients (left) or in G2 patients (right). Statistical comparisons were carried out with paired Student´s t test.
H1C-SL-Rb or H1C-SLD-Rb construct, together with VSV M were co-transfected into HeLa cells, followed by IPs with IgG or the ALYREF antibody at 24 hr post-transfection. The immunoprecipitates were subjected to western analysis to detect ALYREF (D)
C. Statistical analysis of serum titers of LCMV-specific IgM and IgG of WT and KO mice before (D0) and 14 days after (D14) LCMV Armstrong infection. Results represent the data from two experiments and numbers represent biological replicates for each group (WT; n = 7, KO; n = 8).
(e) Representative images of mitochondrial fusion over time (indicated in min). MEFs were co-transfected with mtPAGFP and mtRFP and after 24 h, mtPAGFP was photoactivated in a region of interest and cells were imaged by real-time confocal microscopy. Where indicated, MEFs were starved for 2.5 h. Scale bar, 20 μm. See also Supplementary Movies S1 and S2. (f,g) Quantification of mitochondrial fusion in MEFs of the indicated genotype. Experiments were carried out as in e. Data represent mean ± s.e.m. of four independent experiments.
(f) HA-Beclin-1 WT or S14D was transiently expressed in FIP200−/− MEFs grown under nutrient-rich conditions. Indirect immunofluorescence was performed using antibodies against endogenous LC3B and HA-Beclin-1. Scale bars, 20 μm. (g) Quantification of LC3B puncta from confocal in f. In the HA-Beclin-1- or HA-Beclin-1-S14D-transfected samples, only the HA-positive cells were counted for LC3B puncta. Error bars were processed as in e. Mean value shown; P values determined by Student's t-test using 10 unique fields of view from f. Uncropped images of blots are shown in Supplementary Fig. S4.
Total red blood cell count (RBC), platelet count, hemoglobin (HGB) content and hematocrit (HCT) were determined by ADVIA as indicated in the blood of drug-treated GFP-Chimeras or control mice (n=8 each group) at the termination of the experiment Data are presented as mean ±SEM. Each data point represents one individual mouse. Statistical significance was assessed using one-way ANOVA analysis with Tukey's multiple comparisons test comparing untreated and drug-treated mice within each group (*p<0.05, **p<0.01, ***p<0.001, ****p>0.0001).
(B) qRT-PCR analysis with total RNA isolated from HT-29 cells transfected with control siRNA or IRGM siRNA or doubly transfected with IRGM siRNA and cGAS siRNA or IRGM siRNA and RIG-I siRNA or transfected with three siRNAs as indicated.
I, 2D BNGE/SDS electrophoresis: 1st dimension with digitonin (Dig) and 2nd dimension with SDS, followed by immunoblotting with the indicated antibodies to identify the proteins detected by the commercial anti-SCAF1 antibody. Asterisks indicate missing bands in scaf1Δ1/Δ1.
(A) Unlabeled or Cy5-labeled dsRNA corresponding to the first 200 nucleotides of either the Renilla luciferase (dsRNA-RL) or the GFP (dsRNA-GFP) coding sequences visualised on gel stained with ethidium bromide (EtBr-stained, top) or by in-gel fluorescence (bottom).
C Schematic representation of SLAM-Seq workflow to infer RNA dynamics ("RNA velocity"). In short, organoids were passaged and assigned to different medium conditions. After three days, nascent RNA was metabolically labelled for 2 h using 4sU. Organoids were harvested, dissociated, and fixed. RNA in single cells was alkylated, and cells were subjected to single cell sequencing. Reads were assigned to nascent or old RNA status, depending on diagnostic T-C mutational status.
J Confocal microscopy images of HeLa cells transiently expressing the indicated GFP-CbpCT4 mutants. Scale bars, 10 µm.
Quantitative real-time PCR (RT-qPCR analysis of the relative mRNA abundances of Glut1, Glut3, Hk2 and Gapdh in WT and Pink1 KO neurons (c). ß-Actin mRNA was used for normalization, and the Pink1 KO data are expressed as the change relative to WT.
Brcc3+/+ and Brcc3−/− BMDMs were left untreated (Con), treated with LPS alone (LPS) or pretreated with LPS and then stimulated with ATP, nigericin, MSU, Alum, poly (dA:dT) or flagellin. CBA analysis of IL-1β secretion in the culture supernatants.
(F) Increased binding of β-catenin in the promoter region of the Slc6a20a gene in PtenΔC/ΔC mice (P21), revealed by ChIP (chromatin immunoprecipitation) assay. Note that β-catenin binding was increased selectively in the target region for the primer set #4 indicated in the diagram. TSS, transcriptional start site; P1-4; promoter regions targeted by the four primer sets. (n = 7 mice for WT-P4 and ΔC-P4 and 3 mice for WT-P1/2/3 and ΔC-P1/2/3, *P < 0.05, ns, not significant, Student's t-test). The error bars represent SEM.
(C) Western blot analyses of the degradation of nuclear p65 protein after the knockdown of CDC20. The cytoplasmic and nuclear protein of NC and CDC20si HEK293T cells were extracted after TNF-α treatment for 30 minutes.
(A) Single confocal sections of HeLa cells at metaphase treated for 8 min at 4°C and immunostained to reveal the presence of unattached kinetochores. CENPT labels all KTs, while MAD1 identifies unattached ones. Enlargements of boxed areas displaying both chromosomes aligned and not aligned at the metaphase plate. (B) Quantification of the number of KTs based on attachment, position and MAD1 positivity. Each point per category represents one experiment in which 10 random KTs/cell of 20 cells/sample were counted. P values are obtained by 2-way ANOVA with Dunnett's multiple comparison test. Note that in control cells most of the KT are attached, MAD1-negative and aligned to the metaphase plate, while in SNAP29KD cells half of the KTs that are unattached, MAD1-positive and not aligned to the metaphase plate.
C. Stage II and Stage III patients from the TCGA COAD dataset were partitioned into STING-Hi (top 1/3 of samples, N=87) and STING-Low (bottom 1/3 of samples, N=87). Kaplan Meier curve is shown with P value indicated.
(L) Cell cycle time of E13.5 NPs in the lateral pallia of WT (upper column) and KO cells (lower column), as calculated by EdU cumulative labelling and PH3 staining KO NPs have a 16.1% faster cell cycle time, due to a 30.9% shorter G1-phase compensating a 14.3% longer S-phase. n ≥ 3 brains. In graphs, data are represented as means ± SEM. 2-way ANOVA (*P<0.05, **P<0.01, ***P<0.001).
C. Plasma insulin levels of control and NICDiOE-EC mice during the 2-DG uptake assay. n=5, data represent mean ± SEM, unpaired t-test.
Data derived from the Linnarsson mouse database (Linnarsson) displays the expression of Nid1 and Fbln2 within cells of the central, peripheral and enteric nervous system. Unit color scale: Expression (log(UMI)); UMI, unique molecular identifier. Abbreviations: ENS, enteric nervous system (i.e. enteric neurons and glial cells); OL, oligodendrocytes; Exc N, excitatory neurons in CNS; Hippo, hippocampus; MB, midbrain; HB, hindbrain; Str, striatum; Hyp, hypothalamus; BG, basal ganglia; OB, olfactory bulb; CB, cerebellum; SC, spinal cord; PNS, peripheral nervous system; Glia, glial cells; Vasc, vasculature.
(B) Blue native gel electrophoresis (BN-PAGE) analysis of mitochondrial respiratory chain complexes from synaptoneurosomes as compared to HEK293T mitochondria.
(b) Phase diagrams generated through droplet microfluidics for the coacervation of NSP2 and NSP5, in the presence of 0% v/v (left), 1.5% v/v (middle), and 4% v/v (right) 1,6HD. Phase diagrams were generated from N = 2206, 2035 and 1470 data points for each 1,6-hexanediol concentrations, respectively, and the data were used to construct the LLPS probability plots.
H-J H, Representative images and i, quantification of Ki67 positive cells in pancreatic tumours and in J, metastasized liver sections after treatment with oral doses of vehicle (0.5% methyl cellulose with 0.2% Tween-80) or of BYL-719 (50mg/kg).
(A) CHD1-depleted cells show hypersensitivity to Mitomycin (MMC) treatment. For colony formation assay shCont and shCHD1 PC3 cells were treated with the indicated MMC concentrations for 4 h and surviving fractions were measured by counting colonies after 3 weeks and mean values were represented (n=3, ± SD, p-value=0.0002, ANOVA, ***p ≤ 0.001).
(P.Q) TEM images of WT and KO P8 ONs showing thick astrocytic processes filled with intermediate filaments (red dotted lines and arrowheads) in KO nerves. Scale bars (500nm)
qRT-PCR was performed on total RNA from GSC#9 treated for 4 hours with vehicle (DMSO) or MPZ (20 μM). Histograms showed changes in RNA expression of indicated targets. Data were normalized to two housekeeping genes (ACTB, HPRT1) and are presented as the mean + s.e.m of technical triplicates.
(I) XO8 GSCs and DFCs were treated with 50 nM MG132 for 24 hrs. Cell viability was measured using trypan blue exclusion assay. Error bars represent the mean ± SD from three independent experiments. For statistical analysis, student t-test (two-sided, one type) were used. (*P < 0.05).
E Binding of Usp27x to BimEL can be blocked by the MEK-inhibitor UO126293FT-TetR-3xFlag-Usp27x cells (see C) were treated for 24h with dox to induce expression of Usp27x. In all conditions PMA [16.2 nM], and Q-VD-OPh [10 µM] were added at the time of Usp27x-induction. As a control, cells were pretreated with UO126 [10µM] for 30 minutes to block the PMA stimulated ERK pathway before the addition of dox plus PMA plus QVD. 3xFlag tagged Usp27x was immuno-precipitated from whole cell lysates using anti-Flag resin. Interaction with BimEL was detected by Western blotting using anti-Bim antibodies. As a control Flag-matrix (beads) was used alongside the IP under same conditions but without addition of protein lysates. Western blots are representative of n=4 independent experiments (see also Appendix Fig. S2B).
Inhibition of OST1 and SnRK2.4 by clade A PP2Cs. The data in (G) to cells expressing inactive ABI1D177A instead of ABI1.
(M,M') NR2F1 level as measured on single cells in the VZ (M) and CP (M') of sulci and gyri n ≥ 4 sections from n = 1 fetal brain. (N-O) NR2F1 (red) and TBR2 (green) immunostaining NR2F1 level as measured in single TBR2+ IPs by pixel intensity is shown in graph (O). n ≥ 4 convolutions from n = 1 fetal brain (n = 2 sections). Data information In graphs, data are represented as means ± SEM. Student t-test (M,M',O (*P<0.05, ***P<0.001).
I. Staining of MSCs, osteocytes, and adipocytes with Alizarine Red (osteocytes) or Oil Red (adipocytes).
(d) Autophagosome (denoted by arrowheads) generation on amino-acid withdrawal was analysed by electron microscopy. Cell lines and conditions from c were used and representative images from the indicated amino-acid-starved lines are shown; scale bars, 0.4 μm. (e) Quantification of d. Fold induction was determined by arbitrarily making the nutrient-rich condition 1 (solid bars) for each line. Error bars represent the s.d. of the mean value over an average of 20 fields of view within a representative experiment. NS, not significant.
t-SNE plots showing 17224 CD45- Ter119- Tie2- tdTomato+ single cells from all 5 conditions. Cells are colored by conditions (B) Top 20 PCs were chosen for the clustering.
A. Representative micrographs of tissue sections derived from oral squamous cell carcinomas (OSCC) and esophageal squamous cell carcinomas (ESCC) stained with the indicated antibodies by immunohistochemistry (IHC). Top panels show routine H&E staining. Black squares point to regions magnified in the adjacent panels to the right.
(D) Results of in vivo plasmid-to-plasmid transposition assay using WT or R848M RAG1 and the indicated forms of RAG2 containing or lacking (∆L) the four amino acids at the tip of the 333-342 loop, performed as described in (Zhang et al., 2019); (****, p<0.0001). Three biological replicates for each condition except in the last two columns where data from 11 biological replicates are presented.
I Activation of EGFR in MuSCs on EDL myofibers treated with EGF compared to untreated control. Myofibers were cultured for 48 hours and were treated with 40ng/ml of EGF for 10 minutes before fixing and staining with antibodies for PAX7 and phospho-EGFR. Scale bar=30μm.
Tumor-infiltrating immune cells from tumors were analyzed by FACS NK cells (H) Data information: data are presented by Box plots Min to Max showing all points with median corresponding to the percentages of each cell population among CD45+ cells or the indicated parent population or the number of lung nodules. All experiments are performed with n = 8 mice per group; *p < 0.05, **p < 0.01, and ***p < 0.001; Mann-Whitney test.
(F) Example traces of two single cells exposed to two different doses of galactose. (G, H) Same-cell dose response shows same burst duration (G) but lower amplitude (H) at higher galactose concentration, indicating that galactose levels regulate burst frequency (n = 13 cells). Error bars indicate SEM.
H. Control or GFP-DHHC3 expressing tsA201 cells fixed and stained for PI4KIIα or PI4KIIIβ. Bottom panels show enlarged views of DHHC3, PI4KIIα, or PI4KIIIβ at Golgi regions. I. Quantification of PI4KIIα (left; control: n=42, DHHC3: n=24), or PI4KIIIβ (right; control: n=39, DHHC3: n=26) intensity relative to cytoplasm.
(A) Experimental design for monitoring replication and cohesin in Xenopus egg extracts.
A AGO6 dysfunction reduces levels of Pol V‐dependent transcripts. RNA levels were measured by RT-qPCR. Means ± SD are shown, n = 3.
(D) Experimental scheme for EV isolation either by affinity purification technique using CD63 positive biotinylated beads or by ultracentrifugation from Huh7 cell conditioned medium (Huh7CM).
H: Flow cytometric measurements of mitochondrial ROS production at the specific ROS level in Detroit 551, WS5A and CP2A iPSC-derived NSCs calculated by total ROS (MitoSox Red)/MTG (n=4, technical replicates per clone for control and CP2A; n=3, technical replicates per clone for WS5A).
Volcano plots showing the differential expression of the genes after HNRNPC knock-down in the MCF7 (A) and T47D (B) cells. The ISGs with significant differential expression were marked as red dots. The cutoffs (dashed line) were set at the log2 fold-change > 1.5 or < -1.5 and the P-value < 0.001.
Western blot analysis of LAMP2A expression in lysosomes isolated from WT,Ctns−/− and cysteamine-treated Ctns−/− mouse livers.
F. Sparsely cultured RPE or MCF10A cells were immunoprecipitated with an anti-AMOTL2 antibody.
c-d, Comparison of CD8 Tem mitochondrial mass (c) and mitochondrial potential (d) of young and 45-wk-old Dj-1 KO and WT mice.
EGFP-FAM110A-WT, -∆Pro or -∆C proteins isolated from transfected HEK293 cells using GFP-trap were incubated with mock or with active GST-CK1δ for 20 min at 30°C. Mobility of the proteins was analyzed by immunoblotting (n=2).
(D) mH2A1.2 is expressed in neural progenitor cells and neurons cultured in vitro. Scale bar represents 50 μm. Date information: Representative images from at least three independent experiments.
D Analysis of endogenous Bak localization in cytosol (C) and heavy membrane fraction (HM) of HeLa cells expressing degradation-prone DD-FLAG-Bcl-xL. In the absence of Shield-1 DD-FLAG-Bcl-xL is readily degraded, resulting in unaltered Bcl-xL levels. Addition of 0.5 µM Shield-1 instantly stabilizes DD-FLAG-Bcl-xL resulting in elevated Bcl-xL levels that increase cytosolic levels of endogenous Bak (Fig. E3C, D). Akt1 and Tom20 serve as fractionation controls. n = 3.
B, C Pre-treatment of 100 nM Smaducin-6 or scrambled peptide (Pal-Scram #1) for 30 min inhibits (B) NF-κB-mediated luciferase gene expression when RAW264.7 cells are treated with LPS for 2 h. Luciferase activity in (B) was normalized to β-galactosidase activity.
A-E G1-arrested cell cultures were released in YEPD supplemented with CPT (50 μM). (A, D) Electron micrographs of a representative reversed fork wit a three-way replication fork exposing ssDNA at the junction (D). In (D) the black arrow indicates the ssDNA region, which is magnified in the inset. Scale bars: 200nm, 50nm in the insets
(C-D) Electron microscopy of ultrathin cryosections of HeLa cells transfected with EGFP-ORP5 or EGFP-ORP8 and immunogold stained with anti-GFP (15 nm gold) to detect ORP5 or ORP8 and anti-PDI (10 nm gold) to label the ER lumen. ORP5 and ORP8 localize at ER-PM (arrows) and ER-mitochondria contact sites (arrowheads). m, mitochondria; cER, cortical ER. Scale bar 200nm. (E-F) Quantification of the IEM labelling for EGFP-ORP5, EGFP-ORP8 (E) and GFP-Sec61β (F) in transfected HeLa cells. Results are presented as the percentage of the total number of gold particles (800 per condition, n=35-45 cells) for ORP5, ORP8 and Sec61β in the indicated compartments (non-cER, non-cortical ER; ER-PM, ER-PM contact sites; ER-mito, ER-mitochondria contact sites. % gold particles ±SEM. Data are the mean of three independent replicates. *P<0,001 compared to non-cER and ER-mito [EGFP-ORP5] and to ER-PM and ER-mito [EGFP-ORP8 and GFP-Sec61β], #P<0,001 compared to ER-PM and ER-mito [GFP-Sec61β]).
HeLa cells were transfected with each siRNA for 24 hr, and then treated with Tm for 5 hr to induce the UPR. (A, B) Representative confocal micrographs of control (left-hand panels), Yip1A-knockdown (middle panels), and IRE1-knockdown (right-had panels) cells after 0 hr or 5 hr of Tm treatment (A). The ER structure was visualized with an anti-calnexin antibody. Large vacuoles were observed in control cells (arrowheads), but not in Yip1A- or IRE1-knockdown cells. Scale bars are 10 μm. The percentage of cells with vacuoles was counted, and is shown in the bar graph (B). Data are means ± SD from three independent experiments (N = 100). **: p<0.01.
E Representative Western blot showing mitochondrial contents (VDAC, Tom20, and aconitase) in the total lysates of PC12 cells with Q23 or Q74. Mitochondrial protein levels were normalized to beta-actin. n = 3. *P = 0.024; **P = 0.0008; ***P = 0.03.
Confirmation of Bio-TFL457 delivery to mice cortex. Biotinylated TFL457 (Bio- TFL457, 4 nMole/g, retro-orbitally injected) was detected in coronal sections stained with NeuN and DAPI. Representative confocal microscopy images of cortical areas correspond to single sections. Scale bar, 10 µm.
The indicated derivatives of MDA-MB-231 cells were photographed, either directly or following immunofluorescence analysis using the indicated antibodies. Nuclei were stained with DAPI (scale bars, 20 μm and 10 μm, left and right columns, respectively).
(E-F) Immunoblots (E) and related quantitative data (F, n=3) showing NFATc1 protein levels in HEK293T cells overexpressing wildtype HA-SIRT7 or a SIRT7 enzyme-dead mutant (H187Y).
B Simplified photomicrographs of proximity ligation staining in rat cardiomyocytes transfected with Nox4 siRNA or a scrambled control (siScr), with quantification as in (A). Proximity was tested for the FACL4/Nox4 protein couple. Scale bars: 10 µm. n=3/group (with >30 cells/individual experiment).
A. Representative spinning disk confocal live-cell time series of MT-flux in U2OS cells stably co-expressing PA-GFP-α-tubulin (cyan) and mCherry-α-tubulin (red) treated with indicated conditions. White arrowheads follow the poleward motion of the photoactivated regions due to MT-flux. Scale bars, 10 µm. Time, min:sec.
D) Immunoprecipitation and Western blot were performed in HFFs with and without IFNγ (10 U/ml) using an RH strain expressing type II GRA15-HA and as a control RH expressing GRA43-HA. The blots using antibodies against TRAF6 were made after stripping the first blot that is used for HA blotting. The inputs loaded represent 10% of total lysate prepared for immunoblotting. Following TRAF6 blotting, the membrane was stripped and blotted for TNFAIP3.
(B) A similar analysis was performed for indicated markers in kidney protein extracts as described in liver.
Image series from live cell time lapse analysis of CLC2-GFP at the cell plate in roots of wild type seedlings costained with FM4-64, as indicated. Arrowheads, appearance of CLC2-GFP at the cell plate. Times are given relative to the instance when the cell plate contacted the peripheral plasma membrane, defined as t0. Scale bars, 10 μm. Images are from median focal planes chosen from time lapse series recorded as 3D stacks.
Fluorescence intensity of NE and CYT Tau compared to soluble Tau in the cytosol of cells without accumulations. N=3 experiments, and 5-6 images per experiment. Data shown as mean±SD. One-way ANOVA with Tukey test for multiple comparison. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.
A-B, Single-molecule analysis of DNA replication by DNA combing of siRNA transfected cells pulse-labelled with IdU (10 min) and CIdU (20 min). (A) Distribution of inter-track distances. Bars represent the median. n > 100. Mann-Whitney: *** P < 10-3. One representative experiment out of two biological replicas is shown. (B) Size distribution of CldU track length. Bars represent the median. n > 300. Mann-Whitney: *** P < 10-3. One representative experiment out of two biological replicas is shown. C, Distribution of intertrack distances measured by IdU/CIdU pulse labelling and DNA fiber analysis. Bars represent the median. n > 110. Mann-Whitney: **** P < 10-4. One representative experiment out of two biological replicas is shown.
(C-D) Isolated naïve B cells from CRISPR-targeted chimeras after 3-day culture in stated cytokines. (C) mCherry percentage in targeted primary cell cultures. N = 15 mice. *p<0.05, **p<0.01, ****p<0.0001 using two-way ANOVA. (D) AnnexinV surface stain in targeted cells. N = 3-4 mice. Data information: All data show means ± SEM.