EMBO/sd-character-level-ner huggingface dataset:
Figure 2A. Schematic diagram of sucrose density gradient ultracentrifugation for RNC isolation, and PCR verification of the 12 screened lncRNA in both RNC-RNA and total RNA from two groups of CRC cells, among which the translation level of lnc-AP (marked with a green box) was the most notably dysregulated in both groups of drug-resistant cells (n = 3, biological replicates, mean±SEM, t test). Data information: * for P < 0.05; ** for P < 0.01; NS for no significance. B. PCR analysis of lnc-AP in serum samples from oxaliplatin-resistant (PD, n = 15) and -sensitive (PR, n = 9) CRC patients (mean±SEM, t test). C. In the public database lncCAR, high expression level of lnc-AP was correlated with long overall survival in CRC patients. D. Schematic diagram of two lnc-AP OE plasmids of sORF-Flag fusion and the corresponding initiation codon mutant. E. PCR and WB analysis of HEK293T cells transfected with either lnc-AP-Flag OE plasmid (n = 3, biological replicates, mean±SEM, Dunnett-t test). F. PCR and WB analysis of HCT116/L-OHP and SW480/L-OHP cells transfected with either lnc-AP-Flag OE plasmid (n = 3, biological replicates, mean±SEM, Dunnett-t test). Data information: * for P < 0.05; ** for P < 0.01; NS for no significance. Control referred to the corresponding plasmid vector. G IF detection of pep-AP in HCT116/L-OHP and SW480/L-OHP cells transfected with either lnc-AP-Flag OE plasmid. Scale bar, 10 µm. H. Schematic diagram of two lnc-AP OE plasmids of HA-sORF fusion and the corresponding initiation codon mutant. I. WB analysis of HCT116/L-OHP and SW480/L-OHP cells transfected with either HA-lnc-AP OE plasmid. J. IF detection of pep-AP in HCT116/L-OHP and SW480/L-OHP cells transfected with either HA-lnc-AP OE plasmid. Scale bar, 10 µm.
Figure 4Joints from WT-CHIKV, RH-CHIKV, RHEV-CHIKV, EV-CHIKV and mock-infected WT C57BL/6 mice were harvested at 6 dpi. (A) Absolute numbers of leukocytes present in the joints as determined by immune-phenotyping. Data are presented as the means ± SD (n=5 per group). No significance was obtained between the groups by Kruskal-Wallis test with Dunn's multiple comparisons. (B) Jensen-Shannon divergence heatmap showing the level of divergence between infected groups. (C) Superimposed PhenoGraphs of UMAP transformed FACs data from immune-phenotyping of joint cells from WT-CHIKV, RH-CHIKV, RHEV-CHIKV, EV-CHIKV and mock-infected animals (n=5 per group). (D) PhenoGraph cluster IDs from 1 to 33. Clusters are grouped in proximity based on surface marker phenotypic similarities and do not represent conventional cell types. (E) Cluster ID annotation with heatmap. Cluster IDs are indicated in rows, while surface marker expression levels are indicated by the columns. The color represents the relative mean fluorescence intensity (MFI) of a surface marker. Blue and red represents low and high MFI, respectively. Red square and ovals highlight differences of several PhenoGraph clusters in infected joints at 6 dpi.
Figure 1(A) Representative images of WT, Mfn1 KO and Mfn2 KO MEFs transfected for 48 h with a plasmid encoding mitochondria-targeted RFP (mt-RFP). Scale bar: 5 μm. (B) Percentage of cells displaying globular mitochondria (n=310-668 cells analyzed in 3-6 independent experiments). Data are presented as mean ± SEM. (C) Analysis of aspect ratio (AR) calculated as major axis/minor axis and (D) circularity calculated as 4*π*Area/Perimeter2 (n=300 mitochondria in 3 independent experiments). Data are presented as mean ± SEM. Data information: *p<0.05, one-way ANOVA followed by Tukey's post hoc test. (E) Mitochondrial membrane potential (MMP) was determined by measuring TMRM fluorescence (n= 90 cells analysed in 3 independent experiments). Data are presented as mean ± SEM Data information: *p<0.05, one-way ANOVA followed by Tukey's post hoc test. (F) Oxygen consumption rate (OCR) normalized to the amount of protein in WT, Mfn1 KO and Mfn2 KO fibroblasts. Proton leak was measured after application of oligomycin (1 μM), maximal after CCCP application (10 μM) and oligomycin (n= 3 independent experiments). Data are presented as mean ± SEM. Data information: *p<0.05, one-way ANOVA followed by Tukey's post hoc test. (G) Respiratory control ratio (RCR) was calculated as maximal oxygen consumption rate (OCR)/H+ leak OCR (n= 3 independent experiments). Data are presented as mean ± SEM (H) Spare respiratory capacity was calculated as maximal OCR- basal OCR (n= 3 independent experiments). Data are presented as mean ± SEM. Data information: *p<0.05, one-way ANOVA followed by Tukey's post hoc test. I) WT MEFs and Mfn1 or Mfn2 KO MEFs were incubated with 2-DG (10 mM) for 6 h, and ATP levels were measured (n= 3 independent experiments). Data are presented as mean ± SEM. Data information: *p<0.05, one-way ANOVA followed by Tukey's post hoc test.
Parkin-dependent mitochondrial vesicle biogenesis is a rapid response triggered by ROSQuantification of TMRM fluorescence by flow cytometry in U2OS:GFP-parkin cells treated with 25 µM antimycin A (anti A), 25 µM antimycin A and 10 µM oligomycin (anti A + oligo), and 20 µM CCCP (CCCP) for the indicated time, represented as a fraction of fluorescence intensity compared to DMSO-treated cells.Figure 6A Quantification of TMRM fluorescence by flow cytometry in U2OS:GFP‐parkin cells treated with 25 μM antimycin A (anti A), 25 μM antimycin A and 10 μM oligomycin (anti A + oligo), and 20 μM CCCP (CCCP) for the indicated time, represented as a fraction of fluorescence intensity compared to DMSO‐treated cells. Error bars represent the mean ± s.e.m. (n = 3 experiments). B U2OS:GFP‐parkin cells were treated with DMSO (DMSO), 25 μM antimycin A (anti A), 25 μM antimycin A and 10 μM oligomycin (anti A + oligo), or 20 μM CCCP (CCCP) for two hours, then fixed and immunostained for TOM20 (red). Scale bars, 50 μm. C Quantification ofPDHE2/E3 bp−positive/TOM20−negative structures from (B); both the total number (white bars) and the number colocalizing with GFP−parkin (gray bars) are indicated. Error bars represent the mean ± s.e.m. P−values are given first for GFP−/GFP−parkin−positive vesicles, then for total vesicle number (n = 42-60 cells in three experiments); ns, not significant; ***P < 0.001). D U2OS:GFP−parkin cells were treated with DMSO (DMSO), 25 μM antimycin A (anti A), 25 μM antimycin A and 10 μM oligomycin (anti A + oligo), or 20 μM CCCP (CCCP) for two hours, then fixed and immunostained for PDH E2/E3 bp (red) and TOM20 (blue). A merged image of both mitochondrial markers (mito) is shown at the far right. Arrows indicate PDH E2/E3 bp−positive/TOM20−negative structures colocalizing with GFP−parkin (green). Scale bars, 20 μm.
Figure 7A Evaluation of the evolutionary relationships using the Tol-MirrorTree analyses. Pairwise genetic distances (GD) among designated proteins in representative photosynthetic organisms (Dataset EV2) are indicated by green spots. R, Pearson correlation coefficient; P, significant differences between the Maximum Likelihood trees (one-sided t-test). B UV-visible absorption spectra of purified AtDJA6 and SynDJA6. Recombinant SynDJA6 after elution from Ni-NTA Sepharose beads followed by concentration is shown on the right. C Phenotypes and chlorophyll fluorescence images of 6-week-old WT and 35Spro:SynDJA6-FLAG/dja6 dja5 transgenic plants grown on soil. Scale bars = 1 cm. D Expression of FLAG-tagged SynDJA6 in 35Spro:SynDJA6-FLAG/dja6 dja5 transgenic plants. Total leaf extracts (100 μg proteins) from WT and transgenic seedlings were loaded. FLAG-tagged SynDJA6 is indicated by asterisk.
Figure 4Volcano plot of gene expression in Ronin del/del day 8 vs. Ronin del/del day 4 ESC. GO enrichment analysis of Ronin del/del day 8 vs. Ronin del/del day 4 downregulated genes. BP - biological process, CC - cellular component, MF - molecular function. Expression and binding of Ronin to genes encoding cytoplasmic ribosomal proteins. Binding of Ronin to the promoters of genes encoding cytoplasmic ribosomal proteins. GO enrichment analysis of genes that are directly bound by Ronin. BP - biological process, CC - cellular component, MF - molecular function. Analysis of nascent protein synthesis using OP-puro assay. Ronin fl/del cells treated with protein synthesis inhibitor (cycloheximide) are used as negative control. The nuclei are counterstained with DAPI.   Quantification of the OP-puro signal, normalised to DAPI. Ordinary one-way Anova data represent mean ± SD; n = three independent experiments for each genotype.
Figure 5(A) Schematic illustration of the potential mechanisms by which MIG-6 regulates GLUT1 gene expression. (B) Immunoblotting analysis for HIF1α protein expression in BT549 cells with GFP or MIG-6 knockdown.(D) Real-Time PCR analysis for HIF1α mRNA expression in BT549 cells with GFP or MIG-6 knockdown. The quantified results are presented as mean ± SD (n=4). ***p < 0.001, by Student's t-test. (E) Immunoblotting analysis for HIF1α protein expression in GFP and MIG-6 knockdown BT549 cells in the absence or presence of the proteasome inhibitor MG132. (F) GFP and MIG-6 knockdown BT549 cells transfected with the indicated plasmids were treated with MG132, subjected to immunoprecipitation (IP) with Flag-tag or HIF1α antibody, followed by immunoblotting analysis. MG132 was used to rescue HIF1α protein degradation mediated by MIG-6 knockdown, leading to similar HIF1α protein levels in control and MIG-6 knockdown BT549 cells, which were used as the input to examine the role of MIG-6 in the interaction between HIF1α and HAUSP. (G) Luciferase and MIG-6 knockdown 293 cells were transfected with the indicated plasmids and subjected to MG132 treatment. Afterward, cells were harvested for IP with HA antibody, followed by immunoblotting analysis to determine the level of K48-linked ubiquitination of HIF1α. MG132 was used to preserve the degradative K48-linked ubiquitination signals. (H) Immunoblotting analysis for MIG-6 and HIF1α expression in Luciferase and MIG-6 knockdown 293 without MG132 treatment. (I) Immunoblotting analysis for GLUT1 protein expression upon MIG-6 overexpression in GFP and HIF1α knockdown BT549 cells. (J) Immunoblotting analysis for GLUT1 protein expression upon MIG-6 overexpression in GFP and HAUSP knockdown BT549 cells. (K) Immunoblotting analysis for HIF1α protein expression upon HAUSP overexpression in GFP and MIG-6 knockdown BT549 cells.
The human kinase yeast array.Figure 1A. Schematic representation of the principle of the kinase yeast array and a flow diagram showing the construction of the human kinase array. B. The Human Kinome Tree representing 518 kinases in nine families (modified from (Manning et al, 2002) using CORAL at http://phanstiel-lab.med.unc.edu/CORAL/). Pie charts indicate the fraction of kinases contained in the human kinase yeast array. In total, 266 human protein kinases and 52% of the human kinome is represented in the array. C. Hierarchical clustering of the results of the yeast growth phenotype screen. In each strain, the 266 kinases were binned (0-4) according to the number of conditions in which the kinase caused a growth phenotype. Hierarchical clustering revealed sets of topmost (52), highly (98) and moderately active (116) kinases. Top 30 active kinases causing many growth phenotypes over all conditions-strain pairs are listed. D. Comparison of the fraction of kinases that result a growth phenotype over all conditions-strain pairs. Kinases are grouped into 179 (67%) with a yeast ortholog and 87 (33%) human protein kinases without orthologous yeast kinases. The latter group includes 41 tyrosine kinases and 46 S/T kinases which were separated in the analysis. The former group contains both one-to-many and many-to-many orthologous kinases. Tyrosine kinases strongly inhibited yeast growth and human kinases that are orthologous to yeast kinases were more likely to perturb yeast growth than human kinases without a yeast orthologData are from a single large-scale growth measurement. The boxes extend from 1st to 3rd quartile with the central band representing the median. The whiskers extend to the furthest point up to 1.5 times the interquartile range away from the nearest quartile, with any further points marked as outliers. P-values are calculated using the Mann-Whitney test.
Figure 6Schematic representation of the auditory sensory organ, illustrating the positioning of the electrode used for injectoporation of GFP-tagged clarin-2 construct into cochlear supporting and hair cells. (B-F) Top and side views of representative images of supporting cells (asterisk) and hair cells expressing clarin-2. In supporting cells, GFP-tagged clarin-2 (green) was distributed diffusely throughout the cytoplasm (A,B). By contrast, in all injectoporated hair cells, the majority of clarin-2 was observed in the apical stereocilia (C-E). Scale - 2 µm.
Adipocyte EV transfer FA to melanoma cells to fuel FAO, and this transfer is increased in obesity A) Lipids were extracted from EV secreted by 3T3-F442A pre-adipocytes and differentiated 3T3-F442A adipocytes (respectively preAd-3T3-EV and Ad-3T3-EV), and FA content was measured (n=6).Figure 3Lipids were extracted from EV secreted by 3T3-F442A pre-adipocytes and differentiated 3T3-F442A adipocytes (respectively preAd-3T3-EV and Ad-3T3-EV), and FA content was measured (n=6). Workflow of the assay used to evaluate FA transfer by 3T3-F442A adipocyte EV (3T3-EV) to melanoma cells. Mature 3T3-F442A adipocytes were loaded with BODIPY FL C16. Cells were then washed and fresh medium was added. 72h later, conditioned medium was harvested, and 3T3-F442A EV (3T3-EV) were isolated and added to melanoma cells. Indicated melanoma cells were incubated with EV from 3T3-F442A adipocytes previously loaded with BODIPY FL C16 (3T3-FL C16-EV) and, 24h later, cells were fixed and nuclei were counterstained with DAPI before observation by confocal microscopy. Left panel, indicated cells were incubated with EV from 3T3-F442A adipocytes previously loaded with BODIPY FL C16 and immediately treated, or not, with Etomoxir for 24h. Then, cells were fixed and nuclei were counterstained with DAPI before observation by confocal microscopy. Right panel, quantification of BODIPY FL C16 staining area per cell (n=3). Lipids were extracted from EV secreted by adipocytes from lean (ND) and obese (HFD) mice (n=5) (left panel) or from human adipose tissue samples from patients with varying BMI (right panel) and fatty acid content was measured (n=15). Indicated cells were exposed, or not, to adipocyte EV from primary adipocytes from lean mice fed a normal diet (ND) or obese mice fed a high fat diet (HFD) for 24h. Then, cells were fixed, stained with BODIPY and counterstained with DAPI. Left panel, confocal microscopy observation. Right panel, quantification of BODIPY staining area per cell (n=5 for SKMEL28 and n=6 for 1205Lu).
Figure 1A, Schematic of the ColE9 sequence showing its constituent domains: an intrinsically unstructured translocation domain (IUTD) at the N-terminus is followed by three structured domains involved in translocation (T), receptor (R) binding and cytotoxicity (C). The IUTD houses three linear protein-protein interaction epitopes, two OmpF binding sites (OBS1, OBS2) flank a TolB-binding epitope (TBE). Residue numbers denote position in ColE9 sequence. B, Cartoon of the ColE9 OM translocon. ColE9 (orange) exploits the vitamin B12 transporter BtuB (grey) as its extracellular receptor and the porin OmpF (green) for threading its N-terminal IUTD (solid black line) through to the periplasm where it captures TolB (blue). Star, represents the primary site on the ColE9 DNase domain (K469C) where fluorophores were covalently attached throughout this study. C, Cryo-EM map of the fully assembled ColE9 translocon, with local resolution range 4.5-16 Å. Component proteins are coloured as in panel b. The structure shows extracellular ColE9 creating a protein bridge between the two OMPs, BtuB and OmpF. The β-barrel of BtuB is tilted 35° relative to that of OmpF. TolB is located on the periplasmic side of OmpF, with no associations to BtuB. ColE9 and TolB regions of the map have weaker density than those of the β-barrels. D, Model of the intact ColE9 translocon generated after docking and rigid-body refinement of individual structures of ColE9 residues 85-580 (PDB ID 5EW5), OmpF (3K19), BtuB (PDB ID 2YSU), and TolB (PBD ID 4JML). E, Cryo-EM map of the partial ColE9 translocon with an average resolution of 3.7 Å, which has density consistent with OmpF, TolB, and ColE9 residues 3-314. Map is coloured based on component parts shown in panel b. TolB is much better resolved here than in the full translocon map in panel c, although ColE9 density is weaker. ColE9 and TolB density aligns on the extracellular and periplasmic side of OmpF, respectively. F, The refined structure of the partial translocon, generated by docking and refinement of ColE9 residues 85-580 (PDB ID 5EW5), OmpF (PDB ID 3K19), and TolB-ColE9 TBE (PDB ID 4JML). ColE9 residues 3-67 were built de novo.
Figure 3(A,B) Representative immunofluorescence microscopy images of the distribution of septin 7 (red), Cdc42 (red) and tubulin (green) in LT-HSCs from borg4fl/fl or borg4∆/∆ mice. Nuclei were stained with DAPI (blue), scale bar= 5 µm. (C) Percentage of cells with a polar distribution of septin 7, Cdc42 or tubulin in borg4fl/fl or borg4∆/∆ LT-HSCs. At least 3 biological repeats, at least 50 cells were scored per sample. Error bars indicate mean±SEM, Two-way ANOVA analysis, ***P<0.001, **P<0.01, *P<0.05. (D,E) Representative immunofluorescence microscopy images of the distribution of borg4 (red), Cdc42 (red) or tubulin (green) in LT-HSCs from septin 7fl/fl or septin 7∆/∆ mice. Nuclei were stained with DAPI (blue). scale bar= 5 µm. (F) Percentage of cells with a polar distribution of borg4, Cdc42 or tubulin in septin 7fl/fl or septin 7∆/∆ LT-HSCs. At least 3 biological repeats, at least 30 to 50 cells were scored per sample. Error bars indicate mean±SEM, Two-way ANOVA analysis, **P<0.01, *P<0.05.
Figure 4DC subsets (cDC1, cDC2), Macrophages (MP) and Langerhans cells (LC) were sorted from IMQ treated back skin (5h). c-Jun, Ccl2 and Il23p19 mRNA was analyzed by qRT-PCR. Sort strategy is shown in (B). (10 mice were pooled per condition, 1 experiment). Sort strategy for the separation of MPs (CD45+Lin-CD64+CD172a+CD11b+), and the DC (CD45+Lin-CD64-CD11c+MHCII+) subsets cDC1 (XCR1+CD11b- DC), cDC2 (XCR1-CD11b+CD24-/intCD326-) and LC (XCR1-CD11b+CD24+CD326+) in IMQ-treated (5h) murine back skin. qRT-PCR detection of c-Jun, Ccl2 and Il23p19 in MPs and cDC2 sorted as described in (B) from indicated mice. (MPs: n= 7-9; 3 independent experiments, cDC2: n = 5-10; 2-3 independent experiments and back skin from 2 mice was pooled). Data information: Data are shown as mean ± SEM. P‐values were calculated by unpaired, two-tailed t-test Statistical significance: ns > 0.05, *P < 0.05. See Appendix Table S3 for exact P‐values. Sort strategy for CD11c expressing MPs (CD45+Lin-CD11cint/lowMHCII+CD64+CD11b+CD172a+) is shown. Murine skin was treated for 5h with IMQ. Expression of indicated targets was analyzed by qRT-PCR in cells sorted as described in (D). (n= 3; Back skin from 2 mice was pooled). Data information: Data are shown as mean ± SEM. P‐values were calculated by unpaired, two-tailed t-test Statistical significance: ns 0.05, *P < 0.05. See Appendix Table S3 for exact P‐values.
| | RNR inhibition enhances ara-C efficacy in primary patient AML blasts in a SAMHD1-dependant manner Drug synergy plots for ara-C and HU or dF-dC in primary patient-derived AML blasts.Figure 3Drug synergy plots for ara-C and HU or dF-dC in primary patient-derived AML blasts. Each data point indicates an average delta score from a single patient sample subjected to a dose-response matrix experiment performed in triplicate, n = 16 for HU and n = 9 for dF-dC. Zero, >0, or <0 corresponds to additive, synergy, or antagonism, respectively, whilst >5 indicates strong synergy. Median, upper and lower quartiles, and range of delta scores are indicated by box-and-whisker plots. Pearson correlation of relative SAMHD1 protein abundance and synergy delta scores for ara-C and HU or dF-dC in primary patient-derived AML blasts (n = 23). Immunoblot of primary patient-derived AML blasts treated with control (dX) or Vpx-containing (X) virus-like particles (VLPs): patient A2953 (C), ALG17_001 (E). Accompanying proliferation inhibition analysis of ara-C and indicated RNRi combination in these samples: patient A2953 (D), ALG17_001 (D). Error bars indicate s.d. of single experiment performed in triplicate. Paired drug synergy plot for ara-C and RNRi (HU, n = 7; dF-dC, n = 5) in primary patient-derived AML blasts pre-treated with control (dX) or Vpx-containing (X) VLPs. Zero, >0, or <0 corresponds to additive, synergy, or antagonism, respectively, whilst >5 indicates strong synergy and <5 indicates strong antagonism. Each data point indicates an average delta score from a single patient sample subjected to a dose-response matrix experiment performed in triplicate. Statistical testing was performed using two-way ANOVA.
Figure 6An illustration of a brain slice (sagittal view) of ChAT(+)-cre mouse showing the site of AAV-hM4D(Gi)-mCherry injection. Representative traces showing that the spontaneous firing of mHb neurons from ChAT(+)-cre::hM4D(Gi) mice were reduced when CNO (10 μM) was applied in the ACSF. A summary bar graph showing the reduction of normalized frequencies of spontaneous firing of the mHb neurons transfected with AAV-hM4Di-mCherry before and during CNO treatment (The numbers inside each bar indicate the numbers of recorded cells). In the EPM task, the time spent in the open arms of CNO-treated ChAT(+)-cre::hM4D(Gi) mice was significantly decreased compared to that of the PBS-treated controls (CTL). (nCTL=6, ncKO=9, unpaired Student t-test). E. In the LDB task, the time spent in the light zone by CNO-treated ChAT(+)-cre::hM4D(Gi) mice was significantly decreased compared to that spent by the PBS-treated control mice. (nCTL=6, ncKO=9, unpaired Student t-test). F. In the PA task, the entry latency of CNO-treated mice was significantly decreased compared to that of the CTL group. (nCTL=6, ncKO=9, unpaired Student t-test). G. Nestlet shredding behaviors of the CNO-treated ChAT(+)-cre::hM4D(Gi) mice were also decreased. (nCTL=6, ncKO=9, unpaired Student t-test;). In the 3-chamber test, the subject mouse of the PBS-treated mice and the CNO-treated mice explored freely the three test chambers. The time spent in each chamber during two sessions was measured. The CNO-treated ChAT(+)-cre::hM4D(Gi) mice exhibited a decreased interest for a stranger mouse (S1) against objects (O) compared to the PBS-treated group (left two panels) (nCTL=6, ncKO=9, unpaired Student t-test). I. When CNO-treated ChAT(+)-cre::hM4D(Gi) mice were exposed to a new stranger mouse (S2) against the familiar S1 mouse, they also showed a decreased interest for the S2 mouse compared to the PBS-treated control group (right two panels). The preference index showed decreased sociability of the CNO-treated group in both sessions (nCTL=6, ncKO=9, unpaired Student t-test;).
Figure 6A: Representative flow cytometric analysis showed that CD34+cells were significantly decreased at 5dpi with knockdown of AP2A1, METTL22, LRSAM1, or HAL. The human cord blood CD34+cells were transduced with pLKO-shRNAs targeting human AP2A1, METTL22, LRSAM1, or HAL and cultured for 5 days. Then, the flow cytometric analysis was performed to examine the percentage of CD34+cells in culture.B: Quantification of CD34+cells at 5dpi with knockdown of AP2A1, METTL22, LRSAM1, and HALshowed significant decrease of CD34+ cells compared to control. Error bars represent standard deviation (SD) from duplicates, *p<0.05. C: Flow cytometric analysis showed CD34+/CD90+cells were strikingly decreased at 5dpi with knockdown of AP2A1, METTL22, LRSAM1, and HAL.D: Quantification of CD34+CD90+cells at 5dpi with knockdown of AP2A1, METTL22, LRSAM1, and HALshowed significant decrease of CD34+CD90+cells compared to control. Error bars represent standard deviation (SD) from duplicates, *p<0.05.