EMBO/sd-character-level-ner huggingface dataset:
a) Outline of the screening strategy.figf1(a) Outline of the screening strategy. MS, mass spectrometry. See the text for details. (b) Density gradient fractions of NRK cells starved for 8 h were analysed by transmission electron microscopy (upper panel) and immunoblotted for LC3 or LAMP1 (lower panel). Proteins in fraction 1 were identified by mass spectrometry analysis. Three replicates were carried out from three independent purifications. Scale bar, 2 μm. (c) The dark grey circle shows the percentage of proteins identified from all three purifications; the grey circle shows the percentage of proteins identified from two of the three experiments. (d) Diagram of a SAMCell. Each dot represents a self-assembled cell island that can be transfected with an individual RNAi. (e) NRK cells grown on the SAMCell were transfected with FAM-RNAi by reverse transfection and the transfection efficiency was monitored by microscopy. Left panel, FAM-RNAi-positive cells; right panel, differential interference contrast (DIC) image of the cells in the left panel. (f) Functional associations were analysed by STRING. Stronger associations are represented by thicker lines. Uncropped images of blots are shown in Supplementary Fig. S6.
Parkin-mediated ubiquitination recruits p62 and HDAC6.Figure 4.Parkin-mediated ubiquitination recruits p62 and HDAC6. MEFs were transfected with WT, A240R, and T415N mutant GFP-parkin followed by CCCP treatment for 8 h. Cells were double immunostained with cytochrome c (red) and p62 antibody (blue) in A, and cytochrome c (red) and HDAC6 antibody in C. Arrows indicate mitochondrial aggregates. (B and D) The average percentages of p62- or HDAC6-positive mitochondrial aggregates from three independent experiments are presented with standard deviation as error bar. Parkin-mediated ubiquitination recruits p62 and HDAC6. MEFs were transfected with WT, A240R, and T415N mutant GFP-parkin followed by CCCP treatment for 8 h. Cells were double immunostained with cytochrome c (red) and p62 antibody (blue) in A, and cytochrome c (red) and HDAC6 antibody in C. Arrows indicate mitochondrial aggregates. (B and D) The average percentages of p62- or HDAC6-positive mitochondrial aggregates from three independent experiments are presented with standard deviation as error bar. (E) Wild-type and HDAC6 knockout (KO) MEFs were transfected with parkin-GFP or cotransfected with a Flag-tagged HDAC6 followed by CCCP treatment for 16 h as indicated. Cells are immunostained with Tom20 (red) and Flag (blue) antibodies. Bar, 25 µm. (F) Wild-type MEFs were transfected with control or HDAC6 siRNA and parkin-GFP, and treated with or without CCCP for 16 h. Cell lysates were subjected to immunoblotting analysis using antibodies for Tom20, parkin, HDAC6, and actin.
Figure 6A) Left panel: plasma total cholesterol in the different lipoprotein fractions of WT and Pkp2+/- mice fed a CD (green line) or a 3-month HFD (blue line; n=7). Right panel: plasma total cholesterol quantity in fractions 29-32, corresponding to low, very-low and intermediate-density lipoproteins, of WT and Pkp2+/- mice fed a CD (green line) or a 3-month HFD (blue line; n=7; Mann-Whitney test). Data information: mean ± SEM. * p<0.05; ** p<0.01; *** p<0.001. B) Plasma concentration of oxLDL in WT and Pkp2+/- mice samples, fed a CD and a 3-month HFD (n=6; Two-tailed Student's t-test). Data information: mean ± SEM. * p<0.05; ** p<0.01; *** p<0.001. C) Left panels: representative images of ORO staining of HFD-fed WT and Pkp2+/- cardiac sections. Right panel: quantification of ORO positive area percentage (n=10). For comparison, quantification of ORO positive area of cardiac sections of CD-fed WT and Pkp2+/- mice (n=9; Two-Way Anova) is shown (Appendix Figure S3). D) Representative images of PPARγ (green) immunostaining on HFD-fed WT and Pkp2+/- mice cardiac sections (n=10; Two-Way Anova). Quantification of the PPARγ staining in HFD is shown relative to the values of CD (n=9). E) Representative images of MDA (green) immunostaining on HFD-fed WT and Pkp2+/- mice cardiac sections (n=10; Two-Way Anova). Quantification of the MDA staining in HFD is shown relative to the values of CD (n=9). F) Representative images of CD36 immunostaining (green) on HFD-fed WT and Pkp2+/- mice cardiac sections (n=10; Two-Way Anova). Nuclei are counterstained with Hoechst33342 (blue). Quantification of the staining in HFD is shown relative to the values of CD (n=9). Data information: mean ± SEM. * p<0.05; ** p<0.01; *** p<0.001. G) RV EF and FS percentages, RVID in systole and diastole of WT and Pkp2+/- mice after (right) 3-month HFD feeding (n=10; Two-tailed Student's t-test). Data information: mean ± SEM. * p<0.05; ** p<0.01; *** p<0.001.
Figure 1a, Expression levels of ISX, TWIST1, Snail1, Fibronectin (FN1), VEGF, and E-cadherin (CDH1) mRNA were examined in a ISX-GFP inducible Tet-ON transformants at 8 h after addition of DOX. Data are presented as mean ± SD in graph (p< 0.001 compared to time point 0h; Student's t‐test) of 3 independent experiments, each performed in triplicate. b, Western blotting analysis of the protein levels of ISX, HIF1α, TWIST1, Snail1, Slug, ZEB1, Bmi1, E-cadherin (E-Cad.), Fibronectin, N-cadherin (N-Cad.), Vimentin, and VEGF in A549 and H1299 cells with DOX-inducible ISX expression system 8hs after DOX induction. ISX transcriptionally activates luciferase activity driven by TWIST1 (c) promoter regions in A549 cells. Data are presented as mean ± SD in bar graph (p < 0.001 , Student's t‐test) of 3 independent experiments, each performed in triplicate. d, ISX transcriptionally activates luciferase activity driven by Snail1 (d) promoter regions in A549 cells. Data are presented as mean ± SD in bar graph (p < 0.001 , Student's t‐test) of 3 independent experiments, each performed in triplicate. e, ChIP analysis of ISX binding to the TWIST1 promoter region in A549 and H1299 cells (10% input of each groups were pull down and applied to qPCR). Data are presented as mean ± SD in bar graph (p < 0.001 , Student's t‐test) of 3 independent experiments, each performed in triplicate. f, ISX transactivation activity analyzed by luciferase activity driven by the TWIST1 promoter. Data are presented as mean ± SD in graph (p < 0.001 , Student's t‐test) of 3 independent experiments, each performed in triplicate. g, ChIP analysis of ISX binding to the endogenous promoters of TWIST1 (-180/+35) and Snail1(-160/+40) in A549 and cells (10% input of each groups were pull down and applied to qPCR). Data are presented as mean ± SD in bar graph (p < 0.001 , Student's t‐test) of 3 independent experiments, each performed in triplicate. h, Effect of forced expression and knockdown of ISX on cell migration (wound healing) measured in A549 cells. ISXi, ISX-specific shRNAi Data are presented as mean ± SD in graph (**, p < 0.01, ***, p < 0.001 , Student's t‐test) of 3 independent experiments, each performed in triplicate. Scale bar, 100μm. i, Effect of forced expression and knockdown of ISX on cell invasion (transwell) activity measured in A549 cells. Data are presented as mean ± SD in bar graph (***, p < 0.001 , Student's t‐test) of 3 independent experiments, each performed in triplicate. Scale bar, 100μm.
Figure 4Schematic of the neural differentiation system used in this study, indicating the relevant markers that define different stages of differentiation. Brightfield images and analysis of NPC markers by immunofluorescence staining (Sox1 and Nestin) or RT-qPCR (Nestin and Pax6) (n=3, mean ±SEM). Brightfield images of aggregates. Brightfield images and immunofluorescence staining for Nestin and neuronal marker Tuj1 in NSC cultures grown in EGF/FGF proliferating conditions. White arrows indicate NSCs and green arrows differentiated cells. Two small panels on the right are examples of Tuj1+ neuronal cells in slow/slow NSCs cultures. Immunofluorescence staining for neuronal marker Tuj1 in neuronal cultures grown on poly-ornithine/laminin at 21 days of differentiation.
TBK1 promotes mTORC1 signaling independently of mTORC2 dependent Akt S473 phosphorylation.Figure 3A. mTORC1 signaling remains intact in MEFs lacking mTORC2 function: Rictor-/- MEFs stably expressing vector control (V) or rescued stably with HA-rictor were serum starved (20 hr.), pre-treated with Ku-0063794 [1 μM] (30 min), and stimulated -/+epidermal growth factor (EGF) [25 ng/mL] (30 min). Whole cell lysate (WCL) were immunoblotted as indicated. B. TBK1 is required for mTORC1 signaling in MEFs with reduced Akt S473 phosphorylation: TBK1 expression was reduced using lentivirally-delivered TBK1 shRNA in Rictor-/- MEFs stably expressing vector control (V). Cells were EGF stimulated as above, except Torin1 [100 nM] was used to inhibit mTOR.
a, Multiple-sequence alignment of N-myristoylated glycine and downstream sequences from the indicated proteins (Hs, Homo sapiens; Sc, S.f4a, Multiple-sequence alignment of N-myristoylated glycine and downstream sequences from the indicated proteins (Hs, Homo sapiens; Sc, S. cerevisiae). b, In-gel fluorescence assay showing myristoylated peptides of the indicated proteins in cells expressing IpaJ or catalytic mutant. The peptide-eGFP and IpaJ inputs are indicated. *Demyristoylation of GRASP65 and hVPS15 resulted in slower mobility of the resulting peptide, potentially owing to reduced mobility in SDS-polyacrylamide gel electrophoresis caused by proteolytic reaction. c, In-gel fluorescence assay showing myristoylated ARF1 T31N mutant (a GDP-locked mutant) or ARF1 Q71L (a GTP-locked mutant) after in cells co-expressing IpaJ or catalytic mutants as indicated. Equal loading of ARF1 (middle panel) and IpaJ (lower panel) was determined by western blot analysis. d, Fluorescence microscopy showing ARF1-mCherry and the Golgi apparatus (GM130) after cellular microinjection of recombinant IpaJ or IpaJ C64A, over the indicated period. Scale bar, 10 µm. e, Fluorescence microscopy showing ARF1-eGFP (green) and Golgi membranes (blue, GM130) after infection with Shigella M90T and the indicated mutants (red; mCherry-expressing bacteria). Scale bar, 10µm.
MHC class II‐restricted presentation of NeoR and NucNeoR is blocked by inhibition of autophagy and lysosomal degradation.Figure 2(A) RCC1.24‐NeoR and ‐NucNeoR cells were incubated for 48 h with IFN‐γ and in addition with 7.5 mM 3‐MA or 200 µM leupeptin or 100 µM chloroquine for the last 20 h. Subsequently, the cells were fixed with 0.5% paraformaldehyde and tested with 20‐4/A4 cells in a GM‐CSF release assay. Both NeoR and NucNeoR were presented by a pathway that involves autophagy and lysosomal processing. (B) LCL1.11 cells transfected with NeoR or NucNeoR expression plasmids were tested likewise. Presentation of endogenous NeoR and NucNeoR was blocked by all inhibitors, indicating that NeoR and NucNeoR presentation is dependent on autophagy and lysosomal processing. (C) To exclude toxic side effects of the inhibitors, LCL1.11 cells were incubated for 20 h with 200 ng/mL recombinant NeoR protein in the presence or absence of the inhibitors. Afterwards, residual protein and inhibitors were removed by washing and the cells were co‐cultured with the NeoR‐specific CD4+ T cell clone 20‐4/A4. The presentation of exogenous NeoR on MHC class II was not affected by 3‐MA, but was impaired when lysosomal processing was blocked with chloroquine or leupeptin. (D) LCL1.11 cells were transfected with siRNA specific for Atg12 or GFP, and Atg12 mRNA levels were determined 24 h later by qPCR. The relative amount of Atg12 mRNA was reduced approximately fourfold. (E) At 24 h after siRNA transfection, NeoR and NucNeoR were expressed in these cells and antigen presentation was assessed 36 h later. In cells transfected with Atg12 siRNA, but not in mock‐transfected cells or cells transfected with GFP siRNA, antigen presentation of both NeoR and NucNeoR was greatly reduced.
Maximum likelihood phylogenetic trees of nucleotide sequences of the ORF1a, ORF1b, E and M genes of WHCV and related coronaviruses.Figure 3.Maximum likelihood phylogenetic trees of nucleotide sequences of the ORF1a, ORF1b, E and M genes of WHCV and related coronaviruses. Numbers (>70) above or below branches indicate percentage bootstrap values for the associated nodes. The trees were mid-point rooted for clarity only. The scale bar represents the number of substitutions per site.
DHS-induced DNA damage in cardiomyocytes is caused by direct inhibition of histone deacetylases: (A) Pharmacological inhibition of HDAC1 using romidepsin, MS-275 and pyroxamide in human cardiomyocytes cause extensive DNA damage, shown here by γH2A.Figure 3(A) Pharmacological inhibition of HDAC1 using romidepsin, MS-275 and pyroxamide in human cardiomyocytes cause extensive DNA damage, shown here by γH2A.X staining. (B) Bar-graphs depict quantification of γH2A.X+ hCM nuclei in the indicated conditions. (C) In silico simulation showed docking of sphinganine-analogue DHS1P in the tubular active site of human HDAC1. (D) Sphinganine derivatives DHS1P and S1P show comparable binding affinity to HDAC1, similar to the known HDAC inhibitor TSA. (E) Sphinganine and its derivative DHS1P inhibits class 1 HDACs in the human cardiomyocytes as inferred from the in cellulo HDAC-activity assay, shown here as bar graph. (F) In vitro HDAC activity assay revealed inhibition of nuclear HDAC's and purified HDAC1 activity by DHS1P and S1P, shown here as bar graphs. Assessment of transcription levels measured by EU labeling assay upon treatment with DHS on hCMs Assessment of transcription levels measured by EU labeling assay upon treatment with DHS on hCMs and respective quantifications. (I) Representative micrographs of hCMs indicating the rescue of the DHS induced DNA damage by co-incubation with RNA Pol II inhibitor, triptolide and (J) the respective quantifications of γH2A.X+ CM nuclei represented as bar graph. Scale bars for A,I=10 µm and G = 50 µm. (When not specified, the experiments were conducted in at least 3 biological replicates).
Variability in the magnitude, direction, and timing of signaling changes in response to RAF/MEK inhibition PLSR-derived variable importance in the projection (VIP) scores predicting viability for each of the ten studied cell lines.Figure 2PLSR-derived variable importance in the projection (VIP) scores predicting viability for each of the ten studied cell lines. VIP scores are shown for each cell line-specific model, each signal and measurement time point. The direction of the bars (left or right) shows whether the changes in signal correlated negatively or positively with relative viability. VIP scores of larger than one indicate important variables (signals and time points) that predict the responses (viability). Unsupervised clustering of melanoma cell lines based on the VIP scores >1 from each individual cell line model (excluding pMEK and pERK). Prior to clustering, VIP scores of between 0 and 1 were set to zero and a minus sign was added to VIP scores associated with signals that negatively correlated with viability. Average relative resistance of the cell lines to the five tested RAF/MEK inhibitors (on the right) is computed based on area under the time-dose-response curve plotted for non-apoptotic viability measured by single-cell imaging across seven doses and two time points (48 and 72 h) following treatment. The data for the cell line K2 are not shown because clumping made it difficult to score single-cell phenotypes in this cell line after 72 h.
Figure 5A, B. HeLa cells (A) or SH-SY5Y neuroblastoma cells (B) treated with non-targeting (Ctrl) or Rab1a siRNA were co-transfected with mCherry-FIP200 (red) and empty vector (EV), Myc-C9orf72L or Myc-C9orf72S (green). As positive control EV transfected cells were treated for 3 h with Torin1 (250 nM). Translocation of the ULK1 complex was quantified as the number of mCherry-FIP200 positive puncta per cell (A, HeLa, Mean ± SEM; one-way ANOVA with Fisher's LSD test; ns, not significant; **** P ≤ 0.0001; N (cells from 3 independent experiments) = Ctrl/EV: 81; Ctrl/EV/Torin1: 48; Ctrl/C9orf72L: 86; Ctrl/C9orf72S: 48; Rab1a/EV: 79; Rab1a/EV/Torin1: 68; Rab1a/C9orf72L: 78; Rab1a/C9orf72S: 52; B, SH-SY5Y: Mean ± SEM; one-way ANOVA with Fisher's LSD test; ns, not significant; ** P ≤ 0.01; **** P ≤ 0.0001; N (cells from 2 independent experiments) = Ctrl/EV: 70; Ctrl/EV/Torin1: 56; Ctrl/C9orf72L: 45; Ctrl/C9orf72S: 43; Rab1a/EV: 63; Rab1a/EV/Torin1: 55; Rab1a/C9orf72L: 44; Rab1a/C9orf72S: 37). Rab1a knockdown was confirmed by RT-qPCR (Appendix Fig S2). Scale bar = 10 µm. C. SH-SY5Y neuroblastoma cells treated with non-targeting (Ctrl) or Rab1a siRNA were co-transfected with EGFP-LC3 (green) and empty vector (EV), Myc-C9orf72L or Myc-C9orf72S (red). As positive control EV transfected cells were treated for 3 h with Torin1 (250 nM). Autophagosomes were quantified as the number of EGFP-LC3 positive puncta per cell (Mean ± SEM; one-way ANOVA with Fisher's LSD test; ns, not significant; * P ≤ 0.05, *** P ≤ 0.001, **** P ≤ 0.0001; N (cells from 2 independent experiments) = Ctrl/EV: 102; Ctrl/EV/Torin1: 92; Ctrl/C9orf72L: 97; Ctrl/C9orf72S: 76; Rab1a/EV: 102; Rab1a/EV/Torin1: 107; Rab1a/C9orf72L: 101; Rab1a/C9orf72S: 87). Rab1a knockdown was confirmed by RT-qPCR (Appendix Fig S2). Scale bar = 10 µm.
Figure 7(A) ILC210 were isolated from ex vivo-expanded kidney ILC2s by flow sorting. ILC210 were co-transplanted with islet locally or adoptively transferred into diabetic C57BL/6 mice twice at one day prior to and two days post islet transplantation. Mice were sacrificed at day 80 post-islet transplantation or at the day when grafts were considered rejected. (B) Islet graft survival of five groups of mice was assessed by monitoring blood glucose and calculated using the Kaplan-Meier method. Cumulative data from two independent experiments are shown. Statistical analysis was performed with a log-rank test. ***P<0.001. (C) To track ILC210 in vivo, CD45.2+ ILC210 were co-transplanted with islets in diabetic CD45.1+ C57BL/6 mice. Representative FACS analysis showing the proportion of locally transplanted ILC210 (CD45.2+ST2+) in the total CD45+ cell compartment from islet graft at day 5 and 80 post-islet transplantation or at the day when grafts were considered rejected. (D,E) Proportion and numbers of locally transplanted CD45.2+ST2+ ILC210 in islet grafts of islet transplant mice over time. Data shown are the mean ± SEM (n=4-5 per group) and a one-way ANOVA was performed, **P<0.01, ***P<0.001. (F) The expression of IL-10-GFP was examined in locally transplanted CD45.2+ST2+ ILC210 by flow cytometry. IL-10-GFP (red lines) and controls (gray-filled areas) are shown. Data represent the mean ± SEM of evaluations of MFI from each group (n=4-5 per group) and a one-way ANOVA was performed; ***P<0.001. MFI, mean fluorescence intensity.
Elp3cKO SGNs are lost before birth as a result of early apoptosis.Figure 2(A) Toluidine blue stainings of semi-thin sections from P15 WT and Elp3cKO cochleae reveal a drastic reduction in the size of the spiral ganglion due to SGN loss. Scale bar=100µm, OC: Organ of Corti. (B) Specific labellings of the pre-synaptic marker Ctbp2 (in IHCs) and the post-synaptic marker GluR2/3 (in SGN terminals) indicate a decrease in the number of double-labelled dots in P15 Elp3cKO whole-mounted cochlea compared to WT. The remaining synapses still show coupling of the pre- and post-synaptic markers, suggesting they may be functional. Scale bar=20µm. (C) The mean number of Ctbp2-GluR2/3 double-positive dots in IHCs from the basal region of P15 Elp3cKO cochleae is reduced by 65% compared to control (n=6 animals per genotype; Unpaired two-tailed t-test, ***p<0.001, t=13.48, DF=10, mean±SD). (D) NF-1 and Mafb stainings on P0 cochlear sections indicate that SGN loss occurs before birth in Elp3cKO cochleae. Scale bar=100µm. (E) SGN quantifications on cochlear sections from different embryonic stages reveal a progressive loss of SGNs from E14.5 onwards (n=3-6 animals; one-way ANOVA, Tukey's multiple comparisons test; ***p<0.001; *p<0.05; F=20.43; DF=7; mean±SD). (F) Detection of apoptotic SGNs from E13.5 cochlear sections with cleaved caspase-3 and B-tubIII stainings. Scale bar=100µm. (G) The number of apoptotic SGN per section is significantly increased in E13.5 and E14.5 Elp3cKO compared to WT cochleae, suggesting SGNs are properly specified but die during their differentiation process (n=3-4 animals; one-way ANOVA, Tukey's multiple comparisons test; ***p<0.001; **p<0.01; F=23.74; DF=7; mean±SD).
Deletion of Chk1 in HSC prevents fetal hematopoiesis.Figure 4Representative pictures of E13.5 and E18.5 embryos of the indicated genotypes. Scale bar E13.5 = 1 mm, E18.5 = 5 mm. Quantification of fetal liver cell number at E13.5. Bars represent means ± S.E.M. (N=4 for Chk1fl/+, N=3 for Chk1fl/+ Vav-Cre and N=4 for Chk1fl/- Vav-Cre). Representative pictures of isolated fetal livers at E13.5. Scale bar = 1 mm. Representative H&E-staining of fetal liver sections from E13.5 embryos. Black arrows indicate lack of erythroid cells in the blood vessels of Chk1fl/- Vav-Cre fetal livers. Scale bar = 500 µM. Single cell suspensions of fetal livers from Chk1fl/+, Chk1fl/+ Vav-Cre and Chk1fl/- Vav-Cre embryos were stained with antibodies recognizing ckit, CD71, Ter119 or CD45 to assess primitive and definitive erythropoiesis by flow cytometry. Large 'EryA' erythroblasts (CD71high Ter119high FSChigh), smaller, more mature 'EryB' erythroblasts (CD71high Ter119high FSClow); most mature erythroblast subset is EryC (CD71low Ter119high FSClow). Bars represent means ± S.E.M. ctrl N=5 (pooled N=4 Chk1fl/+ and N=1 Chk1fl/+ Vav-Cre), N=3 for Chk1fl/- Vav-Cre.
Figure 4(A) CARD14 signaling induces CYLD and A20 cleavage. HEK293T cells were transfected with different concentrations of FLAG-CARD14, with or without MALT1 as indicated. CARD11(L232LI) transfection was used as a positive control. Endogenous CYLD and A20 processing was analyzed by immunoblotting. CYLD and A20 cleavage fragments are indicated by an arrow. (B) CARD14 signaling induces CYLD cleavage after R324. HEK293T cells were transfected with different concentrations of FLAG-CARD14sh and either wild-type (CYLDWT) or non-cleavable CYLD (CYLDR324A). CYLD processing was analyzed by immunoblotting. The CYLD cleavage fragment is indicated by an arrow. (C) Expression of catalytically active MALT1 is necessary for CARD14-induced CYLD cleavage. MALT1-deficient HEK293T cells in which MALT1 expression was restored by transfection with different concentrations of either wild-type (MALT1WT) or catalytically inactive MALT1 (MALT1C/A), were co-transfected with FLAG-CARD14sh and CYLD as indicated. CYLD processing was analyzed by immunoblotting. The CYLD cleavage fragment is indicated by an arrow. (D) Psoriasis-associated CARD14 mutation enhances activation of MALT1 proteolytic activity. HEK293T cells were transfected with wild-type (WT) FLAG-CARD14 or different psoriasis-associated CARD14 mutants (20ng), with or without MALT1 (20 ng) as indicated. Endogenous CYLD and A20 processing, as well as IκBα phosphorylation, were analyzed by immunoblotting. The CYLD and A20 cleavage fragments are indicated by an arrow. (E) Pharmacological inhibition of MALT1 proteolytic activity prevents CARD14-induced gene expression in HaCaT keratinocytes. CARD14 expression was induced by doxycycline (dox) treatment in the absence or presence of different concentrations of mepazine as indicated. The next morning, cell culture medium was refreshed and IL-8 and MCP-1 concentration in the cell supernatant was measured 8 h later by ELISA. TNF stimulation (MALT1-independent) was used as a negative control. Values are the mean of triplicates ± s.e. Data are representative of two independent experiments. Significance levels, *P<0.05, **P<0.001, ***P<0.001 by student t test.