EMBO/sd-character-level-ner huggingface dataset:
Figure 7Cells in the posterior compartment (blue) produce the short-range morphogen Hedgehog, which is received by cells immediately anterior to the compartment boundary, in the anterior compartment (dark green). Hh signal releases Ptc-mediated inhibition of Smo, allowing for the stabilization of the transcriptional co-effector Ci, and increased expression of the ENaC channel Rpk, which depolarizes cells immediately anterior to the compartment boundary. Depolarization results in increased membrane abundance of the Hh activator Smo, and increased transduction of Hh signal. This, in turn, increases expression of Rpk, reinforcing high levels of Hh signal transduction in the region immediately anterior to the compartment boundary.
Figure 2IRE1-/- and IRE1+/+ BMDMs were treated with PA (500 µM) or vehicle for 6 hours prior to RNA isolation for miRNA analysis using a micro-array platform. (A) Table showing fold change (log2) of statisticaly significant (p-value <0.050) top PA-upregulated miRNAs in IRE1+/+ and IRE1-/- cells. NS indicates micro RNAs that didn't show statistically significant difference. Fold change was calculated by comparing mean intensity values of micro array signal for vehicle treatment with PA treatment (n=3 biological replicates). IRE1-/- and IRE1+/+ BMDMs were treated with PA (500 µM) or vehicle for 6 hours prior to RNA isolation for miRNA analysis using a micro-array platform. (B) RNA lysates from the same experiment were analyzed by qRT-PCR for miR-2137 expression. IRE1-/- and IRE1+/+ BMDMs were treated with PA (500 µM) or vehicle for 6 hours prior to RNA isolation for miRNA analysis using a micro-array platform. (C) Protein lysates were analyzed by western blotting using specific antibodies for pIRE1S742, IRE1 and β-actin (n=3 biological replicates). IRE1-/- and IRE1+/+ BMDMs were treated with PA (500 µM) or vehicle for 6 hours prior to RNA isolation for miRNA analysis using a micro-array platform. (D) RNA lysates were analyzed by qRT-PCR for sXBP1 expression (n=3 biological replicates). IRE1-/- and IRE1+/+ BMDMs were treated with PA (500 µM) or vehicle for 6 hours prior to RNA isolation for miRNA analysis using a micro-array platform. (E-F) RNA lysates from the same experiment were analyzed by qRT-PCR for (E) BLOC1S1 and (F) SCARA3 expression (n=3 biological replicates).
Lactobacillus acidophilus NCK2187 strain development and characteristicsAgarose gel image illustrating PCR amplicons of ltaS (LBA0447), slpB, and slpX deletions in NCK2187.Figure 1A Agarose gel image illustrating PCR amplicons of ltaS (LBA0447), slpB, and slpX deletions in NCK2187. B SDS-PAGE gel of 5 M LiCl‐purified S‐layer fractions from the parental strains, NCK56 and NCK1909, NCK2030 (LTA+, SlpA+, SlpB−, SlpX−), and NCK2187 (LTA−, SlpA+, SlpB−, SlpX−). C B6 mice were orally gavaged with 109 CFU erythromycin‐resistant NCK56 or NCK2187. Fecal pellets were collected daily and tested for the presence of erythromycin‐resistant strains. n = 3 mice/group. Data are representative of five independent experiments and are shown as mean ± SEM. D ColonicLPL cells were co‐cultured with NCK56 or NCK2187 (1:1), and secreted cytokines were measured in the supernatants. Data are shown as mean ± SEM and are representative of two experiments performed in triplicate. *P 0.05, **P 0.01, ***P 0.001.
Figure 3A. Schematics of astrocytic calcium imaging in cortex from ex vivo slices and representative images of spontaneous calcium signaling (green) of Daam2 cHet and cKO mice injected with AAV-GfaABC1D-GCaMP6f virus. Approximate territory of astrocytes including soma and multiple microdomains is outlined in yellow. Representative traces of GCaMP6f signal in Daam2 cHet and cKO astrocytes are shown in black and red respectively. B-D. Quantification of amplitude (B), frequency (C), and area under curve (AUC) (D) of ∆F/F GCaMP6f signal events in cortex. Daam2-deficient astrocytes have lower amplitude and frequency of signal events in cortex. Data is presented as mean ± SEM. Total number of cells is n=20, 17 from N=3-4 mice of each genotype. Student's t-test was used for statistics, *p<0.05. E. Schematics of astrocytic calcium imaging and representative images of spontaneous calcium signaling (green) of Daam2 cHet and cKO mice injected with AAV-GfaABC1D-GCaMP6f virus in OB. Approximate territory of astrocytes including soma and multiple microdomains is outlined in yellow. Representative traces of GCaMP6f signal in Daam2 cHet and cKO astrocytes are shown in black and red respectively. F-H. Quantification of amplitude (F), frequency (G), and area under curve (AUC) (H) of ∆F/F GCaMP6f signal events in OB. Amplitude and AUC of Daam2-deficient astrocytes are significantly increased in OB. Data is presented as mean ± SEM. Total number of cells is n=16, 15 (F-H) from N=3-4 mice of each genotype. Student's t-test was used for statistics, *p<0.05.
Figure 6(A) Protein stability of an unstable firefly luciferase variant (Fluc_F89E) assessed by anti-Fluc antibodies in ΔIndLon mutants grown in inducing (+) or depleting (-) conditions (48h of depletion). LC, loading control. (B) Lon surveillance of truncated protein variants. As an example, an N-terminal FLAG-tagged derivative of MPN304, which encodes a truncated variant of ArcA (ArcA-Nt), was expressed in the ΔIndLon mutant, and its accumulation monitored over time under non-inducing conditions using anti-FLAG antibodies. Anti-Lon antibodies were used to monitor Lon depletion along the time course experiment (western blot figure on left). MPN304 stability was also assessed by in vivo degradation experiments in Lon depleted mutants (36h of depletion), in which protein expression was blocked with gentamicin (Gm) during the indicated time points after transient induction of Lon for 3h. As control, samples after Gm treatment were also taken from non-induced samples. LC, loading control (western blot figure on right). Immunoblots are representative of two independent experiments. A schematic representation of the arginine deiminase pathway, which is inactive in M. pneumoniae, and the estimated protein copy number of its components are also shown for the ΔIndLon mutant grown in inducing (+) or depleting (-) conditions. (C) MS analysis showing the distribution of log2 fold-changes of the Triton X-100 insoluble fraction compared to the whole cell lysate, under Lon depleting conditions. Proteins associated with the terminal organelle (TO proteins), known to be enriched in the insoluble fractions, are shown as reference. Lon substrate candidates exhibit larger log2 fold-changes compared to the other proteins (Mann-Whitney-Wilcoxon two-sided test, ****p=0.0003), showing enrichment in the insoluble fraction under Lon depleting conditions. (D) Lon enrichment in protein insoluble fractions during heat stress conditions. Lon levels in total (T) and Triton-X100 soluble (S) and insoluble (I) fractions were assessed by immunoblot analyses in untreated wild-type cells or exposed at 45°C during the indicated time points. As controls, the Triton-X100 insoluble protein HMW1 and the soluble ribosomal protein RL-7 were also analysed by using antibodies raised against these proteins. (E) Protein quality control of terminal organelle assembly mediated by Lon. Top of panel E, a schematic representation of the genetic organization at the P65 operon after deleting the N-terminal region of the hmw2 gene in the ΔIndLon mutant. The expected PCR products for the deletion are also indicated. Below, an agarose electrophoresis gel showing the PCR validation of the intended deletion, and SDS-PAGE and immunoblot analyses monitoring the expression of specific terminal organelle proteins, whose stability depend on HMW2, for ΔIndLon or ΔIndLon_Δhmw2Nt cell lysates grown in inducing (+) or depleting (-) conditions (48h of depletion). LC, loading control.
High CDK activity prevents Polη-dependent bypass of replication barriers created by Chk1 loss.Figure 3Labelling protocol used here and in all figures that follow, except otherwise indicated. The lower panels show representative images and schemes of an ongoing fork and of origins that fired during the CldU (1st) or IdU (2nd) pulses. Gray dotted lines represent unlabeled DNA. Percentage of origins was calculated as: (1st+2nd pulse origins)/total number of fibers. IdU track lengths from U2OS cells (Rosc, roscovitine). 180-200 DNA fibers obtained from 2 independent experiments were measured for each condition. The upper panel shows a Western Blot of Polη, 48 h after transfection with siRNA. Actin was used as a loading control. Percentage of origin firing (mean+SD) from DNA fibers in (A) (n=2). The dotted line represents the mean percentage of origin firing in control samples.
Analysis of TRAIL/death receptor pathway.Fig 4A,B) Relative gene expression analysis of IGF1R, Fas, and the death receptors TRAILR-1 and TRAILR-2 after bortezomib treatment with the respective IC50 concentrations for 48 h. Untreated control cells served as reference value (ratio = 1). C) The human apoptosis antibody protein array supported the important role of the IGF binding proteins (IGFBP-1 to IGFBP-6), the TNF receptor family, and the TRAIL receptors in both cell lines. D) Western blot analysis showed the downregulation of Fas and TNF-R1.
Figure 2Micro-CT and statistical analyses of BMD, BV/TV, Tb.N and Tb.Sp in trabecular bone from AdipoqCre; ranklfl/fl (Rankl-/-) and ranklfl/fl (Rankl+/+) mice at 4 weeks, 8 weeks and 16 weeks (n=5 independent biological replicates). Scale bar=1 mm. Data were compared using an unpaired t-test (** indicates P < 0.01), error bars are standard deviations. Micro-CT images of cortical bone from Rankl-/- and Rankl+/+ mice at 8 weeks. Images are representative of five independent biological replicates. Scale bar =1 mm. Micro-CT analyses and statistical analyses of cortical bone from Rankl-/- and Rankl+/+ mice at 8 weeks (n=5 independent biological replicates). Data were compared using an unpaired t‐test, error bars are standard deviations.
Figure 6A-B) Analysis of Tbk1-/- MEFs complemented with the indicated Flag-tagged TBK1ΔC-galectin fusion proteins. S. Typhimurium replication kinetics (A). Infected cells were lysed at the indicated times post inoculation (p.i.). and bacteria were enumerated by their ability to form colonies on agar plates. Mean and s.d. of triplicate MEF cultures and duplicate colony counts. Data are representative of at least two repeats. Statistical differences to Tbk1-/- MEFs expressing TBK1ΔC are shown. *p<0.05, ***p<0.001, one-way ANOVA with Dunnett's multiple comparisons test. Western blot for Flag-tagged TBK1 variants in post nuclear cell lysates. A-B) Analysis of Tbk1-/- MEFs complemented with the indicated Flag-tagged TBK1ΔC-galectin fusion proteins. (B) Percentage of GFP:WIPI1 positive S. Typhimurium at 1 h p.i. in the indicated complemented MEFs. Mean and s.e.m. of three independent experiments. >200 bacteria counted per coverslip. *p<0.05, one-way ANOVA with Dunnett's multiple comparisons test.
Figure 3(A) Tumor spheroid formation. Representative images of tumor spheres formed and quantitative data comparing the average number of spheres formed in the indicated cells treated with PBS or human recombinant ESM1 (rhESM1) or normal goat IgG control or anti-ESM1 neutralizing antibody. (B) Representative images of tumor spheres. Spheres were cultured for 14 days before counting. Histogram shows the mean numbers of spheres cultured. Scale bar: 100 μm. Bars are the mean ± SD of three independent experiments. (C) 22Rv1-P cells stably expressing ESM1 were treated with docetaxel at the indicated concentrations for 48 h. ** (D) Cells with or without ESM1-NLS overexpression were subcutaneously injected (10, 100, 1000 cells per mouse) into NOD/SCID mice. Tumor formation ability and tumor weight were analyzed. Scale bar: 1 cm. Bars are the mean ± SD of indicated (n) independent experiments. (E) Flow cytometry analysis of the ratio of CD44+ and CD133+ cells in the indicated cells with different subcellular localization types of ESM1 overexpression were performed. (F) qRT-PCR analysis of the mRNA levels of the indicated genes in cells with different types of ESM1 overexpression. Differences in mRNA levels compared with vector cells are shown as fold changes presented as the mean ± SD of three independent experiments. *
a, Plasma and tissue amino acid concentrations.figf4a, Plasma and tissue amino acid concentrations. Amino acid concentrations were measured at 0 and 10 h after the caesarean delivery under fasting conditions. 'Total' indicates the sum of the Asp, Thr, Ser, Asn, Glu, Gln, Pro, Gly, Ala, Val, Cys, Met, Ile, Leu, Tyr, Phe, Lys, His and Arg concentrations; 'Essential' indicates the sum of Thr, Val, Met, Ile, Leu, Phe, Lys, His and Arg concentrations; 'BCAA' indicates the sum of the Val, Ile and Leu concentrations. Tissue amino acid concentrations are expressed as mmol kg-1 of wet weight. Bars represent mean ± s.d. of three mice. Single and double asterisks indicate a significant difference between mutant and control (wild-type plus heterozygous) mice at P 0.05 and P 0.01, respectively. b, Activation of AMPK in Atg5-/- mice. Hearts were isolated from unfed neonates at 0 h (lanes 1, 3, 5) and 10 h (lanes 2, 4, 6), and force-fed mice at 10 h (lane 7) after the caesarean delivery. Homogenates were prepared in the presence of phosphatase inhibitors and analysed by immunoblotting using anti-AMPK and anti-phospho-AMPK antibodies. c, ECGs from neonatal mice at the indicated time after caesarean delivery. ST elevation was observed in all Atg5-/- mice (n = 4) at 8-9 h after delivery.
PINK1 processing is regulated by MPP, PARL, m‐AAA and ClpXP.Figure 1(A) Immunoblots of mitochondrial fractions from siRNA‐transfected HEK293T cells±CCCP. (B) Immunoblots against indicated proteases in siRNA‐transfected cells. (C) Quantification of full‐length PINK1 (n=4) and knockdown efficiency (n=2) compared with non‐targeting (NT) siRNA from experiments performed as in B. *P0.05; **P0.01; ***P0.001. CCCP, carbonyl cyanide m‐chlorophenyl hydrazone; MPP, mitochondrial processing peptidase; PARL, presenilin‐associated rhomboid‐like protease; PINK1, phosphatase and tensin homologue‐induced kinase 1; siRNA, short interfering RNA.
Figure 10Three-day-old seedlings of Col-0, MPK3SR, MPK3SRarr1/12, and arr1/12 were transferred to 0.5 μM NA-PP1-containing 1/2 MS medium for 2 d to inhibit MPK3TG in MPK3SR and MPK3SRarr1/12 and then the plants were transferred to 0.2 μM NA-PP1-containing 1/2 MS medium supplemented with or without 50 mM or 75 mM NaCl. Pictures were taken and the elongated root length was determined 3 d later Three-day-old seedlings of Col-0, MPK3SR, MPK3SRarr1/12, and arr1/12 were transferred to 0.5 μM NA-PP1-containing 1/2 MS medium for 2 d to inhibit MPK3TG in MPK3SR and MPK3SRarr1/12 and then the plants were transferred to 0.2 μM NA-PP1-containing 1/2 MS medium supplemented with or without 50 mM or 75 mM NaCl. Pictures were taken and the elongated root length was determined 3 d later (A-B). Changes in survival rates (C) and fresh weights (D) were examined 10 d later. The fresh weight of NaCl treated seedlings/fresh weight of NaCl untreated seedlings ratios were labelled in the columns in (D). Scale Bar, 1 cm.
Figure 7A Schematic of genome-wide experiments performed on naïve and activated CD4+ T cells. B Volcano plot of MISO results for all annotated skipped exons. ΔAS exons (in red) have Bayes factor > 10 and a ΔPSI absolute value > 0.10. Within the ΔAS exons, negative ΔPSI values indicate activation-excluded exons, whereas positive ΔPSIs reflect activation-included exons. Unchanged alternative exons (AS, not Δ, dark gray) have a Bayes factor < 10 and a ΔPS| absolute value < 0.10. C Proximity of exons to CTCF. Genomic coordinates corresponding to alternatively spliced (ΔAS, red), unchanged alternative (AS, not Δ, gray), expressed constitutive (black), or all annotated constitutive exons (white) were intersected with empirically defined CTCF binding sites in naïve and activated CD4+ T cells. Exons were queried for CTCF sites that directly overlap or are located within 1.5 kb upstream or downstream, as indicated. p= Fisher's exact test. D CTCF binding sites (CBS) with overlapping 5hmC in CD4+ T cells were examined for activation-induced changes in 5hmC as determined by MedIP-seq. Results for exon proximal CTCF sites are shown, as are all intragenic CTCF sites and random intragenic bins. Input adjusted changes in methylation > 10% are indicated. E Exon proximal CTCF binding sites with activation-induced differential 5hmC were assessed for reciprocal changes in 5mC. The percent of sites with differential 5hmC and 5mC were sorted according to the directional change in methylation. The significance of differences between sites adjacent to different exon classes is indicated (p=one-sided Fisher's exact test). F ΔAS proximal CTCF binding sites with reciprocal 5hmC/5mC were examined for the impact on alternative splicing. Sites with relative increased 5hmC and decreased 5mC (shown in orange) were considered as potential sites of CTCF gain, whereas sites with decreased 5hmC and increased 5mC (shown in blue) were considered as sites of CTCF loss. Exons were sub-categorized based on CTCF location (upstream or downstream) and assessed for directional changes in splicing as either activation-included (stippled boxes) or activation-excluded (solid boxes). Pie charts show the relationship between affected exon splicing categories and reciprocal methylation at upstream or downstream CTCF binding sites as percent of exons (bottom left). Median +/- standard error of ΔPSI values for exons with up- or downstream CTCF and increased or decreased relative 5hmC are plotted (right), mean values are given above.
Screening of active members of the myotubularin family identifies Jumpy as a negative regulator of autophagy.Figure 1(A) Domains and members of catalytically active myotubularins. Asterisks, Jumpy mutations found in patients with centronuclear myopathy. C330S, R336Q and Y462C, Jumpy mutants used in this study. (B) RAW 264.7 cells transfected for 48 h with control (sc), MTMR6 or Jumpy siRNA, were pretreated for 30 min with 100 nM Bafilomycin A1 (BafA1), 10 μg/ml E64d and 10 μg/ml pepstatin, then incubated for 30 min in full or starvation media in the presence of BafA1, E64d and pepstatin. Cells were lysed and analysed by immunoblotting with anti-LC3 or anti‐actin. Densitometric LC3‐II/actin ratios are shown underneath the blot. Graph: LC3‐II/actin ratio for Jumpy siRNA in full medium is equal to or exceeds LC3‐II/actin ratio for control siRNA (sc) in starvation medium. (C, D) RAW 264.7 cells were transfected for 36 h with control (scramble) or Jumpy siRNA, transfected once more with corresponding siRNA and mRFP‐GFP‐LC3 DNA construct overnight and incubated for 2 h in full or starvation media. Cells were fixed and LC3 puncta analysed by confocal fluorescence microscopy. (C) Representative confocal images of RAW 264.7 cells in full or starvation media after transfection with control (scramble) or Jumpy siRNA (siJumpy). Red and yellow arrows indicate GFP−RFP+ and GFP+RFP+ puncta, respectively. (D) Quantitation of number of LC3 puncta per cell, total puncta per cell (GFP+RFP+ and GFP−RFP+ puncta) GFP+RFP+ puncta per cell and GFP−RFP+ puncta per cell. Data, mean±s.e.m. n=5 independent experiments, 30 cells per experiments. *P0.05, **P0.01, ***P0.001 (t‐test). Scale bars, 5 μm. (E, F) C2C12 myoblasts were transfected for 48 h with control (scramble) or Jumpy siRNA, followed by a second transfection overnight with corresponding siRNA and mRFP‐GFP‐LC3 DNA construct. Cells were fixed and LC3 puncta were counted by confocal fluorescence microscopy. (E) Representative confocal images of C2C12 cells in full media after transfection with control (scramble) or Jumpy siRNA (siJumpy). (F) Quantification of the number of LC3 puncta per cell. Data, mean±SEM for n=3 (independent experiments), 30 cells per experiments. *P 0.05 (t-test). Scale bars, 10 μm. (G, H) C2C12 cells were transfected for 48 h with control (sc) (lanes 1, 2, 5, 6) or Atg5 siRNA (lanes 3, 4, 6, 7) followed by a second transfection for 48 h with same siRNA and control (lanes 1, 3, 5, 7) or Jumpy siRNA (lanes 2, 4, 6, 8). Cells were incubated for 1 h with or without 100 nM Baf A1, 10 μg/ml E64d and 10 μg/ml pepstatin in full media, lysed and analysed by immunoblotting with anti‐LC3 or anti‐actin (G). (H) Densitometric LC3‐II/actin ratios for samples treated with BafA1 and protease inhibitors from G (lanes 5-8). Inset shows Atg5 knock‐down by immunoblotting.
Figure 2A. Results of real-time RT-PCR analysis of Trpv1, Trpv2, Trpv3 and Trpv4 expression in mouse pre-adipocytes and differentiated brown adipocytes. Expression levels of mRNA were normalized to those of 36B4. Data are presented as mean ± SEM, n = 6. * P < 0.05 vs. pre-adipocytes. Unpaired Student's t-test. B. The number of 6 day-differentiated mousebrown adipocytes treated with TRPV2 agonists, 100 µmol/L 2APB or 10 µmol/L LPC. Mean ± SEM, n = 6, * P < 0.05 and ** P < 0.01 vs. dimethyl sulfoxide (DMSO) group. One-way ANOVA followed by 2-tailed t-test with Bonferroni correction. C and D. Comparison of the numbers of 6 day-differentiated mouse brown adipocytes (C) and triglyceride levels (D) in the cells from WT and TRPV2KO mice with different differentiation media. 1/10 suppl. indicates differentiation medium ten-time-diluted with DMEM. Mean ± SEM, n = 8, ** P < 0.01 vs. control group, ##P < 0.01 vs. WT group. One-way ANOVA followed by 2-tailed t-test with Bonferroni correction.
Figure 2male C57BL/6J mice fed with chow diet supplemented with 0, 0.5%, 1% SUC for 8 weeks. The muscle grip strength male C57BL/6J mice fed with chow diet supplemented with 0, 0.5%, 1% SUC for 8 weeks. running time in low speed male C57BL/6J mice fed with chow diet supplemented with 0, 0.5%, 1% SUC for 8 weeks. four-limb handing time male C57BL/6J mice fed with chow diet supplemented with 0, 0.5%, 1% SUC for 8 weeks. running time in high speed. male C57BL/6J mice fed with chow diet supplemented with 0, 0.5%, 1% SUC for 8 weeks. serum concentration of RBC male C57BL/6J mice fed with chow diet supplemented with 0, 0.5%, 1% SUC for 8 weeks. HGB in whole blood male C57BL/6J mice fed with chow diet supplemented with 0, 0.5%, 1% SUC for 8 weeks. ex vivo gastrocnemius muscle force male C57BL/6J mice fed with chow diet supplemented with 0, 0.5%, 1% SUC for 8 weeks. ex vivo gastrocnemius muscle force male C57BL/6J mice fed with chow diet supplemented with 0, 0.5%, 1% SUC for 8 weeks. fatigability male C57BL/6J mice fed with chow diet supplemented with 0, 0.5%, 1% SUC for 8 weeks. glucose consumption male C57BL/6J mice fed with chow diet supplemented with 0, 0.5%, 1% SUC for 8 weeks. lactate production were tested.
Figure 8A. Induction of autophagy in murine MCA205 fibrosarcomas. Cells were treated with 3,4 DC alone or in combination with chloroquine, and were harvested 6 hours later for immunoblot detection of LC3 lipidation. B. Schematic overview on the in vivo treatment of MCA205 fibrosarcomas with mitoxantrone (MTX) and 3,4-DC, alone or in combination. (C-E). Cytofluorometric characterization of tumor infiltrating lymphocytes (TIL), in particular FOXP3+ regulatory T cells (Treg), CD8+ cytotoxic T lymphocytes and the ratio of CD8+ T cells over Treg. Growth kinetic of MCA205 fibrosarcomas that were wild type and were evolving in immunocompetent C57Bl/6 mice treated as indicated Growth kinetic of MCA205 fibrosarcomas that were either wild type or Atg5KD (H-I) and were evolving in immunocompetent C57Bl/6 mice or immunodeficient nu/nu mice (G), treated as indicated (J-L) Immunocompetent C57Bl/6 mice were subcutaneously inoculated with TFEB/TFE3 double knockdown MCA205 cells or its scramble control cells (K). When tumors became palpable, mice were treated as indicated Tumor growth curves from mice subjected to 3,4-DC administration alone or in combination with MTX are shown (J,L). Asterisks indicate significant effect of MTX with respect to untreated controls (mean value ±SEM, *p<0.05, **p<0.01, ***p<0.001), while hash symbols refer to the comparison of the effects of MTX plus 3,4-DC to MTX alone (#p<0.05, ##p<0.01, ###p<0.001)(n=6-14).