EMBO/sd-character-level-ner huggingface dataset:
Figure 2Western blotting of the phosphorylated- NF-kB, -Akt, -JNK in PLP-150Q mouse corpus callosum at 3 months of age showing that LAQ (5 or 25 mg/kg) treatment for 2 months could dephosphorylate these proteins. Ratios of the phosphorylated proteins to their total proteins obtained from 3 independent experiments were presented on the right. One-way ANNOVA with Tukey's test. NF-kB: ***P=0.00067(left) and 0.00086(right); JNK: ***P=0.00013(left) and 0.00047(right); Akt: **P=0.0014 and ***P=0.00012. Data are mean ±SEM. In vitro phosphorylation assay of N-terminal MYRF (nMYRF). GST fusion proteins containing nMYRF were incubated overnight with brain lysates from PLP-23Q or PLP-150Q mice that were treated with vehicle or 5 mg/kg LAQ (left panel). GST fusion proteins containing wild type N-terminal MYRF, S259A, or S261A were incubated with PLP-150Q mouse tissue lysates (right panel). The beads were then centrifuged and analyzed by western blotting with anti-GST (lower panels) and anti-phosphor-serine (upper panels). Note that LAQ treatment could eliminate mutant HTT (PLP-150Q)-mediated MYRF phosphorylation and that S259A substitution prevents MYRF phosphorylation. Co-immunoprecipitation of nMYRF and N-terminal HTT (1-212 aa) containing 150Q in HEK293 cells. Five μM LAQ treatment decreased the phosphorylation of immunoprecipitated MYRF and the amount of co-immunoprecipitated HTT. Λ-PPase served as the positive control for the dephosphorylation of MYRF. The ratios of the pho-serine MYRF or precipitated HTT to immunoprecipitated MYRF are shown under the blots, which were obtained from 3 independent western blotting experiments. One-way ANNOVA with Tukey's test. ***P=6.51x10-5. Data are mean ±SEM. Co-transfection of nMYRF with N-terminal mutant HTT in HEK293 cells and immunoprecipitation of nMYRF with or without LAQ (5 μM) treatment. Coomassie blue staining confirms the presence of immunoprecipitated N-terminal MYRF bands (arrow). Transcriptional activity of wide type N-terminal MYRF, S259A, or S261A with the MBP promoter reporter in HEK293 cells was detected using a luciferase assay. Compared to wide type MYRF or S261A, S259A (non-phosphorylated) remained transcriptional activity that was not inhibited by N-terminal mutant HTT. The ratios were obtained from 3 independent experiments. One-way ANNOVA with Tukey's test. ***P< 0.001. Data are mean ±SEM. Co-transfection of wide type N-terminal MYRF, S259A, or S261A with N-terminal mutant HTT in HEK293 cells and immunoprecipitation of MYRF. Compared to wide type MYRF and S261A, less S259A (non-phosphorylated) was precipitated with mutant HTT. The ratios of the pho-serine MYRF or precipitated HTT to immunoprecipitated MYRF are shown under the blots and were obtained from 3 independent experiments. One-way ANNOVA with Tukey's test. ***P=2.30x10-5 . Data are mean ±SEM.
Figure 3A-C, Micrographs showing examples of sections of E15 cerebral cortex 2 days after IUE of the plasmids indicated in (A-C) with magnifications showing the indicated immunostainings. Scale bars: 100 µm and 20 µm for magnifications. Filled arrows show electroporated cells expressing Pax6 and Ki67 proteins. Open arrows show electroporated cells negative for Pax6 and Ki67 staining. VZ: ventricular zone; SVZ: subventricular zone; IZ: intermediate zone; CP: cortical plate; V: ventricle. D, Bar and dot plot showing the distribution as percent of GFP+ cells/bin after IUE and staining as in A-C. A cortical column reaching from the ventricular surface (bottom in A) to the pial surface (top in A) was divided into equal sized bins with bin1 at the ventricular surface and bin 5 at the pial surface. E-G, Bar and dot plots of the proportion of proliferating (Ki67+) cells (E), the number of NSCs (Pax6+) (F) and the proportion of proliferating (BrdU+) TAPs (Tbr2+)(G) as in A-C showing a significant increase in proliferating NSCs upon Trnp1 IUE compared to control or ∆1-16Trnp1 proteins (E- F) and a significant decrease in proliferating TAPs upon Trnp1 IUE and increase upon ∆1-16Trnp1 IUE compared to control (G) at E15, 2 days after IUE.
Figure 3Peritoneal macrophages (A) were stimulated with LPS (20 ng/mL, 20 min) Cell lysates were subjected to co-immunoprecipitation (IP) with CD14 antibody, followed by immunoblotting (IB) with TLR4 antibody. Raw264.7 cells (B) were stimulated with LPS (20 ng/mL, 20 min) in the presence/absence of ATX inhibitor (50 μM, 30 min) or vehicle (Veh, DMSO 0.1%). Cell lysates were subjected to co-immunoprecipitation (IP) with CD14 antibody, followed by immunoblotting (IB) with TLR4 antibody. The physical interaction of TLR4-CD14 was examined by FRET analysis. HEK293 cells were transfected with TLR4-EYFP (yellow) and CD14-ECFP (blue) (C) followed by ATX inhibitor (50 μM, 30 min) or vehicle treatment (Veh, DMSO 0.1%). Cells were washed with PBS and fixed, then visualized with filter sets for CFP and YFP. FRET images were visualized with the FRET filter set, expressed as corrected FRET efficiency and displayed in quantitative pseudocolor indicating the distance between proteins (arbitrary linear units of fluorescence intensity). Presented are the representative images from at least three independent experiments. Scale bar indicates 5 μm. The physical interaction of TLR4-TLR4 was examined by FRET analysis. HEK293 cells were transfected with TLR4-EYFP and TLR4-ECFP (D), followed by ATX inhibitor (50 μM, 30 min) or vehicle treatment (Veh, DMSO 0.1%). Cells were washed with PBS and fixed, then visualized with filter sets for CFP and YFP. FRET images were visualized with the FRET filter set, expressed as corrected FRET efficiency and displayed in quantitative pseudocolor indicating the distance between proteins (arbitrary linear units of fluorescence intensity). Presented are the representative images from at least three independent experiments. Scale bar indicates 5 μm.
Trpml1-/- mice develop chronic pancreatitis.Figure 3(a, b) Plasmaamylase(a) and pancreaticMPO(b) in 3 mice of each line. (c) Edema was evaluated in pancreas of mice treated with PBS or caerulein by H&amp;amp;E staining. Shown are examples of images and the average damage in the columns, which show the mean±s.e.m of 4 experiments with each mouse line. (d) Intracellular trypsin activity was measured in isolated pancreatic acini before and after stimulation with 10 nM CCK for 30 min. Shown are example images and the averages. (e) Isolated acini before and after stimulation with 10 nM CCK for 30 min were stained for the lipid ceramide. The figure shows example images before stimulation and the averaged fluorescence intensity. (f) Isolated acini before and after stimulation with 10 nM CCK for 30 min were stained with the autophagy marker LC3. The figure shows example images before stimulation and the columns depict the number of autophagosomes as determined by ImageJ. Results in (c-f) are given as the mean±s.e.m of 4 experiments from 4 mice of each line and at least 10 acini in each experiment.
Figure 3MCF10A cells were transfected with LIMT-specific siRNAs (or with control siRNA) and their migration (A, left panels) or invasion (B, left panels) were determined. Likewise, cells were stably transfected with plasmids encoding for shRNAs against LIMT (middle panels) or with plasmid encoding for either LIMT or eGFP (Control). Also shown is quantification of cell migration and invasion upon lncRNA knockdown and over-expression (OX). P-values of one-way ANOVA are presented. Each assay was repeated at least 3 times. (C and D) MCF10A cells were treated for 30 minutes with U0126 (a MEK inhibitor), or with no agent, and thereafter EGF was added (10 ng/ml) and incubated with cells for additional 30 minutes (C). Protein extracts were used to assess levels of phosphorylated ERK (C). (C and D) MCF10A cells were treated for 30 minutes with U0126 (a MEK inhibitor), or with no agent, and thereafter EGF was added (10 ng/ml) and incubated with cells for additional 4 hours (D). Isolated RNA was used to determine abundance of LIMT using real-time qPCR (D). Expression values are presented as fold-change relative to time zero. Beta-2-microglobulin was used for normalization. P-value of t-test from 4 repeats is shown. All values represent mean ± S.D. of replicates.
Nucleolar proteins recruited to mitotic chromosomes.Figure 4A. MKI67 (HPA000451). B. NOC2L (HPA044258). C. EMG1 (HPA039304). D. BMS1 (HPA043081). E. BRIX1 (HPA039614). F. RSL1D1 (HPA043483). Data information: Protein of interest is shown in green, microtubules in red and DAPI in blue. Images of interphase cells were acquired from a different experiment and staining intensities cannot be compared between interphase and mitotic cells. Scale bar 10 μm.
Figure 4A. RNA secondary structure was predicted based on MFE and partition function through RNAfold WebServer (left panel, and the colour scale represents positional entropy). The thermodynamic ensemble of RNA structures (right panel). B. RIP analysis using EZH2 antibody for the co-transfection of Flag-EZH2 and NDIME exons 1-3 (P = 0.0177) or exon 4 (P = 0.4335) in HEK293T extracts compared with IgG. C. Verification of the efficiency of overexpression of exons 1-3 or exon 4 of NDIME in NDIME-knockout cells at day 5 of neural differentiation via qRT-PCR. Three different primers P1, P2, and P3 were used to analyze NDIME or exons of NDIME expression in Ctrl, NDIME-/-, NDIME-/-+Rs26-Exon123, and NDIME-/-+Rs26-Exon4 cells (compared with control cells). The position of primers P1, P2, and P3 are shown in the schematic diagram. FACS , in Ctrl, NDIME-/-, NDIME-/-+Rs26-Exon123, NDIME-/-+Rs26-Exon4 cells at day 5 of neural differentiation. E. , expression of neural-lineage genes , in Ctrl, NDIME-/-, NDIME-/-+Rs26-Exon123, NDIME-/-+Rs26-Exon4 cells at day 5 of neural differentiation. F. Microscopy of the control, NDIME-/-, NDIME-/-+Rs26-Exon123, and NDIME-/-+Rs26-Exon4 cells at day 5 of neural differentiation. Scale bars, 100 µm. G. NESTIN and GFP immunostaining of the cells in (F) at day 5 of neural differentiation. Scale bars, 100 µm. H. Enrichment of H3K27me3 at the Mef2c promoter of the cells in (F) at day 5 of neural differentiation. I. Western blot analysis of expression of MEF2C in the cells in (F) at day 5 of neural differentiation.
Figure 5 A Oxygen consumption rate of female mice fed a diet with 0.0075% Rosi for 2.5 weeks, determined by indirect calorimetry and normalized to body weight (n=9/10). Grey bars represent the dark phase   B Oxygen consumption rate of female mice shown in (A) adjusted for body weight by ANCOVA. 3-day averages of VO2 were calculated for each mouse (n=9/10). *P=0.025 in ANCOVA with Bonferroni posttest (Cited4-/- vs. Cited4+/+)   C Oxygen consumption rate of female mice fed a diet with 0.0075% Rosi for 3.5 weeks and injected with 1 mg/kg CL-316,243 (CL) at the indicated time point, as determined by indirect calorimetry (n=9/10). *P=0.013 (-1 h), *P=0.045 (3 h), *P=0.015 (5 h), *P=0.016 (6 h), *P=0.019 (8 h), *P=0.046 (10 h) in repeated measures 2x2 ANOVA with Holm-Sidak posttests (Cited4-/- vs. Cited4+/+)   D Maximal CL-induced oxygen consumption rate of female mice shown in (C) adjusted for body weight by ANCOVA. Averages of VO2 throughout 3-8 hours post CL injection were calculated for each mouse (n=9/10). *P=0.049 in ANCOVA with Bonferroni posttest (Cited4-/- vs. Cited4+/+)   E Oxygen consumption rate of male mice treated as in (A), determined as in (C) (n=9/10). ANCOVA with Bonferroni posttest (Cited4-/- vs. Cited4+/+)   F Oxygen consumption rate of male mice treated as in (C), determined as in (C) (n=9/10). *P=0.044 (9 h), *P=0.013 (12 h) in repeated measures 2x2 ANOVA with Holm-Sidak posttests (Cited4-/- vs. Cited4+/+)   G Maximal CL-induced oxygen consumption rate of male mice shown in (F) and calculated as in (D) (n=9/10). ANCOVA with Bonferroni posttest (Cited4-/- vs. Cited4+/+)
Figure 8Shade (low R:FR) displaces phytochrome photoequilibrium towards the inactive form, allowing PIFs to promote the expression of shade avoidance related genes, such as HFR1. PIF transcript or/and protein levels are induced in response to warm temperatures, resulting in enhanced expression of growth-promoting genes. HFR1 abundance is also increased by warm temperature. HFR1 modulates these responses by heterodimerizing with PIFs and inhibiting their DNA-binding ability. As a result, HFR1 attenuates hypocotyl elongation of A. thaliana seedlings in response to shade or warm temperature. In C. hirsuta, higher HFR1 activity inhibits more effectively PIF action than in A. thaliana. In addition, PIF abundance is attenuated in C. hirsuta. Both changes alter the PIF-HFR1 balance in C. hirsuta, resulting in lower PIF transcriptional activity. As a consequence, shade- and warm temperature-induced hypocotyl elongation are repressed and DIS is delayed in this species.
Figure 1A, Immunoblot analysis of protein lysates from HeLa cells stably expressing TFEB-FLAG treated with 250 μM NaAsO2 for 1h run under either non-reducing or reducing conditions. B, Immunoblot analysis of protein lysates from HeLa cells treated with 250 μM of NaAsO2 for the indicated times and run under non-reducing conditions. C, Immunoblot analysis of protein lysates from MEF cells treated with EBSS, 250 nM Torin-1 or 100 μM NaAsO2 for 1h and run under non-reducing conditions. For refeed condition, starved cells were incubated in complete medium for the indicated times. D, Immunoblot analysis of protein lysates from ARPE-19 cells treated with 250 μM NaAsO2 or EBSS for 1h and run under non-reducing conditions. E, Immunoblot analysis of protein lysates from Raw 264.7 cells treated with 1 μg/ml LPS for the indicated times and run under non-reducing conditions. Immunoblots are representative of at least three independent experiments. F, Quantification of oligomer versus monomer ratios from Raw 264.7 cells treated with 1 μg/ml LPS, as shown in (E). Quantified results are fold change of oligomer versus monomer ratios from its corresponding vehicle treated cells. Data are presented as mean ± SD using one-way ANOVA (**)p< 0.01, from two independent experiments.
Figure 6(A) 786-0 cells expressing shGDH1 with restored expression of WT or ED GDH1 were supplemented with or without 500 μM methylated αKG for 48 hours, and then the cells were collected to extract total protein. WB was performed using the indicated antibodies. (B) In 786-0 cells expressing shGDH1 with restored expression of WT or ED GDH1, the ratio (ED/WT) of H3K9me3, H3K27me3 and H3K36me3 targeting RP genes was calculated. The outer circle in the chord diagram represents the ratio of ribosomal protein-coding genes targeted by each histone lysine methylation compared with the control. The inner circles represent the sum of the indicated ratios. The IgG was used as a control. (C) The relative enrichment of KDMs targeting ribosomal protein-coding genes in 786-0 cells. The outer circle represents the enrichment value of ribosomal protein-coding genes by each KDM. The inner circles represent the sum of the indicated values. The IgG was used as a control. (D) 786-0 cells were transfected with siRNAs targeting KDM4A or KDM6A for 56 hours, and the cells were fixed with 1% formaldehyde. ChIP assays were performed using antibodies against H3K9me3 or H3K27me3. qPCR tested the enrichment of the above histone H3 lysine modifications on the promoters of the indicated RP genes. (E) 786-0 cells were transfected with siRNAs targeting KDM4A or KDM6A and then were supplemented with or without methylated αKG (500 μM) for 48 hours. The cells were collected to extract total RNA, and qPCR was used to test the mRNA level of the indicated RP genes. (F) Isothermal titration calorimetry (ITC) was used to test the binding affinity of αKG (1 mM) to recombinant KDM4A (0.05 mM) or KDM6A (0.05 mM) (left), and then statistical analysis was performed (right). The data are represented as the means ± SEM of three independent experiments. Unpaired t-test was used (N.S.: not significant). (G) An in vitro demethylation assay was performed with recombinant KDM4A (0.05 mM) or KDM6A (0.05 mM), and the indicated histone H3 lysine methylation peptides under 50-μM αKG conditions (no αKG was used as a blank control). Various concentrations of 2HG from 0 to 200 μM was used to compete with αKG in the reaction system. (H) 786-0 cells were supplemented with or without 2HG (500 μM) for 48 hours, and the cells were fixed with 1% formaldehyde. ChIP assays were performed using antibodies against H3K9me3 or H3K27me3. qPCR was used to test the enrichment of the above histone H3 lysine modifications on the promoters of the indicated RP genes.
Phosphorylation of T166 in M18BP1 regulates metaphase localization and binding to CENP-C A) Representative immunofluorescence images showing M18BP1-1 mutant localization.Figure 5A) Representative immunofluorescence images showing M18BP1-1 mutant localization. M18BP1-depleted metaphase extract was supplemented with in vitro translated full-length Flag-M18BP1-1 mutants and sperm chromatin. In addition, extract was either mock-depleted or CENP-C depleted to assess CENP-C dependent metaphase localization. M18BP1-1 species and CENP-C depletion status are indicated at left, immunolocalized protein is indicated above. Scale bar, 10 μm. Insets are magnified 3X. B) Representative Western blot showing co-immunoprecipitation of CENP-C with phosphorylation site mutants of M18BP1-1. M18BP1-depleted metaphase or interphase extract was supplemented with in vitro translated Flag-M18BP1-1 mutants. M18BP1-1 mutation and cell cycle state indicated above, immunoblotted species indicated at right. Mock precipitations using rabbit reticulocyte lysate without any in vitro translated protein served as a negative control. C) Quantification of (A). Flag-M18BP1-1 mutant centromere intensity normalized to WT localization in the mock-depleted condition. Error bars represent SEM from three independent experiments. D) Model showing phosphorylation-dependent binding of Xenopus M18BP1 to CENP-C during mitosis (top) and the transition to CENP-A-dependent binding in interphase (bottom).
Molecular restoration of tumor-intrinsic type I IFN suppression inhibits bone-metastatic outgrowth and modulates osteoimmunity (A) Bone metastasis-free survival in WT C57BL/6 mice harboring RM1 parental, RM1 BD Irf- and RM1 BD REV (LP) tumors (n = 5 per group; **p = 0.Figure 4(A) Bone metastasis-free survival in WT C57BL/6 mice harboring RM1 parental, RM1 BD Irf- and RM1 BD REV (LP) tumors (n = 5 per group; **p = 0.0043 by log-rank (Mantel-Cox) test). (B) Schematic of enforced Irf7 expression in RM1 BD Irf- cells under exogenous promoter control. (C) ELISA of IFN-α production by RM1 parental, RM1 BD Irf- base vector (BV) and RM1 BD Irf7 over-expressing (OE) with 24 h poly I:C stimulation (n = 4). (D) Bone metastasis-free survival in WT C57BL/6 mice subsequent to IC inoculation of RM1 BD Irf- BV (n = 9) and RM1 BD Irf7 OE (n = 7) cells (**p = 0.0028 by log-rank (Mantel-Cox) test). (E) Bone metastasis-free survival in Ifnar1 -/- mice harboring RM1 BD Irf- BV and RM1 BD Irf7 OE tumors (n = 4 per group) and representative bioluminescent imaging of tumor burden in leg bones (also shown in Appendix Fig S4F). (F) Immunohistochemical (IHC) staining for cerulean (anti-eCFP; green) on naïve WT bone, RM1 parental, RM1 BD Irf- BV and RM1 BD Irf7 OE tumor-bearing bones derived from WT animals; and RM1 BD Irf7 OE tumor-bearing bones derived from Ifnar1 -/- animals at survival assay endpoints. Blue represents DAPI nuclear staining. Scale bar, 100 μm. (G-I) FACS analysis of dormant (claret+) RM1 BD Irf- and RM1 BD Irf7 OE cells from bone at (G) day 11 post-IC injection (n = 4 per group) and (h) day 17 (n = 4-6 mice group/time point) with (I) D17 active (claret-) colony quantitation (mean +1) determined by multiphoton imaging and IMARIS interrogation (n = 9 bones from 3 mice per condition). Median shown. Upper and low box hinges denote first and third quartiles. Whiskers mark value limits. (J) FACS analysis of IFN-γ+ and TNF-α+ CD8+ T cells (%) post-ICS induction of T cell (spleen-derived from tumor-bearing mice) activation by RM1 parental, RM1 BD Irf- BV and RM1 BD Irf7 OE cells (n = 6 per condition/group). (K) Mean quantitation of TRAP-stained osteoclasts differentiated with M-CSF ± RANKL in co-culture with RM1 BD Irf- BV and RM1 BD Irf7 OE cells with representative wells shown (n = 3 per condition M-CSF; n = 6 per condition M-CSF + RANKL; also shown in Appendix Fig S4J). Scale bar represents 100 μm.
Figure 1Flow cytometry analysis of PD-L1 expression in (A) KPC and (B) Pan02 cells following treatment with gemcitabine and RT. Gemcitabine was diluted in DMSO (vehicle) and DMSO was used for the 'untreated' control group (mean ± SD, n=3, One-way ANOVA, Bonferroni test). Western blot analysis of indicated proteins in both KPC (C) and Pan02 (D) cells following radiation and chemotherapy ± AG490, a JAK/Stat kinase inhibitor. Actin represents loading control. Flow cytometry analysis of PD-L1 after gemcitabine and RT (as described above) in KPC (E) and Pan02 (F) following Stat1 downregulation by siRNA (mean ± SD, n=3, Student's t-test). MFI, mean fluorescence index;
Figure 3A) Circular representation of the M. florum L1 chromosome enhanced with 5'-RACE data generated in this study. Outer to inner circle: genomic coordinates (kbp); genes encoded on the positive (blue for coding sequences, turquoise for RNAs) and negative (red for coding sequences, fuchsia for RNAs) DNA strands; raw 5'-RACE signal (0-1,000 read starts scale) observed at each genomic position for the positive (black) and negative (gray) DNA strands; putative transcription start sites (TSSs) identified on the positive (green) and negative (orange) DNA strands from significant 5'-RACE peaks. B) M. florum promoter motif determined using the MEME software (Bailey & Elkan, 1994) from the the DNA sequence located upstream the 605 putative TSSs identified by 5'-RACE. A total of 422 sites across the genome were included in the motif. The position of the -10 box (TAWAAT) and the extended element (EXT) is indicated. C) Venn diagram illustrating the number of TSSs associated with a conserved promoter motif (see panel B) found by MEME, MAST or both software compared to the total number of putative TSSs passing filters (605). Eight additional putative TSSs were added to the initial set according to the MAST search. D) Localization and orientation of TSSs associated with a MEME or MAST promoter motif. p-gTSS, parallel intergenic TSS; a-gTSS, antiparallel intergenic TSS; p-iTSS, parallel internal TSS; a-iTSS, antiparallel internal TSS. For gTSSs, the orientation was defined according to the closest downstream gene, while the overlapping gene was used in the case of iTSSs. E) Relative frequency distribution of the spacing between TSSs and their associated promoter -10 box. F) Nucleotide identity at the transcription initiation site (+1) for gTSSs and iTSSs associated with a promoter motif. G) Aggregate profile showing the mean RNA-seq read coverage observed at and around all motif-associated TSSs identified in this study. The calculated SEM is also shown. The aggregate profile was centered on the TSSs coordinates (relative position 0 bp), indicated by a gray dashed line.
Figure 4(A) Cortices from E14.5 brains stained with antibodies against the radial glial cell marker PAX6 (yellow), intermediate progenitor marker TBR2 (red) and DAPI (blue). Scale bar = 50 μm. (B and C) Quantification of the number of (B) radial glial cells (PAX6+) and (C) intermediate progenitors (TBR2+) within a 250 μm-width column of E14.5 cortices. WT N=3, Cep63T/T N = 3, Cep63T/T;Usp28-/- N = 3, Sas4cKO N = 3, Sas4cKO;Usp28cKO N = 3; one-way ANOVA with post-hoc analysis. (D) Cortices from E14.5 embryos stained with antibodies against Cleaved-Caspase 3 (CC3, white), TP53 (red) and DAPI. Scale bar = 50 μm. (E and F) Quantification of the number of CC3+ apoptotic cells (E) and cells with TP53 activation (F) within a 250 μm-width column of E14.5 cortices. WT N = 5, Cep63T/T N = 3, Cep63T/T;Usp28-/- N = 3, Sas4cKO N = 4, Sas4cKO;Usp28cKO N = 4; one-way ANOVA with post-hoc analysis.