EMBO/sd-character-level-ner huggingface dataset:
(a) Widefield immunofluorescence images of PFA fixed HeLa cells showing endogenous FLCN and LAMP1 staining in (top) normal growth or (bottom) starvation conditions. Orange arrows highlight association of FLCN with LAMP1 positive membranes in the peri-nuclear region. White dotted line shows cell periphery in starved condition. Scale bar = 10μm.(b) (left) Quantification of FLCN/LAMP1 association in growth and starvation conditions. A cell is scored as positive if 5 or more discrete FLCN/LAMP1 puncta were observed at 100x magnification using a widefield microscope. Data represents 60 cells from 3 independent experiments, error bars show S.E.M, *** = p<0.001. (right) Western blot shows levels of phosphorylated of S6K and 4EBP in whole cell extracts under the same conditions.
(B-F) Suppression of X-ray-induced autophagy in PPM1D−/−thymocytes. (D) The indicated thymocytes were X-ray irradiated (5 Gy), and 3 hr later, thymocytes were subjected to EM analysis. An autophagic vacuole (arrowhead) can be seen in an irradiated PPM1D+/+thymocyte.
G, Fluorescent in situ hybridization for cd74a, tnfa, and ctsc at 7 dpi, respectively. mpeg1 was used to label all MC cells. The blue boxed region is highlighted in the zoom-in image to the right. Scale bar = 50 μm. A stands for arium, V stand for ventricle and OFT stands for outflow tract. White dashed lines outline the heart and the yellow dashed lines indicate approximate resection plane.
(A, B) Production of cytokines TNF-α, IL-1, IL-6, CXCL15 (A), or chemokines Ccl5, Cxcl10, Icam1 (B) in the A20-/-Socs6+/+, A20+/+Socs6+/+, A20-/-Socs6-/- and A20+/+Socs6-/- BMDMs after Mtb stimulation. Data are shown as the mean ± s.e.m. of n = 3 biological replicates.
(A Representative images of HUVECs (A; n=6) sprouting in a fibrin gel with VEGF (25ng/ml) in the presence or absence of COCO. (D Quantification of tube surface area of micrographs shown in (A, (D: *P=0.0119 (- vs COCO 30ng/ml); *P=0.0108 (- vs COCO 60ng/ml)
Bar graph comparing the number of the cell:cell adhesion proteins identified in each protein interaction dataset. All scale bars 10μm. All error bars represent s.e.m.
C. Representative ATPase activity assay (ATP regenerating system). At the indicated time, 200 nM ConA is added to the assay.
(E) Immunoblots of cell lysates to analyze phosphorylated TAK1 by mouse peritoneal macrophages isolated from WT or Galectin-9 KO mice stimulated with AG (1 μg/ml) for indicated times; GADPH of cell lysates served as the loading control. Data are representative of at least n=3 independent experiments.
(E, F) Exercise capacity of MCK-KO and WT mice from the high intensity regiment (n = 5 mice, means ± SEM, two-tailed student's t-test).
A panel of selected breast cancer and near normal cell lines was reverse-transfected with 5 nM pooled CEP55 siRNAs and cell viability determined after 6 days. Cell viability relative to its own respective control transfected with scramble siRNA was calculated. Graph represents the mean± SEM of three independent experiments.
B-G. intracellular ATP levels (E-G) were measured in N2a-IP3R3 cells expressing Sig1R-FLAG (B and E), SOD1 (C and F), or both (D and G). Mean ± SEM from three independent experiments is plotted. *: p < 0.05.
A. PCR analysis of the human H49K variant of the ATPIF1 and rtTA constructs in wild-type (wt), ACTA1-rtTA (T), ATPIF1H49K-TRE (H) or double transgenic (T/H) mice.
A. Numbers of ATAC peaks called separately in ESC+2i and ESC+serum including their localization according to compartmentalization. B. Numbers of significant changed ATAC peaks in ESC+2i vs. ESC+serum and their localization according to compartmentalization. Increased chromatin accessibility sites (IAS), decreased accessibility sites (DAS). C. Average profile of normalized ATAC read counts over differential peaks in ESC+2i and ESC+serum changed ATAC peaks. The signal is plotted over +/- 1kb on the center of the ATAC peak.
E. Rabl2Q80L retained ciliary GPR161 after SAG stimulation. RPE1 cells infected as in (B) were serum-starved for 24 h, followed by DMSO (mock) or SAG treatment for 24 h. Arrows indicate cilia that are displayed in insets. At least 100 cilia were scored in each experiment and condition.
(B) Depiction of the Vav1 mutants used in these experiments. The E201A mutation is shown as a gray circle. The activity of each mutant is represented on the right. Green box, WT activity; red box, gain-of-function; blue box, loss-of-function.
A-C. Consequences of effector depletion on the endogenous Arl8b/Rab7 hybrid compartment. A. Representative confocal images of fixed HeLa cells harbouring GFP-tagged endogenous Arl8b (G-eArl8b, green), transfected with the indicated siRNAs and immunolabeled against endogenous Rab7 (eRab7, magenta). B. Colocalization (Mander's overlap) between endogenous Arl8b and Rab7 in response to effector depletion, n siC=10, n siRILP=9, n siSKIP=9 images (4≥ cells per image) analysed from 2 independent experiments. C. Immunoblot analysis for depletion efficiency of SKIP and RILP, with actin as loading control.
B) Fisher exact test to identify significantly enriched GO-terms in the PD-associated proteins in urine. Importantly, the enrichment score of the Fisher exact test does not indicate if the proteins were up- or downregulated in PD patients but rather that the regulated proteins - independent of the directionality - compared to the total urinary proteome are associated with the enriched term. All GO-terms that were significant in both cohorts are displayed (FDR < 5%
(D) Comparison of the ATP-dependent DNA unwinding activities of WT and mutant TmMR proteins. Lane 2, no protein; lane 3 and 4, WT TmMR; lane 5 and 6, Rad50 (Δ54-56) mutant; lane 7 and 8, R87E mutant; lane 9 and 10, K95E mutant; lane 11 and 12, R94E/K95E mutant; lane 13 and 14, K115E mutant. For each TmMR, reaction contains 1:2 or 1:5 ratio of DNA: protein.
DC(Ctrl) and DC(NLRC3-OE) were stimulated with LPS (100 ng/ml) for specified time. CFSE proliferation assay (D) among naive 2D2 CD4+ T cells stimulated with MOG(35-55) plus DCs treated with LPS for 48 hours.
F) Cells generated in (D) were transfected with siCtrl (siC) or siADAR1 (siA). Protein lysates were prepared 78h post-transfection, followed by SDS-PAGE and immunoblotting using the indicated antibodies (n=2).
A RIP experiments were performed using anti-myc antibodies and extracts from Trt1-myc expressing strains followed by qRT-PCR to detect G-rich TERRA, polyA+ TERRA, ARRET, TER1 and ACT1. Values represent fractions of input RNA detected in immunoprecipitated material expressed as fold increase over untagged (unt) wt strain. Bars and error bars are averages and SD from three independent experiments. * P < 0.05 (relative to unt; two-tailed Student's t test).
A, B, Representative images of scaf1-/- and scaf1+/+ females (A) and males (B) fed with the indicated diets.
B Intensity of the nucleosome band (nucl) is plotted. The value at 4 µM of yNap1-H2A-H2B (lane 13) was set to 100%. For non-specific DNA binding, the value in the absence of H2A-H2B (lane 5) was set to 100% intensity and disappearance of the free DNA AF647 band was plotted. Each curve is representative of three independent experiments performed on different days. Standard deviations are shown.
E, Dynamics of MC subpopulations during heart regeneration as shown by the proportion of each subpopulation in total nonCM or total MC.
A. Superimposition of Rb+/Ba2+ and Ba2+ bound EAAT1CRYST structures with tranD depicted in pink and green, respectively, and TM4 removed for clarity. Black mesh depicts anomalous-difference map in the absence of Rb+ contoured at 4σ around the tranD, and inset shows details of Ba2+ coordination at Na3. Residue numbering corresponds to EAAT1WT, and the coordination distance (angstrom) is parenthesis.
mRNA expression analysis of epithelial cell differentiation markers Sftpc (AEC II), Hopx (AEC I), Foxj1 (ciliated cells), Muc5ac (secretory cells) and p63 (basal cells) in BALO at days 0, 10, and 21 of culture (n=3 biological replicates with pooled cells from 4 cultures per replicate)
22 weeks old Foxd1Cre::Pdgfrb+/J mice were treated with imatinib (daily gavage, 50mg/kg bodyweight for 21 days; Foxd1Cre::Pdgfrb+/J + imatinib) and were compared to control Foxd1Cre::Pdgfrb+/J mice receiving vehicle (water; Foxd1Cre::Pdgfrb+/J + water). Aged-matched wt mice are indicated as dashed lines. Hemoglobin serum levels were significantly improved in imatinib treated mice.
A. Scheme to assess the influence of the highly connected nodes on germ layer acquisition.
HEK293T-ACE2-GFP stable cells were transiently transfected with mouse (m) IFITM plasmids or vector control for 24 h. C) Graph depicts normalized infection percentage measurements from 2-3 independent experiments, each performed in triplicate, from cells infected Bars represent averages with individual data points shown as circles. Error bars represent SD. #p<0.05 compared to vector control by ANOVA followed by Tukey's multiple comparisons test.
C HSF1WT or HSF1S121A was co-expressed with either GFP or GST-AMPKαCA in HSF1-deficient HEK293T cells. HSF1 Ser121 phosphorylation was examined by immunoblotting.
(D) TEM images of upper crypt goblet cells from control and Atg5VC transverse colons. The yellow dashed lines outline the mucin granules in the apex of the cells. Bars=2 μm.
(B) Illustration of the Mic60 redistribution upon re-expression of Mic10 in Mic10 depleted mitochondria.
Blob plot depicting selected marker genes in cell clusters. Dot size encodes the percentage of cells expressing the gene, and color encodes the average per cell gene expression level.
B In vitro Ub chain formation assay by Ubc7 with indicated Hrd1 (left) and Doa10 (right) variants in the presence of indicated Cue1 variants. Rates for reactions of mono-Ub to di-Ub and di-Ub to tri-Ub with fluorescently-labeled Ub are shown on a logarithmic scale. Values are reported as means ± standard deviation (n = 3). Significances for pairwise comparisons were determined by One-way ANOVA Test; * p < 0.05. For clarity only significances related to the "no E3" control of given reaction set are shown.
A HeLa cells were transfected with plasmids encoding CB-GFP or CB-TOP2A-GFP. Lysates of asynchronously growing cells were immunoblotted with the indicated antibodies.
D Immortalized Brca1F/−MEFs were infected with retroviruses expressing TRF2 shRNAs and/or Cre recombinase, together with a lentivirus expressing PARP1 shRNA, followed by selection with puromycin for 72 h. Cell extracts were prepared 48 h later and analysed by Western blotting as indicated. SMC1 was used as a loading control. *non‐specific band.E Quantification of the frequency of end‐to‐end chromosome‐type fusions of cells treated as in (D) represented as a percentage of fusions observed after TRF2 depletion. A minimum of 1,500 chromosomes were scored for each sample. Error bars represent SD of three independent experiments. The P‐value was calculated using an unpaired two‐tailed t‐test. NS, P > 0.05.
F. RT-qPCR confirmation of transcriptomics results for selected transcripts (MX1, MX2, IFITM1, ISG15 and IFIT1) in A549 cells treated as outlined in (A). GAPDH transcript levels were used for normalization. Data represent means and standard deviations from n=3 biological experiments (individual data points shown). Statistical significance was determined by unpaired 2-tailed t-test on ΔCt values (*p-value < 0.05; n.s. not significant).
We included 126 studies in our curated cancer metabolomics dataset, studies that described profiling in multiple cancer types were counted once for each type, giving rise to 136 "cancer vs control" cohorts spanning 18 different cancer types, 71 assessing blood, 39 tissue and 26 urine. We also included 18 diabetes mellitus type 2 studies to determine if our vote-counting method could identify distinct metabolic signatures in cancer versus diabetes.
(A) The number of AV in neurites decreased significantly in neurons exposed to the SIV-infected microglia supernatant for 3 or 24 hr, however; pretreatment with rapamycin blocked this effect. *** P<0.001, for n = 6 experiments. Scale bar, 20 µm.
A Permanent LAD occlusion was perfomed at ZT5 or ZT13. TTC staining (white, infarct; red, vital myocardium) and quantification of infarct size normalized to the left ventricle (LV). Student`s t-test; n = 4 mice for ZT5 and n = 5 for ZT13 MI; *P = 0.0041.
B qPCR analysis of CAT genes expression in the third and fourth rosette leaves from 36-day-old plants. The expression of CAT genes in the wild-type plant is given as 1. Three independent experiments were conducted. Data are represented as means ± SD, n=3. Student's t test, *p<0.05.
Rapamycin treatment recovers survival of TSC1 knockdown in P493-6 cells. Relative viable cell number counts of P493-6 (-Tet) cells expressing scrambled control shRNA or TSC1-specific sh-RNA 14 days after seeding equal number of viable cells (Trypan blue exclusion), in the presence of 30pM rapamycin where indicated (mean ± st.dev., n=3 biological replicates).
(B) Decreased synaptic PRR7 immunostaining following glutamate stimulation in DIV 21 hippocampal neurons. Epoxomicin (50 nM) added 20 min prior to stimulation did not affect this decrease (Cont=100±3.6%, Glut=32.3±1.3%; +epoxomicin Cont=99.8±4.6, Glut=42.9±1.1). (n=14-17 dendrites, >1200 puncta for each group). Statistical analyses for (A) and (B) were performed using the Mann Whitney test.
B Proportion of protein aggregation following renaturing with the ds/ss/ds oligonucleotides shown in (A).
(C) Quantitative RT-PCR analysis of selected transcripts in the Sst-Cre::ReteGFP (darker colors) and Ret-eGFPHi:IB4Neg (lighter colors) subsets displayed as differential expression compared to the Ret-eGFPHi:IB4Neg subset. Almost all the genes that are up-regulated in the Ret-eGFPLo:IB4Neg compared to the Ret-eGFPHi:IB4Neg subset are also enriched in the Sst-Cre::ReteGFP population (n=3)
C Percentage of indicated mutant animals with different levels of SQST‐1::GFP aggregates. S: strong. M: medium. N: none. >30 animals were examined in each group.
B Co-immunofluorescence staining of pre- versus post-menopausal tissue for E-cadherin (cyan), PDGFRβ (yellow) and F-actin (pink). DAPI is shown in grey. The arrowheads in (v) depict fibroblasts in direct contact with the myoepithelial layer. For 2D and 3D confocal imaging: n=13 premenopausal and n=10 postmenopausal specimens. Scale bars: wholemount and optical sections:100 μm (panels i-iv); enlargements, 30 μm (panel v).
E Envelope V1 to V3 region of HIV-1 RNAs in (D) was reverse-transcribed and PCR-amplified. The PCR products were proceeded to TA-cloning. At least 60 single clones were picked from each group and sequenced. These sequences and the standard HIV-1 sequence HXB2 were aligned. Genetic diversity index of each sequence was calculated for each group. F The schematic of CAF-1 body-mediated HIV-1 latency. Detailed information for the schematic was indicated in discussion.
I. RT-qPCR analyses of GSNOR mRNA in input and heavy polysome fraction pooled together in H2O2-treated samples (for either 8 or 24 h). H3A mRNA expression level was used as housekeeping gene control. Results represent the means ± SD of n ≥ 6 independent experiments shown as % normalized by H3A. ***, p<0.001 Vehicle: PBS.
D Endogenous, reciprocal co-IP of FOXP3 and TRAF6 in murine iTreg lysate. Naïve CD62Lhigh/CD25-/CD4+ T cells were obtained by FACS and activated with anti-CD3/CD28 antibodies (1ug and 2ug/ml, respectively) in the presence of IL-2 and TGFβ (100U/ml and 5ng/ml, respectively) for four days before lysis. Antibodies against TRAF6 (left) or FOXP3 (right) were used to pull down target proteins and their interaction partners, which were resolved under denaturing conditions and visualized by immunoblotting.
A and B) The bleach was applied to elongated membrane clusters of the LS‐TVN approximately 27 h post‐infection. The UIS4‐mCherry signal typically did not recover in the bleached area (A), although it was found to recover in some elongated membrane clusters (B).
(J, K) The H&E staining (J) and Masson's trichrome (K) of the histological sections from implanted NC and CDC20sh hBMSCs scaffold hybrids. Scale bar, 100 μm.
G) Representative TEM images of longitudinal sections of quadriceps muscle from young and old control and Mfn2KO mice and quantification of mitochondrial area and density (n=3 mice per group) (Scale bar: 0.4 µm). H) Representative TEM images of aberrant mitochondria highly present in Mfn2KO old mice. Data represent mean ± SEM. #p<0.05 Mfn2KO vs. control mice, *p<0.05 old vs. young mice.
B. BAZ2A associates with chromatin of ESC and NPC. Chromatin-bound (Chrom.) and soluble (Sol.) fractions of equivalent cell number of ESC+2i and NPCs were analyzed by western blot for BAZ2A levels. Total, total protein. Tubulin and histones are shown as loading and fractionation control.
Wild type mouse cortical neurons depleted of Nprl with shRNA had mitochondria that were insensitive to amino acids (R+L Each colored line refers to a single field of view containing 5-15 cells, and each pair of solid and dotted lines (same color) refers to the same field of view. Knockdown efficiency was monitored by western blotting of phosphorylated ribosomal S6 protein, a downstream target of the mTORC1-S6K pathway. Western blots are representative of 3 independent assays. Statistical analyses were performed using Student's two-tailed unpaired t-test
A-H) Analysis of Tbk1-/- MEFs stably expressing the indicated TBK1 alleles and infected with S. Typhimurium at 1 h p.i. Confocal microscopy of MEFs expressing the indicated GFP fusion proteins or immunolabeled for WIPI2 and infected with mCherry expressing S. Typhimurium (C-H).
B The flow cytometry figures of each group. The reactivation efficiency was indicated by the GFP-positive cells percentages which were labelled on the top right corner.
(J) Equal amount (2*105 cells) of doxorubicin-induced (250 nM for 24 hours, right flank) and abemaciclib-induced (4 μM for 8 times 24 hours, left flank) senescent MDF-3MR cells were subcutaneously injected into the same wild-type mice. At 1 dpt, 7 dpt and 15 dpt, the above-mentioned mice were injected with coelenterazine and bioluminescence from the injected MDF-3MR cells was visualized/quantified by the IVIS spectrum in vivo imaging system and quantified (n=4 mice/group). Upper panel, scheme of experimental design. Data information: Data are means ±SD. Two-way ANOVA *p<0.05, **p<0.01, ***p<0.001, N.S.=not significant.
(I) Lungs were resected and subjected to hematoxylin-eosin (H&E), trichome (collagen) and α-SMA IHC staining (scale bars, 200 μm),
Percentage of GFP+ cells indicative for CRE expression and recombination in the peripheral blood after tamoxifen administration by gavage (5 x 2 mg, 200 μl/mouse; daily). Bars represent means ± S.E.M. (Day 0 N=9/10, WT/Chk1fl/fl, Day 7 N=8/7, Day 14 N=3/3).
C) The efficiency of the removal of the duplicated region at the DNA level was assessed via qPCR analysis by normalizing the signal obtained from the duplication junction to that of the total Dmd signal. Dup18-30 untreated, n = 3-4; Dup18-30 treated, n = 5-9.
(C) Flag‐tagged DAPK (60 ng) was incubated with GST-WT beclin 1 or with GST-T119A beclin 1 (1000 ng) in the presence of Ca2+, calmodulin and ATP for 30 min. GST‐beclin 1 levels were visualized by Ponceau S staining, and phosphorylation on Thr 119 was detected by a phosphoThr 119 antibody (Western blot).
A U6 RNA was used as a nuclear RNA control (relative value set to 1 in the nuclear fraction), and glycine tRNA was used as the cytoplasmic RNA control (relative value set to 1 in the cytoplasmic fraction) (n=4 biological replicates), U6 and tRNA-Gly was used as an endogenous control.
(F) Co-IPs of PRR7 with c-Jun and FBW7 from lysates of HEK293 cells overexpressing PRR7 and FBW7. FBW7 expression increased the amount of c-Jun pulled down with PRR7 (n=4 gels for each panel, representative blots). Lower bar graphs- quantitation for Western blots show c-Jun/FBW7 binding is 41.4±13.9% greater in the presence of PRR7 (normalized for FBW7 expression). Similarly, c-Jun/PRR7 binding is 97.4±38.1% greater in the presence of FBW7 (normalized for PRR7 expression). Statistical significance was calculated using unpaired t-tests.
(A) Mapping of the Atg16L1-binding site on FIP200. Yeast two-hybrid mapping experiments showing that Atg16L1 binds to both the N-terminal and C-terminal coiled-coil regions of FIP200.
(a) Dose-dependent binding of IL-17A to fungal Crh11p (red circle) and Gas1p (light blue circle) proteins, but not to the fungal polysaccharides β-1,3-D-glucan (open circle), mannan (blue circle) and zymosan (grey circle). The lack of binding of IL-17E to Crh11p (orange circle) serves as a negative control. The optical density at 450 nm was corrected by subtracting the optical density at 570 nm (normalized OD) in the enzyme-linked immunosorbent assay. Data are mean±s.d., n=3. P0.001, Crh11p and Gas1p without versus with IL-17A (one-way analysis of variance Bonferroni post-test).
F-I. TEM images of 2 week aged retinas corresponding to either wildtype or ADAM17-/- mutants grown on either DMSO or AD4. Scale bars: 10μm.
A HeLa cells were co−transfected with FUNDC1−Myc and FLAG−ULK1. Cells were lysed and immunoprecipitated with anti−Myc antibody 24 h after transfection.
A Visualization of interaction between MAPKKK5K375M and MKK1 / MKK2 / MKK4 / MKK5 by BiFC analysis in Arabidopsis protoplasts. Venus fluorescence indicates interaction. Scale bar=10µm.
(I) Immunoblots of NMHCIIA and Gp96 levels from Gp96 IP of HeLa cells left untreated (U) or treated with LLO (0.5 nM, 15 min), SLO (1.5 μg/ml, 30 min) (SLO) or aerolysin (0.2 nM, 40 min) (AL).
(D) Indirect immunofluorescence analysis of endogenous LC3 (green) and WIPI1 (white) in old I90 cells transfected with nons (a, b) and bag3 siRNA (c, d) for 96 h. DAPI (blue) was used as a nuclear marker. Representative pictures are shown. Bar: 20 μm. Diagrams show percentage of cells counted as in Figure 3E.
A-C. Immunoblot analyses of Hog1 phosphorylation. Yeast strains (A) KT235; (B) KT209; and (C) KT234 were stimulated with the indicated concentrations of NaCl for 10 min, and phosphorylated Hog1 (Hog1-P) and total Hog1 in cell lysates were detected by immunoblotting. The relevant genotypes of the strains are indicated in the top row of each panel, and are schematically shown in the diagrams at left. The second row from the top indicates the genes carried by a single-copy plasmid that are expressed from their own promoters. vec, vector; WT, wild-type.
(b) HeLa cells stably expressing FLAG-HA-tagged FBXL2 were transfected with either siRNAs targeting FBXL2 (four different siRNAs) or a non-silencing siRNA (NS). Cells were lysed, and proteins were immunoblotted as indicated. NT, non-transfected.
Determination of the levels of mtDNA (ND1/ACTB) by qPCR of mutant cybrids transfected with MERRF mitoTev-TALE
E. Mitochondrial shape (BacMam Mitochondria-RFP; in red) in H295R/TR N-FlagFATE1 cells shown by fluorescence confocal microscopy in basal conditions and after Dox treatment. FATE1 (green), DAPI (blue). A higher magnification of merged green and red signal is shown in the insets. Scale bars, 10 μm.F. Mitochondrial fragmentation index in H295R/TR N-FlagFATE1 cells after Dox treatment (red histogram) compared to cells cultured in basal conditions (white histogram) (mean ± SEM; n=111 for basal and 101 for Dox-treated cells). ***p<0.001.
B-D Jejunal spheroids were cultured in differentiation medium with DMSO only or with 1 µM dmPGE2 and pharmacological inhibitors of EP1 (EP1i, SC 51322), EP2 (EP2i, PF 04418948), EP3 (EP3i, L-798,106) or EP4 (EP4i, L-161,982) at a concentration of 10 µM or an equivalent volume of DMSO vehicle. **p<0.01, ***p<0.001, ****p<0.0001 compared to the DMSO only group by one-way ANOVA and Dunnett's post-test. (B) Representative bright field images. Scale bars, 200 µm.
C, D. Menin-null PIME cells were treated with various concentrations of MI-463 for 48 hrs, followed by detecting Pbk expression with WB (C) and qPCR (D). qPCR data was from three independent experimets (n=3). ns, not statistically significant difference (two-tailed unpaired student's t-test).