EMBO/soda-nel huggingface dataset:
Negative regulation of the NEMO signaling by the ubiquitin E3 ligase MARCH2 (2020)
Figure 3

Figure 3. Deficiency of MARCH2 leads to augmented innate immune responses upon microbial infection in immune cells.

A-C BMDMs isolated from MARCH2+/+ or MARCH2-/- mice were infected with PR8-GFP (A, MOI=3), VSV-GFP (B, MOI = 5), or HSV-GFP (C, MOI = 3), and virus titer was measured in a plaque assay.

D, E BMDMs isolated from MARCH2+/+ or MARCH2-/- mice were infected with viruses or treated with poly(I:C) (80 µg/ml) or poly (dA:dT) (1 µg/ml). The concentration of secreted IFN-β (D) and IL-6 (E) in the supernatants was measured in an ELISA.

F, G PBMCs isolated from whole peripheral blood from MARCH2+/+ or MARCH2-/- mice were infected with VSV-GFP (MOI =1). (F) The virus titer was measured in a plaque assay. (G) Concentration of IFN-β and IL-6 in the supernatant was measured in an ELISA.

H, I PMs isolated from MARCH2+/+ or MARCH2-/- mice infected with CVB-GFP (MOI = 3). (H) Virus titer was measured in a plaque assay. (I) Concentration of IFN-β and IL-6 in the supernatant was measured in an ELISA.

J, K BMDMs (J) or PMs (K) isolated from MARCH2+/+ or MARCH2-/- mice were infected with Salmonella typhimurium or L. monocytogenes, or treated with LPS or zymosan. The concentration of IL-6 and IL-12 in supernatant was analyzed in an ELISA.

L, M RAW 264.7 cells transfected with control siRNA (si-control) or MARCH2-specific siRNA (si-MARCH2) were infected with PR8-GFP (MOI = 1), VSV-GFP (MOI = 0.5), poly (I:C) (20 µg/ml) or HSV-GFP (MOI = 1) poly (dA:dT) (1 µg/ml) and IFN-β (L), and IL-6 (M) levels in the supernatant were measured in an ELISA.

N RAW 264.7 cells transfected with control siRNA (si-control) or MARCH2-specific siRNA (si-MARCH2) were infected with S. typhimurium, or L. monocytogenes, or treated with LPS (100 ng/ml) or zymosan (100 µg/ml) and IL-6 secretion into the cell supernatant was measured in an ELISA

Data information: *P < 0.05, **P < 0.01, ***P < 0.001 (two-tailed Student's t-test). Data are representative of three independent experiments, each with similar results, and expressed as the mean ± S.D. of two biological replicates.

Source data are available online for this figure.
Negative regulation of the NEMO signaling by the ubiquitin E3 ligase MARCH2 (2020)
Figure 4

Figure 4. MARCH2 negatively regulates innate immunity by interacting with NEMO.

A-D HEK293T cells were transfected with a firefly luciferase reporter plasmid encoding the IFN-β promoter (A, B) or the NF-κB promoter (C, D), plus a TK renilla plasmid and an increasing dose of flag-tagged MARCH2 plasmid (50, 100, 200, 400 ng) plus expression plasmids for RIG-I 2CARD, RIG-I, MDA-5, MAVS, TBK1, TRAF 6, TRAF 2, NEMO or TRIF, or stimulated with poly (I:C), for 24 h. Results are expressed relative to those of renilla luciferase alone (Internal control).

E, F Interaction between MARCH2 and NEMO in response to viral infection. HEK293T cells (E) or Raw264.7 cells (F) were infected with NDV-GFP in a time-dependent manner in the presence of MG132 (proteasome inhibitor, 10 μM). Cell lysates were subjected to immunoprecipitation with an anti-NEMO antibody, followed by immunoblotting with an anti-MARCH2 antibody.

G Confocal microscopy was conducted to examine time-dependent colocalization of MARCH2 and NEMO in HeLa cells upon NDV infection (MOI = 1) in the presence of MG132 (10 μM). Arrow indicates the co-localized NEMO and MARCH2 protein.

H-I Interaction between MARCH2 and NEMO in response to bacterial infection. Raw264.7 cells were infected with L. monocytogenes (H) or S. typhimurium (I) in a time-dependent manner in the presence of MG132. Cell lysates were subjected to immunoprecipitation with an anti-NEMO antibody, followed by immunoblotting with an anti-MARCH2 antibody.

J Confocal microscopy was conducted to examine time-dependent colocalization of MARCH2 and NEMO in HeLa cells expressing TLR2 upon zymosan treatment (100 μg/ml) in the presence of MG132 (10 μM). Arrow indicates the co-localized NEMO and MARCH2 protein.

Data information: Scale bar (G, J), 10μm. ns, not significant. *P < 0.05, **P < 0.01, ***P < 0.001 (two-tailed Student's t-test). Data are representative of at least two independent experiments, each with similar results, and are expressed as the mean ± S.D. of three biological replicates.

Source data are available online for this figure.
Negative regulation of the NEMO signaling by the ubiquitin E3 ligase MARCH2 (2020)
Figure 6

Figure 6. MARCH2 interacts with CC1 domain of NEMO via TM domain.

A, B (A) Schematic representations of the domain constructs of MARCH2. (B) HEK293T cells were transfected with Strep-tagged MARCH2 domain constructs together with GST-tagged NEMO. Whole cell lysates were subjected to a Strep-PD assay, followed by immunoblotting with an anti-GST antibody.

C, D (C) Schematic representations of the domain constructs of NEMO. (D) HEK293T cells were transfected with GST-tagged NEMO domain constructs together with Strep-tagged MARCH2-TM. Whole cell lysates were subjected to a Strep-PD assay, followed by immunoblotting with an anti-GST antibody.

E, F (E) Schematic representations of the deletion mutant constructs of NEMO. (F) HEK293T cells were transfected with HA-tagged NEMO domain constructs together with GST-tagged MARCH2. Whole cell lysates were subjected to a GST-PD assay, followed by immunoblotting with an anti-HA antibody.

G HEK293T cells transfected with His-tagged ubiquitin and HA-tagged NEMO WT or NEMO lacking the CC1 domain together with Strep-tagged empty vector or MARCH2 were treated with MG132 (10 μM) for 6 h before harvest. Lysates were subjected to immunoprecipitation with an anti-HA antibody, followed by immunoblotting with an anti-His antibody. WCL were subjected to immunoblotting with anti-HA, anti-Strep, or anti-β-actin antibody.

H, I (H) Schematic representation of the domain constructs of NEMO. (I) HEK293T cells transfected with GST-tagged NEMO mutants together with Strep-tagged empty vector or MARCH2 were subjected to immunoblotting with anti-GST, anti-Strep, or anti-β-actin antibody.

J HEK293T cells transfected with HA-tagged ubiquitin and GST-tagged NEMO mutants together with Strep-tagged empty vector or MARCH2 were subjected to pulldown with GST beads, followed by immunoblotting with anti-Lys48 (K48)-linkage specific polyubiquitin (K48-ub), anti-Lys63 (K63)-linkage specific polyubiquitin (K63-ub), or anti-Strep antibody. Whole cell lysates were determined by immunoblotting with the indicated antibodies.

Data information: Data are representative of three independent experiments, each with similar results.

Source data are available online for this figure.
Negative regulation of the NEMO signaling by the ubiquitin E3 ligase MARCH2 (2020)
Figure 7

Figure 7. MARCH2 targets Lys 326 of NEMO for K48-linked polyubiquitination.

A HEK293T cells were transfected with GST-tagged NEMO-WT and its point mutants with or without Strep-tagged MARCH2. Whole cell lysates were subjected to immunoblotting with the indicated antibodies.

B HEK293T cells were transfected with GST-tagged NEMO-WT and its point mutants together with Strep-tagged MARCH2 in the presence of MG132. Whole cell lysates were subjected to a GST-PD assay, followed by immunoblotting with an anti-K48 linkage specific polyubiquitin antibody.

C In vitro ubiquitination assay for NEMO-K326R. HEK293T cells were transfected with GST-tagged NEMO, GST-tagged NEMO-K326R or Strep-tagged MARCH2 individually. Immunoprecipitates were incubated with a reaction mixture containing ubiquitin, E1, and UbcH6 (E2), followed by immunoblotting with an anti-K48-linkage specific polyubiquitin antibody.

D-G HEK293T cells transfected with GST-tagged empty vector, NEMO-WT, or NEMO-K326R, with or without Strep-tagged MARCH2 were infected with VSV-GFP. (D-F) Viral replication was determined by fluorescence microscopy (D), fluorescence absorbance (E), and a plaque assay (F). (G) IFN-β and IL-6 in the cell supernatants were measured in an ELISA.

H, I IFN-β (H) and NF-κB (I) luciferase reporter assay.

Data information: ns, not significant. *P < 0.05, **P < 0.01, ***P < 0.001 (two-tailed Student's t-test). Data are representative of at least two independent experiments, each with similar results, and expressed as the mean ± S.D. of three biological replicates.

Source data are available online for this figure.

Expanded View Figures legend
The Arabidopsis Cdk1/Cdk2 homolog CDKA;1 controls chromosome axis assembly during plant meiosis (2019)
Figure 1

Figure 1. Changes in CDKA;1 distribution and meiotic defects in hypomorphic cdka;1 mutants in male meiocytes.

(A) Confocal laser scanning micrographs showing the localization of a functional CDKA;1:mVenus fusion protein in the wildtype (WT) and cartoons on top highlighting the changes in abundance of CDKA;1:mVenus in the nucleus and cytoplasm during the course of meiosis. The region colored in beige represents the cytoplasm, in green the nucleoplasm, and in white the nucleolus. Scale bar: 10 μm.

(B) Quantitative analysis of the signal distribution of the nuclear versus cytoplasmic fraction of CDKA;1:mVenus during prophase I of meiosis as revealed by live cell imaging (Movie EV1). Twenty cells at each time point were used for the analysis. Error bars represent mean ± SD and two biological replicates were performed.

(C) Immunolocalization of CDKA;1 (green) and ASY1 (red) on spread chromsomes in leptotene and zygotene of wild-type plants expressing a functional PROCDKA;1:CDKA;1:Strep construct. The last lane shows a magnification of the region marked by the red rectangle. Arrowheads indicate synapsed regions of homologous chromosomes where CDKA;1 is no longer present. Scale bar: 5 μm.

(D) Chromosome spread analysis of the wildtype and the hypomorphic cdka;1 mutant CDKA;1T161D. (a, h) zygotene or zygotene-like stages; (b, i) pachytene or pachytene-like stages; (c, j, k) diakinesis or dikinesis-like stages; (d) metaphase I; (e, i, m, n) end of meiosis I with two (e, m) or three (i) pools of chromosomes; (f) metaphase II; (g) tetrad. Red arrowheads indicate the initiated formation of a phragmoplast. White arrowheads depict mitochondria. Scale bars: 10 μm.
The Arabidopsis Cdk1/Cdk2 homolog CDKA;1 controls chromosome axis assembly during plant meiosis (2019)
Figure 2

Figure 2. ASY1 is a phosphorylation target of CDKA;1.

(A) ASY1:GFP and ASY1T142D:GFP localization in late G2 and leptotene of male meiocytes of the wildtype and CDKA;1T161D mutants. ASY3:RFP, highlighting chromsomes, was used as a marker for the staging of meiosis. Scale bar: 5 μm.

(B) Kinase assays of CDKA;1-SDS, -TAM, and -CYCA3;1 complexes using ASY1 puried from baculovirus-infected insect cells as a substrate. The upper panel shows the autoradiograph. The control reaction without CDKA;1-cyclin complex indicates a background activity co-purified from insect cells. The lower panel indicates protein loading by coomassie briliant blue (CBB) staining. Arrowheads indicate ASY1 proteins and asterisks depict the relevant cyclin used which also gets phosphorylated in the assay. Numbers indicate the relative intensities of ASY1 bands.

(C) The upper panel shows a phos-tag gel analysis of ASY11-300 and ASY11-300/T142V;T184V with and without CDKA;1-SDS kinase complexes using an anti-MBP antibody. The lower panel denotes loading of CDKA;1 using an anti-Strep antibody. Arrowheads represent the proteins as indicated.
The Arabidopsis Cdk1/Cdk2 homolog CDKA;1 controls chromosome axis assembly during plant meiosis (2019)
Figure 3

Figure 3. Phosphorylation of ASY1 is essential for its chromosomal localization. (A) Schematic representation of ASY1 with the five predicted consensus Cdk phosphorylation sites. The sites found to be phosphorylated in vitro by CDKA;1-SDS complexes are highlighted in red (Appendix Fig S4A).

(B) Localization patterns of different ASY1:GFP variants together with ASY3:RFP (for staging of zygotene and pachytene) in a asy1 mutant background during prophase I. Bar: 5 μm.

(C) Signal distribution profiles of ASY1:GFP and ASY1T142V:GFP at leptotene as shown in (B). The regions used for analysis are highlighted by white lines in respective panels in (B). The many small peaks with low amplitude in ASY1T142V:GFP indicate diffused localization as opposed to the clear peaks seen in the wildtype.

(D, E) Immunolocalization of ASY1 (D) and ZYP1 (E) in ASY1:GFP (asy1) and ASY1T142V:GFP (asy1) plants using anti-GFP and anti-ZYP1 antibodies, respectively. DNA was stained with DAPI (blue). Scale bars: 5 μm.
The Arabidopsis Cdk1/Cdk2 homolog CDKA;1 controls chromosome axis assembly during plant meiosis (2019)
Figure 4

Figure 4. A negative charge at T142 in the HORMA domain of ASY1 promotes its interaction with ASY3.

(A) Yeast two-hybrid interaction assays of ASY3 with different ASY1 variants. Monomeric GFP (mGFP) fused with AD (activating domain) and BD (binding domain) were used as controls. Yeast cells harboring both the AD and BD plasmids were grown on synthetic medium supplied with glucose in the absence of Leu and Trp (-L/T, left panel), on Synthetic Drop-out (SD) medium in the absence of Leu, Trp, and His (-L/T/H, middle panel), and on SD medium in the absence of Leu,Trp, His and Ade (-L/T/H/A, right panel). Yeast cells were incubated until OD600 = 1 and then diluted 10-, 100- and 1000-fold for the assays.

(B) GST pull down of ASY3 with different ASY1 variants. The numbers above the bands show the relative intensity of the bands. The input and pull down fractions were analyzed by immuno-blotting with the anti-GST (ASY3) and anti-MBP (ASY1) antibodies.

(C) Quantification of the pull down fractions of ASY1 as shown in (B). The band intensity in the pull down of ASY11-300/T142V;T184V at a Triton X-100 concentration of 0.5% was defined as 1. The relative amount of ASY1 in the pull down fractions was normalized by the band intensity of the pulled-down ASY3 fraction. The average band intensity of ASY1 at different concentrations of Triton X-100 used was plotted. Asteriks indicate significant difference (two-tailed t-test, p < 0.05). Error bars represent mean ± SD and two biological replicates were performed..
The Arabidopsis Cdk1/Cdk2 homolog CDKA;1 controls chromosome axis assembly during plant meiosis (2019)
Figure 5

Figure 5. Phosphorylation of ASY1 counteracts the action of PCH2 in early prophase.

(A) Localization patterns of ASY1T142V;T184V:GFP together with ASY3-RFP in the wildtype and pch2 mutants. Please note that images of ASY1T142V;T184V:GFP in pch2 mutants were taken with increased sensitivity for a better visibility. Scale bar: 5 μm.

(B) Seed sets (mean ± SD, n = 5) of WT, asy1, pch2, asy1 pch2, ASY1T142V;T184V:GFP (asy1) and ASY1T142V;T184V:GFP (asy1 pch2) plants. Asteriks indicate significant differences (two-tailed t-test, P < 0.01) and ns depicts no significant difference. Scale bar: 2 mm.

(C) Localization patterns of ASY1:GFP, ASY1T142V;T184V:GFP, and ASY11-570:GFP in the wildtype (WT) and/or in pch2 mutant plants at early prophase I. Scale bars: 5 μm.

(D) Localization pattern of PCH2:GFP together with ASY1:RFP in the male meiocytes of wildtype. Arrowheads indicate the chromosomal regions where the ASY1 removal was concomitant with the localization of PCH2. Scale bar: 10 μm.
The Arabidopsis Cdk1/Cdk2 homolog CDKA;1 controls chromosome axis assembly during plant meiosis (2019)
Figure 6

Figure 6. Phosphorylation of ASY1 affects its self-assembly.

(A) Schematic graph of ASY1 full length protein (aa 1 to 596). The HORMA domain is depicted in orange, the presumptive closure motif is highlighted in blue. The lines below indicate the constructs used for yeast two-hybrid interaction assays.

(B) Interaction assays of different ASY1 fragments (with and without the closure motif).

(C) Interaction analysis of the ASY1 closure motif (ASY1571-596) with different ASY1 HORMA domain variants.
Striatal infusion of cholesterol promotes dose-dependent behavioral benefitsand exerts disease-modifying effects in Huntington's disease mice (2020)
Figure 4

Figure 4 Striatal infusion of the high dose of cholesterol increases endogenous cholesterol catabolism and synthesis in the striatum of HD mice.

(A) Schematic pathway of cholesterol synthesis and cholesterol precursors and catabolites in the brain. Green arrows indicate enzymes with downregulated mRNA in HD, and red arrows indicate cholesterol precursors or metabolites that were decreased per ID-MS; SREBP2-dependent genes are in bold. Here, lanosterol, 7-lathosterol, desmosterol, and 24S-OHC (highlighted in the boxes) were measured by ID-MS. References: 1) Bobrowska, 2012; 2) Lee, 2014; 3) Valenza, 2005; 4) Samara, 2014; 5) Boussicault, 2016; 6) Kacher, 2019; 7) Valenza, 2007a; 8) Valenza, 2007b; 9) Shankaran, 2017; 10) Valenza, 2010; 11) Valenza, 2015.

(B) 24S-OHC level measured by mass spectrometry in the infused and contralateral striatum of wt, R6/2‐ACSF, and R6/2 chol-high mice at 12 weeks of age after a 4-week striatal infusion of cholesterol (N=4/group). All values are expressed as % above the mean of wt, and these data are shown as scatterplots with means±standard error. Each dot corresponds to the value obtained from each animal.

(C-E) Lanosterol (C), lathosterol (D), and desmosterol (E) level measured by mass spectrometry in the infused and contralateral striatum of wt, R6/2 ACSF, and R6/2 chol-high mice at 12 weeks of age after 4-week striatal infusion of cholesterol (N=4/group). All values are expressed as % above the mean of wt, and these data are shown as scatterplots with means±standard error. Each dot corresponds to the value obtained from each animal.

(F-I) Nuclear (white arrows) and perinuclear (yellow arrows) localization of endogenous SREBP2 in the striatum of wt, R6/2 and R6/2-chol mice. Representative confocal image (F and H) and relative quantification (G and I) of SREBP2 (red) in wt and R6/2 mice and in the infused and contralateral striatum of R6/2 chol-high mice (N=4). Hoechst (Ho, blue) was used to counterstain nuclei. Scale bar in F and H: 10 µm. Graphs in (G and I) represents the intensity of SREBP2 normalized on nuclei (%). Data are shown as scatterplots with means±standard error. Each dot corresponds to the value obtained from each image. Statistics: Student's t-test (**p<0.01; ****p<0.0001).

J, K Representative high-magnification confocal images of immunostaining against SREBP2 (red) and NeuN (J) or GFAP (K) (green) on coronal sections of brains from R6/2 chol-high mice. A cell in the infused striatum, positive for NeuN and GFAP respectively, is shown. Hoechst (Ho, blue) was used to counterstain nuclei. Scale bars: 1 µm.
Striatal infusion of cholesterol promotes dose-dependent behavioral benefitsand exerts disease-modifying effects in Huntington's disease mice (2020)
Figure 5

Figure 5 Striatal infusion of the high dose of cholesterol rescues muHTT aggregation in the striatum of HD mice.

(A-G) Immunolabeling of muHTT aggregates (red) in R6/2 ACSF and R6/2 chol-high mice (N=3/group). Zoom of representative confocal images of immunostaining against muHTT aggregates (red) showing muHTT aggregates positive for EM48 antibody in the infused and contralateral striatum (A) and relative quantification of number (B) and size (C) of aggregates. Hoechst (Ho, blue) was used to counterstain nuclei. 18 images/animal were analyzed from 9 sections throughout the entire striatum. Representative confocal images of immunostaining against muHTT (red) and DARPP32 (D) or GFAP (F) (green) showing muHTT aggregates positive for EM48 antibody in the infused striatum in neurons or astrocytes and relative quantification (E, G). Hoechst (Ho, blue) was used to counterstain nuclei. All values are expressed as % above the mean of aggregates in the contralateral striatum of R6/2 ACSF or of R6/2 chol-high. The data in (B, C, E, and G) are shown as scatterplots with means±standard error. Each dot corresponds to aggregates counted in all the images from 3 animals. Scale bars: 10 µm (A) and 5 µm (D, F). Statistics: one‐way ANOVA followed by Newman-Keuls multiple comparison tests (*p<0.05; **p<0.01; ****p<0.0001).

(H) TEM images of EM48 pre-embedding immunogold labelling showing muHTT aggregates in the striatal neuron cell bodies of R6/2 ACSF mice and R6/2 chol-high mice (upper panels). In the lower panels, muHTT aggregates are clearly visible and the magnifications show the area indicated by the black square in the upper images. Arrows indicate large muHTT aggregates with a fibrous structure in the nucleus, and arrowheads indicate single 10-nm gold particles in MSN. Nucleus (N), nucleolus (n), mitochondrion (m), rough endoplasmic reticulum (rer), Golgi apparatus (g), and white arrows indicate the nuclear envelope (N=3 animals/group). Scale bars: 700 nm and 300 nm.

(I-M) Quantification of aggregated and total muHTT in the infused striatum of HD mice after 4-week cholesterol infusion by TR-FRET analysis using different antibody pairs. Schematic representation of employed TR-FRET assay (I). Preliminary assessment of the sustainability of the assay in wt and R6/2 striata (N=5/group) using 4C9-4C9 and 2B7-MW1 antibodies in combination to detect, respectively, muHTT aggregates (J) and total muHTT (K). Quantification of muHTT aggregates (L) and soluble and other muHTT species (M) in the infused striata of R6/2 ACSF and R6/2 chol-high mice. Data in (J-M) are shown as scatterplots with means±standard error. Each dot corresponds to the value obtained from one striatum. Statistics: student's t-test (*p<0.05).
Striatal infusion of cholesterol promotes dose-dependent behavioral benefitsand exerts disease-modifying effects in Huntington's disease mice (2020)
Figure 6

Figure 6 Striatal infusion of the high dose of cholesterol promotes lysosomal clearance and autophagy in the striatum of HD mice.

(A-D) Representative confocal images showing p62 (A, red) or LAMP1 (C, green) in the infused and contralateral striatum on brain coronal sections from wt, R6/2 ACSF, and R6/2 chol-high mice (N=3/group). Hoechst (Ho, blue) was used to counterstain nuclei. Quantification of dots for p62 (B) and density for LAMP1 (D) in the contralateral and infused striatum of R6/2 ACSF and R6/2 chol-high mice. 20 images from 3 sections in the middle of the striatum for each animal were acquired and analyzed. The data in (B and D) are shown as scatterplots with means±standard error, and each dot corresponds to the value obtained from each image. Statistics: one‐way ANOVA followed by Newman-Keuls multiple comparison tests (***p<0.001; ****p<0.0001). Scale bars in (A, C): 10 µm.

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Inactive variants of death receptor p75NTR reduce Alzheimer's neuropathology by interfering with APP internalization (2020)
Figure 1

Figure 1. Neurons lacking p75NTR transmembrane Cys259, the death domain or the whole receptor are equally resistant to Aβ neurotoxicity in vitro

(A) Representative photomicrographs of MAP2 (green) and cleaved caspase-3 (red) immunocytochemistry of embryonic cortical neurons from wild type (upper panels) or C259A mice treated with vehicle or 10µM Aβ oligomers for 14h. Scale bar 15µm.

(B) Quantification of active (cleaved) caspase-3 in cultured cortical neurons of the indicated genotypes treated for 14h with vehicle or Aβ oligomers (2.5, 5, or 10 µM). Data are expressed as percentage cleaved caspase-3 and MAP2 double positive cells over the total number of MAP2 positive cells. Error bars indicate SD. N=3 independent experiments each performed in duplicate. *, P<0.05; **, P<0.01 vs. vehicle (Student's t-test).

(C) Quantification of neurite length in in cultured cortical neurons of the indicated genotypes treated for 24h with vehicle or Aβ oligomers (2.5, or 10 µM). Data are expressed as mean neurite length in individual neurons, relative to wild type vehicle (set to 100 units). Error bars indicate SD. N=3 independent experiments, n≥35 neurons per experiment. *, P<0.05 vs. vehicle (Student's t-test).