EMBO/soda-nel huggingface dataset:
SNX10-mediated LPS sensing causes intestinal barrier dysfunction via a caspase-5-dependent signaling cascade (2021)
Figure 7

Figure 7. SNX10 deficiency protects against IL-10 deficiency-induced chronic colitis and maintains the intestinal barrier function in mice

SNX10 conditional knockout (SNX10 cKO) mice with or without IL-10 KO background and littermate wild type (WT) mice with or without IL-10 KO background were compared (n = 6 animals, each group).

A Body weight of mice was measured every two weeks from the 4th week to 16th week. Data are means ± SD. One-way ANOVA followed by Bonferroni post hoc test was used for statistical analyzes. **p < 0.01 and ***p < 0.001 vs WT group; ##p < 0.01 and ###p < 0.001 vs IL-10 KO group.

B, C Length of colons from WT, SNX10 cKO, IL-10 KO and SNX10 cKO IL-10 KO mice was measured (B) and analysed (C) at the 16th week.

D Clinical score was evaluated every two weeks from the 4th week to 16th week. Data are means ± SD. One-way ANOVA followed by Bonferroni post hoc test was used for statistical analyzes. ***p < 0.001 vs WT group; ###p < 0.001 vs IL-10 KO group.

E, F Representative H&E images of colon tissues of WT, SNX10 cKO, IL-10 KO and SNX10 cKO IL-10 KO mice at the 16th week were shown (E) and analyzed (F). Scale bar: 100 μm.

G The content of TNF-α, IL-6, IL-12/IL-23 p40 and IL-17A in the serum of WT, SNX10 cKO, IL-10 KO and SNX10 cKO IL-10 KO mice at the 16th week were measured by ELISA.

H The relative mRNA levels of Tnf, Il6, Il23a and Il17a in colon epithelial tissues of WT, SNX10 cKO, IL-10 KO and SNX10 cKO IL-10 KO mice at the 16th week were measured.

I Representative immunohistochemistry images of E-cadherin, Snail and Slug in colon tissues from WT, SNX10 cKO, IL-10 KO and SNX10 cKO IL-10 KO mice at the 16th week were shown. Scale bar: 100 μm.

J Protein expression of E-cadherin, p-Lyn and Lyn in colon epithelial tissues was determined by immunoblots.

K The relative mRNA levels of Cdh1 (encoding E-cadherin) in colon epithelial

tissues were measured.

L The gut permeability in vivo was evaluated using FITC-dextran.

Data information: In (C and F-L), data are means ± SD. One-way ANOVA followed by Bonferroni post hoc test was used for statistical analyzes. *p < 0.05; **p < 0.01; ***p < 0.001.

Expanded View Figure Legends
Irgm2 and Gate-16 cooperatively dampen Gram-negative bacteria-induced caspase-11 response (2020)
Figure 1

Figure 1: IFN-inducible protein Irgm2 restrains Caspase-11-dependent response to Gram-negative bacteria

Unless otherwise specified, BMDMs were either infected with various Gram-negative bacterial strains (MOI25) or stimulated with outer membrane vesicles (OMVs) for 16 hours.

(A) siRNA-treated BMDMs were infected for 16 hours with S. Typhimurium (orgA-) and LDH and IL-1β release were measured.

(B) Cell death (LDH) and IL-1β release evaluation in WT, Irgm2-/-, GBPChr3-/- and Casp11-/- BMDMs infected for 16 hours with different Gram-negative bacteria (MOI 25).

(C) Western blot examination of processed caspase-1 (p20) and gasdermin-D (p30) in supernatants and pro-caspase-1 (p45), pro-gasdermin-D (p55) and GAPDH in cell lysates of WT and Irgm2-/- BMDMs infected for 16 h with different Gram-negative bacterial strains.

(D) IL-1β and cell death (% LDH) evaluation in immortalized WT, Irgm2-/-, Casp11-/- and Casp11-/-Irgm2-/- (referred as sgIrgm2) BMDMs after 16 hours of E. coli, S. Typhimurium orgA- and OMV treatment.

(E) Cell death (% LDH) evaluation in IFNγ-primed WT, Irgm2-/- and Casp11-/- BMDMs 4 hours after electroporation or not with 1µg of E. coli LPS.

(F-H) Survival of WT, Casp11-/- and Irgm2-/- mice primed with 100µg poly(I:C) for 6 hours and injected (i.p.) with 5mg/k-1 LPS or 5 and 25 µg of OMVs (n=6 animals per condition).

Data information: Data shown as means ± SEM (graphs A, B, D and E) from n=4 independent pooled experiments; **p ≤ 0.01, ***p ≤ 0.001 for the indicated comparisons using t-test with bonferroni correction. Image (C) is representative of one experiment performed three times. (F-H) are representative of three independent experiments; *p ≤ 0.05; **p ≤ 0.01, ***p ≤ 0.001, Log-rank Cox-Mantel test for survival comparisons (F-H).
Irgm2 and Gate-16 cooperatively dampen Gram-negative bacteria-induced caspase-11 response (2020)
Figure 2

Figure 2: Irgm2 regulates GBP-independent caspase-11 targeting to Gram-negative bacteria.

Unless otherwise specified, BMDMs were treated with 2,5 µg/2.105 cells of OMVs or infected with either S. Typhimurium orgA- (S.Tm orgA-) or F. tularensis spp novicida (F. novicida) with an MOI of 25 for various times.

(A) Measure of LDH and IL-1β release in WT, GBPChr3-/- and Casp11-/- BMDMs were Irgm2 was knocked down 16 hours after exposure to 2,5 µg/2.105 cells of OMVs. Si Scramble (siScr.) refers to RNAi pools with non-targeting sequences.

(B) Cell death (LDH) and IL-1β release evaluation in Irgm2-silenced WT and GBPChr3-/- BMDMs infected for 16 hours with either S.Tm orgA- or F. novicida (MOI 25). Si Scramble (siScr.) refers to RNAi pools with non-targeting sequences.

(C) Florescence microscopy and associated quantifications of GBP-2 (green) recruitments to intracellular S. Tm orgA--mCherry (MOI 10, red) in IFNγ-primed WT and Irgm2-/-BMDMs. Nucleus was stained with Hoechst (blue). Confocal images shown are from one experiment and are representative of n=3 independent experiments; scale bars 5 µm. For quantifications, the percentage of GBP-associated bacteria was quantified and 'n=' refers to the number of intracellular bacteria counted in each experiment; quantifications from n=3 independent experiments were then plotted and expressed as mean ± SEM.

(D) Confocal fluorescence microscopy images and associated quantifications of caspase-11-C254G-GFP (green) recruitment to S.Tm-mCherry (orgA-, red) in IFNγ-primed iWT, iIrgm2-/-, iGBPChr3-/- and iGBPChr3-/-/sgIrgm2 BMDMs after 8 hours of infection. Nucleus (blue) was stained with Hoescht; scale bar 5µm. For quantifications, the percentage of bacteria positive for caspase-11-C254G-GFP was determined by combining the bacterial counts from n=3 independent experiments and expressed as mean ± SEM. ***p ≤ 0.001 for the indicated comparisons using t-test with bonferroni correction.

Data information: Data shown as means ± SEM (graphs A, B) from n=4 independent pooled experiments; ***p ≤ 0.001 for the indicated comparisons using t-test with bonferroni correction. Images (C, D) are representative of one experiment performed three times.
Irgm2 and Gate-16 cooperatively dampen Gram-negative bacteria-induced caspase-11 response (2020)
Figure 3

Figure 3: Irgm2 cooperates with GATE16 to dampen Gram-negative bacteria-induced non-canonical inflammasome activation

(A) GFP-Trap coupled to mass spectrometry strategy used. The volcano plot represents 3 independent combined experiments. Threshold selection of enriched proteins (red dots) in Irgm2-GFP fraction was set at 2 fold enrichment (x axis) and p-value <0.05 (y axis). Blacks and Grey dots indicate proteins that did not reach a p-value <0.05 using t-test with bonferroni correction. Labelled proteins represent the top 6 enriched proteins in the Irgm2 fraction.

(B) Green Florescent Protein (GFP)-Trap assay of the presence of Gate16 in Irgm2-GFP-enriched fraction from the lysates of IFNγ-primed iIrgm2-/- BMDMs complemented with lentiviral constructs coding for a fusion of Irgm2 withGFP (Irgm2-GFP) or GFP alone. Arrows show the presence or not of Gate16, Irgm2-GFP and GAPDH in the Irgm2-GFP-enriched fractions, flow-through and total cell lysates. * means non-specific band

(C) LDH and IL-1β release from siRNA-treated WT BMDMs, and then exposed to 2.5 µg/2.105 cells of OMVs for 16 hours.

(D) LDH and IL-1β release from WT, Irgm2-/- and Casp11-/- BMDMs silenced for Gate16 and treated for 16 hours with 2,5µg/2.105 cells of OMVs. Si Scramble (siScr.) refers to RNAi pools with non-targeting sequences.

(E) Western blot examination of caspase-11 and processed caspase-1 (p20)and gasdermin-D (p30) in supernatants and pro-caspase-1 (p45), pro-gasdermin-D (p55), pro-caspase-11, Gate16 and GAPDH in cell lysates of Gate16-silenced WT, Irgm2-/- and GBPChr3-/- BMDMs exposed to 2,5.105µg/2.105 cells of OMVs for 16 hours. Si Scramble (siScr.) refers to RNAi pools with non-targeting sequences.

(F) Representative confocal fluorescence microscopy images and associated quantifications of caspase-11-C254G-GFP (green) recruitment to S.Tm-mCherry (orgA-, red, MOI 10) in IFNγ-primed iWT BMDMs silenced for Gate16 after 4 and 8 hours of infection. Nucleus (blue) was stained with Hoechst; scale bar 5µm. For quantifications, the percentage of bacteria positive for caspase-11-C254G-GFP was determined by combining the bacterial counts from n=3 independent experiments and expressed as mean ± SEM. **p ≤ 0.01, ***p ≤ 0.001 for the indicated comparisons using t-test with bonferroni correction. Si Scramble (siScr.) refers to RNAi pools with non-targeting sequences.

Data information: Data shown as means ± SEM (graphs C, D) from n=4 independent pooled experiments; ***p ≤ 0.001 for the indicated comparisons using t-test with bonferroni correction. Image (B) is representative of one experiment performed two times and (E) represents one experiment out of three. (A) represents one experiment out of three independent experiments.
Irgm2 and Gate-16 cooperatively dampen Gram-negative bacteria-induced caspase-11 response (2020)
Figure 4

Figure 4: GATE16 inhibits non-canonical inflammasome activation in human myeloid cells.

(A) LDH and IL-1B release from siRNA-treated primary human Monocyte-Derived Macrophages (hMDMs) infected with S. Typhimurium orgA- (MOI25) for 16 hours. When specified, the caspase-4/5 inhibitor Z-LEVD (25µM) or the NLRP3 inhibitor MCC950 (10µM) were added to the experiments.

(B) LDH and IL-1B release from siRNA-treated primary human Monocyte-Derived Macrophages (hMDMs), primed with IFNγ (10UI/mL) and PAM3CSK4 (100ng/mL) and then stimulated with Nigericin (20µM) for 4 hours. When specified, the caspase-4/5 inhibitor Z-LEVD (25µM) or the NLRP3 inhibitor MCC950 (10µM) were added to the experiments.

(C, D) LDH and IL-1B release from siRNA-treated WT or GBP1/2/5-/- U937 Monocytic cell line, primed with IFNγ (10UI/mL) and PAM3CSK4 (100ng/mL) and then infected with (C) with S. Typhimurium orgA- (MOI25) for 10 hours or (D) electroporated with 1µg of E. coli LPS for 4 hours. Φ indicates that no LPS electroporation was performed. Data shown as means ± SEM from n=4 independent pooled experiments; ***p ≤ 0.001 for the indicated comparisons using using t-test with bonferroni correction.

Data information: Data shown as means (graphs A, B) from n=3 different donors; each donor is represented with a coloured circle; * p ≤ 0.05, **p ≤ 0.01 for the indicated comparisons using one-way ANOVA with multiple Bonferroni correction.

Expanded View Figure Legends
Long non-coding RNA TINCR suppresses metastatic melanoma dissemination by preventing ATF4 translation (2020)
Figure 2

Figure 2. The lncRNA TINCR RNA is downregulated in metastatic melanoma.

A Box and whiskers (from 5th to 95th percentile) plots depicting TINCR RNA levels in the TCGA SKCM RNA-seq dataset of melanoma samples. TINCR expression is higher in Primary A melanomas, and its reduction in Primary-B melanomas and all groups of metastatic samples is statistically significant (Mann-Whitney test, **p< 0.01). Median, minimum, maximum, 25th, 75th whiskers of expression values are shown.

B Scatter-plot showing correlation between the expression of TINCR RNA and normalized rank-sums s of the down-regulated (upper panel) and up-regulated (lower panel) genes in the primary melanoma signature. TINCR expression strongly correlates (Spearman's correlation coefficient of 0.79, Spearman rank p-value: **p<0.01) with the expression of the down-regulated genes.

C Dot plot showing qRT-PCR analysis of TINCR expression in primary (8) and metastatic (8) melanoma samples from surgical specimens or patient-derived xenografts (4 primary and 8 metastatic PDXs). TINCR RNA levels relative to the L32P housekeeping gene are shown. Paired primary and metastasis PDXs are marked with an asterisk. The difference between primary and metastatic samples is significant (Mann-Whitney test, **p< 0.01). Median, minimum, maximum, 25th, 75th whiskers of expression values are shown.
Long non-coding RNA TINCR suppresses metastatic melanoma dissemination by preventing ATF4 translation (2020)
Figure 5

Figure 5. TINCR depletion activates ATF4 translation independently of eIF2α phosphorylation.

A Western blot analysis of integrated stress response activation in WM902B and MMC70 cells. Cells were harvested 96 h after shRNA infection and western blot performed using the indicated antibodies.

B Representative comparison of translational efficiency (TE) in control (shSCR) and shTINCR WM902B cells 72h post infection. The genes with statistically significant TE up- and downregulation (FDR< 0.1) are highlighted in red and blue respectively. ATF4 is circled among the upregulated genes.

C Heatmap representing top translationally upregulated and down regulated genes in TINCR WM902B cells. The plotted values are the log2 fold TE change values in each biological replicate (rep1 and rep2) relative to control (shSCR).

D Bar-graph representing ribosome protected fragments (RPF) normalized coverage (mean±sd) mapping to ATF4 CDS in control (shSCR) and TINCR (shTINCR) depleted WM902B cells from two biological replicates is shown.

E UCSC genome browser tracks of ribosome footprint data illustrating RPF density distribution across ATF4 mRNA in shSCR and shTINCR WM902B cells.
Long non-coding RNA TINCR suppresses metastatic melanoma dissemination by preventing ATF4 translation (2020)
Figure 6

Figure 6. Identification of TINCR interacting mRNAs through TINCR interactome analysis (RIA-seq)

A Schematic representation of biotinylated antisense DNA probes complementary to TINCR transcript. Odd (black) and even (red) probes were used for RNA pulldown.

B Q-PCR validation of TINCR and ATF4 RNA recovery in TINCR antisense pulldown. Enrichment relative to input (mean±sd) from three independent pulldown experiments is shown. Statistical analyses were performed using Student t -test (**: p<0.01, ***: p<0.001).

C UCSC genome browser tracks of TINCR-RIA-seq sequencing reads illustrating coverage and enrichment across ATF4 transcript in WM902B and MMC70 cells.

D Gene set Enrichment analysis (GSEA) of TINCR interacting RNAs (FDR<0.25) using Gene Ontology (GO) database.

Expanded View Figure Legends
CFTR Interactome Mapping Using the Mammalian Membrane Two-Hybrid High-Throughput Screening System (2022)
Figure 3

Figure 3 - FLIPR assay results for interactors positively affecting CFTR Cl- channel activity.

A-B Assays were performed in HEK293 cells for both A) F508del-CFTR and B) wt-CFTR. For the wildtype measurements, we compared the maximum Fsk response (Maximum F /F0; where F0 represents baseline RFU right before the stimulation). For the F508del-CFTR measurements, since this mutant does not respond well to Fsk alone (Fsk + VX-770 potentiator is required to obtain a detectable signal) we compared the CFTR inh172 (inhibitor) response (F1/'F-CFTRinh172'; where F1 represents peak RFU immediately before inhibitor addition and 'F-CFTRinh172' is the minimum RFU reached at the end of the CFTR inh172 measurement cycle). For each of wt-CFTR and F508del-CFTR, data for three proteins not detected as interactors (first three bars following CFTR only) are shown as negative controls. Fluorescence signals were normalized to the CFTR-only control and the statistical significance of differences assed by unpaired t-test (*p<0.05, **p<0.01 and ***p<0.001). Samples run in n=3 replicates, and all values are mean ± s.d.
Distinct roles of VE-cadherin for development and maintenance of specific lymph vessel beds (2018)
Figure 1

Fig. 1: Genetic deletion of VE-cadherin in lymphatic endothelial cells results in edema and hyperplasia.

Pregnant dams were injected with 4-OHT at days E10.5 and E11.5 for analysis at day E12.5 or E10.5, E11.5, and E12.5 of development for later times of analysis.

(A) Developmental loss of VE-cadherin leads to severe edema. Fetuses of the genotypes listed in the top row were analyzed at the developmental stages in the first column. Numbers in brackets denote embryos with signs of edema. Last column lists the percentage of viable fetuses in the VE-cadherin deleted cohort.

(B) Fetuses were explanted at day E14.5. Arrowheads indicate prominent edema along the back. The images are representative for 42 (Cdh5lox/lox) and 25 (Cdh5lox/lox; Prox1-CreERT2) analyzed animals.

(C) Skin wholemount preparations stained for PROX1, PECAM1 and VEGFR-3. Maximum intensity projections (MIPs) of tiled confocal stacks (Cdh5lox/lox 2075µm x 2075µm, z=50µm; Cdh5lox/lox; Prox1-CreERT2 3765µm x 3765 µm, z=61µm). Scale bars = 100 μm. The data are representative for 6 (Cdh5lox/lox) and 6 (Cdh5lox/lox; Prox1-CreERT2) analyzed animals from 3 litters.

(D) Enumeration of the PROX1-expressing (PROX1+) nuclei in three different VE-cadherin deleted wholemount preparations analogous to Fig. 1B. Nuclei were counted in a MIP corresponding to an area of 1.5 mm2 and normalized to the number of nuclei in Cdh5lox/lox control preparations.

(E) The area covered by lymphatic vessels was determined in the samples evaluated in (D) and is depicted as relative area compared to Cdh5lox/lox controls. The calculations and measurements were obtained from three animals and three confocal stacks per biological group. PROX1-positive nuclei were counted using the particle analysis tool of Fiji.

Data information: The data in (D and E) represent mean ± SD. *** p < 0.001. Two-tailed unpaired Student's t test.
Distinct roles of VE-cadherin for development and maintenance of specific lymph vessel beds (2018)
Figure 2

Fig. 2: Despite efficient postnatal deletion of VE-cadherin, the discontinuous distribution of PECAM1 is retained in dermal lymphatic vessels.

Deletion of VE-cadherin in lymphatic endothelial cells of newborn pups was induced by two optimal doses of 4-OHT at days P2 and P4. For analysis 6 weeks later ear punches were subjected to wholemount immunostaining of the dermal vasculature.

A-H (A, B, E, F) Overview MIPs of ear skin wholemount preparations immunostained for the indicated antigens. VEC, VE-cadherin; PRX, PROX1. (C, D, G, H) Magnified views of the areas in the red boxes in (A, E). Areas delimited by the stippled yellow boxes in (C) and (G) are enlarged in the insets (yellow solid boxes). Yellow arrows indicate widened lumen, yellow arrowheads denote oak leaf-shaped LECs with discontinuous junctions. Scale bars in (A and E) correspond to 100 μm and in (C and G) to 50 µm. The data are representative for 6 (Cdh5lox/lox) and 5 (Cdh5lox/lox; Prox1-CreERT2) analyzed animals from 2 litters.
Distinct roles of VE-cadherin for development and maintenance of specific lymph vessel beds (2018)
Figure 3

Fig. 3: Dermal lymphatic vessels respond differentially to VE-cadherin loss, depending on the age at the time of induction.

A-L VE-cadherin deletion was initiated either in newborn pups (A, B, E, F) by two applications of 4-OHT at days P2 and P4 or in mice at 11 weeks (C, G) and 42 weeks (D, H) of age (three applications of Tamoxifen at 2 day intervals via oral gavage). Ear skins were prepared 6 weeks (A, B, E, F) or 5 weeks (C, D, G, H) after induction and immunostained for the indicated proteins. Shown are MIPs of confocal tile scans (approx. 750 µm x 750 µm) covering 40 µm in depth. White arrows in (F) highlight distorted and partially fragmented lymphatic vessels and white arrowheads denote aberrantly pointed vessels (F). (I-L) Lymphatic valves in the dermis of the ear were maintained for 5 weeks despite deletion of VE-cadherin at 11 weeks of age (yellow arrows in K, L). PEC1, PECAM1; PRX1, PROX1;VR3, VEGFR-3. Scale bars correspond to 100 μm. The data represent wholemount stainings from 6 (A, E), 5 (B, F), 6 (C, G, K, L), 3 (D, H) and 6 (I, J) analyzed animals.
Distinct roles of VE-cadherin for development and maintenance of specific lymph vessel beds (2018)
Figure 4

Fig. 4: VE-cadherin is indispensable for the maintenance of mesenteric lymphatic vessels at all ages.

A-F Deletion of VE-cadherin was induced in mice at various age (4 weeks (A), neonate (B), 6 month (C, D, E) or 1 year (F)) by either two applications of 4-OHT at days P2 and P4 (neonates) or three applications of Tamoxifen at 2 day intervals via oral gavage (all other ages). The mesentery was prepared as indicated 4, 6, 8 or 11 weeks later and the wholemount preparations were immunostained for the proteins specified in color (left). (B) Inset in top panel shows a magnification of the area outlined by the white dashed line (for further magnification see expanded view Fig. 1A and B). Arrows indicate the previous position of lymphatic valves; white arrowheads indicate PECAM1+ capillaries supplying the intestinal fat tissue lining the vessels (magnified in expanded view Fig. 1C); yellow arrowheads denote island of LECs that display intracellular VEGFR-3 staining. (C) Arrows denote LECs that have converted from growth as tubes to sheets; white arrowheads, thinning of lymphatic vessels; yellow arrowheads, aberrant sprouting of LECs. The area outlined by a white box is depicted magnified in (D) and provides details on LECs growing as a sheet of cells. (E) Formation of ectopic sprouts from the valve area of degenerating lymphatic vessels. (F) Fully deteriorated lymphatic vessel with emanating sprouts that initiate sheet-like growth. PEC1, PECAM1; PRX1, PROX1; VR3, VEGFR-3. Scale bars = 100 μm. The data represent n=5 (A), n=5 (B), n=3 (C-E) and n=6 (F) animals.
Distinct roles of VE-cadherin for development and maintenance of specific lymph vessel beds (2018)
Figure 5

Fig. 5: Deletion of VE-cadherin results in fragmentation and distension of the intestinal lacteals.

A-E Deletion of VE-cadherin was induced in mice at various ages (neonatal (C), 16 weeks (D) or 1 year (E)) by either two applications of 4-OHT at days P2 and P4 (neonates) or three applications of Tamoxifen at 2 day intervals via oral gavage (all other ages).Control mice of the same age were subjected to an identical regimen (A, B). Intestinal wholemount preparations of the jejunum and ileum were generated 5 - 8 weeks after Tamoxifen induction and immunostained for the indicated proteins, scale bars = 100 μm. The data represent wholemount stainings from 3 (D), 5 (B,E) and 6 (A, C) animals analyzed for each genotype.
Distinct roles of VE-cadherin for development and maintenance of specific lymph vessel beds (2018)
Figure 7

Fig. 7: Loss of VE-cadherin from lymphatic endothelial cells results in increased circumferential actin and a reduced number of focal adhesions.

A, B Primary dermal LECs were isolated as described in appendix Fig. S8 A. PdLECs were used for up to four passages and control and VE-cadherin-deleted pdLECs had been cultured for the same number of passages. Cells were seeded at a density of 2.5 x 104 cells on gelatine-coated 15mm glass coverslips and co-stained after 3 days of culture for the actin cytoskeleton (Phalloidin) and either Vinculin (A) or FAK (B) for the visualization of focal adhesions. PROX1 staining in (A) identifies LECs, nuclei in (B) were counterstained with Hoechst. Scale bars correspond to 50 µm.

(C, D) Bar diagrams show the number of focal adhesions/cell, which were enumerated using Image J from the Vinculin and FAK stainings shown in (A and B). Data are mean values + S.E.M. derived from three cell lines and 9 viewfields of 256 µm x 256 µm per biological group. *** p ≤ 0.001. Two-tailed unpaired Student's t test.