EMBO/SourceData huggingface dataset:
Figure 7(A, B) Expression of ABAPT11 during SA treatment. (A) Seven-day-old wild-type (WT) seedlings were treated with 100 µM SA for 0, 10, 20, 30, 40, 50, and 60 min before collection for RNA preparation and RT-qPCR to detect the transcript level of ABAPT11. (B) Transcript levels of ABAPT11 in wild-type (WT) and npr1 mutant 7-day-old seedlings with or without 100 µM SA for 20 min. ACTIN2 was used as an internal control. Data are means ± SD from triplicated replications in an experiment; the pattern was consistent in three biologically independent experiments. Significance analysis using one-way ANOVA followed by Tukey's multiple comparison test is shown in different lower-case letters (P < 0.05).(C) Accumulation of ABAPT11 proteins in the SA response. Seven-day-old seedlings expressing ABAPT11-YFP under its own promoter were treated with 100 µM SA for 0, 20, 40, and 60 min before collection for immunoblotting with an anti-GFP antibody. Coomassie blue staining of total proteins was used as the loading control. Quantitative relative protein levels (YFP / loading) from three biologically independent experiments are shown in the bottom graph. Significance analysis using one-way ANOVA followed by Tukey's multiple comparison test is shown in different lower-case letters (P < 0.05).(D) The S-acylation level of BSK1 in the ABAPT11 mutant during SA treatment. BSK1-GFP was expressed in WT or abapt11-1 mutant cells (with or without 100 µM SA treatment for 40 min) for S-acylation measurement in a biotin-switch assay. Representative immunoblots are shown in the top graph. The immunoblotting signals were quantified by ImageJ and the S-acylation levels were calculated from relative signals ([pulldown+/input+] - [pulldown-/ input-]). The relative S-acylation level of the wild-type sample without SA treatment was set to 1. Data are means ± SD from three biologically independent experiments. Significance analysis using one-way ANOVA followed by Tukey's multiple comparison test is shown in different lower-case letters (P < 0.05).(G, Confirmation of the SA-induced BSK1 translocation which is mediated by ABAPT11. BSK1-GFP was expressed in WT or abapt11-1 mutant cells, with or without 100 µM SA treatment for 40 min. Total proteins (T) were separated into soluble (S) and pellet (P) fractions using ultra-centrifugation. Representative immunoblots using an anti-GFP antibody are shown in (G).
Figure 8(A, B) Effect of BL and SA on hypocotyl development in ABAPT11 mutants. Seeds were germinated on regular medium (Control), medium containing 80 nM BL, medium containing 40 µM SA, or medium containing both 80 nM BL and 40 µM SA. Hypocotyl length was measured 8 days after germination. Representative images from four independent experiments are shown in (A). Quantitative data of hypocotyl lengths are shown in (B). Data are means ± SD from 20 seedlings in four independent experiments. Significance analysis using one-way ANOVA followed by Tukey's multiple comparison test is shown in different lower-case letters (P < 0.05).D) Effect of BL and SA on root development of ABAPT11 mutants. Four days after germination, seedlings of WT, abapt11-1, and abapt11-2 were transferred onto a regular medium (Control), medium containing 15 nM BL, medium containing 20 µM SA, or medium containing both 15 nM BL and 20 µM SA. Quantitative data of root elongation 5 days after transferring are shown in (D). Data are means ± SD from 20 seedlings in four independent experiments. Significance analysis using one-way ANOVA followed by Tukey's multiple comparison test is shown in different lower-case letters (P < 0.05).
Figure 1(B) Representative H/E images of epidermis from control and iDKO mice at 15 DPI. (C) Quantitative analysis of epidermal cell rounding from (B). n=3 biological replicates per genotype.(E-F) Representative images (E) and quantification (F) of whole-mount immunostaining of β-catenin. Control: n=7 biological replicates; iDKO: n=5 biological replicates.(L) Representative immunofluorescent images of the toe pad for filaggrin (Flg). K14 marks basal keratinocytes and DAPI marks nuclei. (M) Quantification of the average size of filaggrin+ deposits at 52 DPI as shown in (L). n=3 biological replicates per genotype.
Figure 2(H) Volcano plots at 25 or 52 DPI.(J) RT-qPCR of the indicated genes in epidermis at 25 (n=4 biological replicates per genotype) or 52 (control: n=10 biological replicates; iDKO: n=6 biological replicates) DPI.
Figure 3(D) ChIP-seq tracks of Ovol1, Ovol2, and histone marks for the select loci. Boxed areas indicate Ovol1/2-binding peaks, and red bars underneath indicate presence of Ovol1/2 sequence motifs.(F-G) Luminex measurements of the indicated cytokines in skin lysates (F; n=7 biological replicates per genotype) or serum (G; n=8 biological replicates per genotype) of control and iDKO mice at 4-6 MPI.
Figure 4(A) Representative whole-mount immunofluorescent staining of LCs in mouse back skin at 52 DPI. (B) Quantification of primary dendrites in LCs as shown in (A). Control: n=5 biological replicates; iDKO: n=4 biological replicates.(C-D) Flow cytometry analysis of isolated epidermis at 52 DPI. Control: n=8 biological replicates; iDKO: n=7 biological replicates. MFI, mean fluorescence intensity.(J-M) Flow cytometry analysis of LNs at 52 DPI. n=4 pairs for (J). Control: n=10 biological replicates; iDKO: n=5 biological replicates for (K-M).(N) RT-qPCR of the indicated genes in LNs from 4-6 MPI. n=4 biological replicates per genotype.(O) Luminex measurements of G-CSF in serum of control and iDKO mice at 4-6 MPI. Left, results obtained using remote service at Eve Technologies (n=3 biological replicates per genotype). Right, results obtained in-house using a custom panel (n=8 biological replicates per genotype).(P) Luminex measurements of IL-17 in serum of control and iDKO mice at 52 DPI. n=5 biological replicates per genotype.
Figure 5(F) Representative H/E images of control and iDKO skin at the indicated times showing changes in dWAT. (G) Quantification of fat cell area in (F). n=4 biological replicates per genotype. For , data is represented as mean ± SD and statistical analysis was done with the Holm-Sidak method. NS, non-significant; *, p≤0.05; **, p≤0.005; ***, p≤0.0005; ****, p≤0.00005.(H-L) Results from metabolic cage analysis of 4-5-MPI control and iDKO mice (n=5 biological replicates per genotype) at RT (white band) or after CE (blue band). Gray bands designate night. Shown are data on food intake (H), movement (I), body weight (J), energy expenditure (K), and RER (L).
Figure 6(C-D) Representative H/E images (C) and quantitative analysis (D) of epidermal cell rounding, in control and iDKO mice at 4 MPI. n=4 biological replicates for PBS-iDKO; n=5 biological replicates for all else.(E) Representative immunofluorescent images of back skin for Flg, K14, and DAPI.(G) Quantification of LN weight. Control-PBS: n=10 biological replicates; iDKO-PBS: n=4 biological replicates; Control-Dex: n=9 biological replicates; iDKO-Dex: n=7 biological replicates. (H-J) Flow cytometry analysis of LNs at 4 MPI. Control-PBS: n=8 biological replicates; iDKO-PBS: n=4 biological replicates; Control-Dex: n=5 biological replicates; iDKO-Dex: n=4 biological replicates for (H,J) and Control-PBS: n=7 biological replicates; iDKO-PBS: n=4 biological replicates; Control-Dex: n=5 biological replicates; iDKO-Dex: n=3 biological replicates for (I).(L) Body weight over time represented as a percentage of the starting weight. Control-PBS: n=13 biological replicates; iDKO-PBS: n=4 biological replicates; Control-Dex: n=9 biological replicates; iDKO-Dex: n=5 biological replicates.
Figure 1(A) Tamoxifen schedule: pulse of the pregnant dam at E9.5, harvest at E10.5. Representative fluorescent immune-staining of E10.5 Sox7iECKO mutants and sibling control embryos. The endothelium is delineated by endomucin (EMCN) (blue) and endothelial cells by ETS-related gene (ERG) (red), SOX7 positive cells are shown in white. (Top right graph) The percentage of SOX7 positive endothelial cells in different vascular beds, quantified from 10 sequential transverse sections in 2 control and 3 Sox7iECKO mutants. (Bottom right panel) Graph indicating the levels of Sox7 transcript in sibling controls and Sox7iECKO mutants. Mean ± SEM; scored sibling control, n=9; Sox7iECKO mutants, n=7; Mann-Whitney U-test. P<0.0005 (***).(C) Brightfield images and whole-mount immunostaining of Sox7iECKO mutant and sibling control skin at E13.5, after Cre induction at E9.5 and E10.5. Dermal lymphatic structures are marked by Neuropilin 2 (NRP2) (membranous white), lymphatic endothelial cells by Prospero-related homeobox 1 (PROX1) (red), and blood vessels by EMCN (green). Dash line represents the midline of the embryo. Scored sibling controls, n=7; Sox7iECKO mutants, n=7(D-H) Quantification of (D) Distance of the lymphatic vessel front from the midline (μm) (E) lymphatic branch points/area (F) lymphatic vessel width (μm) (G) PROX1+ nuclei/area and (H) PROX1+ nuclei/lymphatic vessels across the whole skin. Total area = 4200 x 1500 μm on both sides of the midline, in sibling controls (n=3) and Sox7iECKO mutants (n=5-6). (F,H) Average of width and PROX1+ nuclei was obtained from 7 random representative leading lymphatic vessels, of fixed length at 200 μm, from both sides of the midline in each skin. (I,J) Quantification of (I) PROX1+ nuclei in each LEC cluster with each colour dot representing a different embryo and (J) disconnected lymphatic endothelial cell (LEC) clusters (<100 μm) and vessel branches (>100μm)/area. Total area quantified = 4200 x 2200 μm on both side of midline in sibling controls (n=3) and Sox7iECKO mutants (n=5). PROX1+ nuclei were quantified from n=29 and n=122 LEC clusters, respectively.
Figure 2(C) Arterial endothelial cells express Vegfc transcripts. Transverse section of a E10.5 wild-type embryo stained by single molecule FISH (smFISH) for Gapdh (green) and Vegfc (magenta) and immuno-stained for PECAM (white). The first panel shows a merged version of the pseudo colour signals for the 3 markers. Inset 1 shows the expression of Vegfc in endothelial cell of the dorsal aorta (black and white, pink arrows). Inset 2 shows the high expression levels of mesenchymal Vegfc expressed in the dorso-lateral aspects of the cardinal vein.(E-F) qPCR on human arterial endothelial cells (HUAECs) transfected with SiSOX7 or SiCTRL for (E) 30 h and (F) 17 h. (G) qPCR on human venous endothelial cells (HUVECS) transfected with SiSOX7 or SiCTRL for 17 h. D
Figure 3(A-D) SOX7 transcriptionally activates Notch effector, HEY1, to repress Vegfc. (A) qPCR on FAC-sorted PECAM+CD45-endothelial cells of Sox7iECKO mutants and sibling controls at E14.5 after Cre induction with tamoxifen at E11.5 and E12.5. Expression is normalised to the endothelial marker Pecam and Cdh5. Scored sibling controls, n=9; Sox7iECKO mutants, n=8. (B-C) qPCR on (B) human arterial endothelial cells (HUAECs) and (C) human venous endothelial cells (HUVECs) transfected with SiSOX7 or SiCTRL for 17 h. In addition to HEY1, DLL4 levels were also downregulated in the human cell line experiments. Expression is relative to HPRT and GAPDH. Data from one siRNA experiment performed in triplicates. (D) HEY1 represses human VEGFC promoter activity. HeLa cells were co-transfected with human or mouse VEGFC-luc and either EV (empty vector) or HEY1 expression constructs as indicated. VEGFC luciferase activity was measured and normalised to Renillla luciferase activity, which was then made relative to the promoter-less vector, pGL3-basic, which was set to 1. Biological replicates, n=3 independent repeats of the same experiment. Mean ± SEM; t-test. P<0.05 (*); P<0.005 (**); P<0.0005 (***).SOX7 physically interacts with transcription repressor, HEY1. (G) Co-immnuno-precipitation analysis of SOX7 and HEY1. HEK293 cells were transfected with indicated plasmids and harvested 24 h after transfection, immunoprecipitated by anti-GFP or Ig control before immunoblot analysis to determine the presence of bait/prey (top) and the input (bottom). N=2.
Figure 4(A) SOX7 represses human VEGFC promoter activity. HeLa cells were co-transfected with human or mouse VEGFC-luc, EV (empty vector), or SOX7 expression constructs as indicated. VEGFC luciferase activity was measured and normalised to Renillla luciferase activity, which was then made relative to the promoter-less vector, pGL3-basic, which was set to 1. Biological replicates, n=3. Mean ± SEM; t-test. P<0.05 (*).(G) Quantitation of Vegfc transcript using single molecule FISH in vivo. (colored panel) Representative low magnification images of the trunk transverse sections of E10.5 sibling controls and Sox7iECKO mutants showing the similar relative position of the DA and the CV across genotypes. Sections were stained by single molecule FISH (smFISH) for Gapdh (green) and Vegfc (magenta) and immuno-stained for PECAM (white). Individual molecules of Vegfc transcript (pink arrows) are detectable within the cytoplasm of PECAM positive cells of the dorsal aorta (Middle panel). (Left graph) Quantitation of Vegfc transcript level in the PECAM positive endothelial cells of the dorsal aorta, in sibling controls and Sox7iECKO mutants at E10.5. The mean grey scale intensity of the Vegfc probes in control was 5845. ­­­­­­­­­The mean grey scale intensity of the Vegfc probes in Sox7iECKO was 8956. Mean probe intensity was significantly higher in Sox7iECKO compared to the control (P<2e-16). (Right graph) No differences were observed for the average probe area, meaning that distribution of the probes across the dorsal aorta remained unchanged between control and Sox7iECKO. Scored sibling controls, n=5; Sox7iECKO mutants, n=7. Mean ± SEM; t-test. P<0.0005 (***), Graph: box, interquartile range, central band, median, whiskers, indicate maximum and minimum. Scale bars; low magnification 150 μm, high magnification, 30 μm
Figure 5(A) At 2 days post Cre induction, E11.5 Sox7iECKO mutants showed ectopic expression of PROX1+ (red) in medial ventral regions of the cardinal veins (CVs), close to dorsal aorta (DA) (yellow arrowheads).(D) Whole-mount immunostaining of control and Sox7iECKO embryonic skin at E12.5 after Cre induction at E9.5, E10.5. Dermal lymphatic structures are stained with NRP2 (membranous white), lymphatic endothelial cells, PROX1 (red), and blood vessels, both EMCN (green) and SOX7 (white nuclei). Arrowheads indicate EMCNlow/-PROX1+NRP2+/low individual LECs on blood vessels (type I), and arrow shows EMCN+PROX1+ NRP2low/- LEC within the vessel wall (type II). (E) Graph shows the total events of PROX1+ single LECs or LEC clusters cells associated with EMCN+ blood vessel plexus within each embryo. PROX1+ cells were quantified from n=4 controls and n=5 Sox7iECKO mutants.
Figure 1A Normal lung cells (IMR90 and MRC5) and lung cancer cells (A549 and H1975) were treated with various concentrations of PM2.5 for 24 h, and cell viability was assessed by a trypan blue assay. The values are the mean ± SD of three independent experiments.B H1975 and A549 cells were treated with various concentrations of PM2.5 for 24 h, and their cell lysates were analyzed for phosphor-ERK (pERK), phosphor-AKT (pAKT), phosphor-STAT3 (pSTAT3), phosphor-EGFR (pEGFR), ERK, AKT, STAT3, and EGFR by Western blotting. β-actin served as the loading control. The results shown are from one of three similar experiments (left panel). The relative expression levels of pEGFR, pAKT, pSTAT3, and pERK were quantified by normalized with β-actin and are shown at the right panel. The values are the mean ± SD of three independent experiments. *p < 0.05, as analyzed with one-sample t-test and compared with untreated cells.
Figure 2A, B H1975 and A549 cells were treated with PM2.5 at 50 μg/ml for 90 days, and the proliferation of the treated cells was assessed by trypan blue assays.C, D H1975 and A549 cells were exposed to PM2.5 at 50 μg/ml for different lengths of time, and the cell lysates of treated cells were assessed for phosphorylated EGFR (pEGFR) and EGFR by Western blotting. β-actin served as the loading control.E, F H1975 and A549 cells were treated with PM2.5 at 50 μg/ml for 90 days. The anchorage-independent growth was assessed by a soft agar colony formation assay. The number of colonies was scored, and the data are presented as the relative colony formation ability.
Figure 3H1975 cells were exposed to 50 μg/ml PM2.5 for 90 days. Both unexposed and exposed cells were injected subcutaneously into the flank of each mouse (n = six per group). The tumor volume and excised tumor weight were measured. The results shown in (A) are presented as the means ± SDs of six mice. *p < 0.05 and **p < 0.01, compared with untreated group. (A) p values were determined by two-way repeated measures ANOVA with pairwise comparison of post-hoc analysis with Benjamini-Hochberg (BH) correction.H1975 cells were exposed to 50 μg/ml PM2.5 for 90 days. Both unexposed and exposed cells were injected subcutaneously into the flank of each mouse (n = six per group). C. IHC staining of excised tumors for Ki-67, IL18, and TMPRSS2 is shown in (C). Scale bars, 50 μm.
Figure 4H1975 cells were treated with 50 μg/ml PM2.5 for 90 days, and the expression levels of TMPRSS2 mRNA were determined by real-time RT‒PCR (A) respectively. (A) p values were determined by one-sample t-test.C The proteins from whole cell lysates (WCL) and nuclear fractions (CN) were analyzed for AhR, Lamin B (nuclei marker), and Vimentin (cytoplasm marker) by Western blotting. The data shown represent the mean ± SD of three independent experiments. *p < 0.05 and **p< 0.01, and ***p < 0.001, compared with untreated cells. #p < 0.05, compared with PM2.5 treated cells.E Western blot analysis of TMPRSS2 expression in cells treated with CH223191 at 10 μM for 48 h. The relative expression level of TMPRSS2 was quantified by normalized with β-actin and are shown at the right panel. : The data shown represent the mean ± SD of three independent experiments. *p < 0.05 and **p< 0.01, and ***p < 0.001, compared with untreated cells. #p < 0.05, compared with PM2.5 treated cells.
Figure 5A Expression of TMPRSS2 was examined by Western blots of four lung cancer cell lines (H460, A546, H1299, and H1975) and two normal fibroblasts (MRC5 and IMR90). The results shown in (A are from one of three similar experiments. β-actin was used as the loading control.H1975 cells were infected with sh-TMPRSS2 (sh-TMPRSS2-1 and sh-TMPRSS2-2) or empty vector (Vector). The stable clones of TMPRSS2 knockdown cells were analyzed and their ability to perform anchorage-independent growth in soft agar (C). The data shown in (C) represent the means ± SDs from three independent experiments. (C) p values were determined by one-sample t-test.
Figure 6A-C H1299 cells were transfected with TMPRSS2-HA or the empty vector (Vector). After 48 h, the transfected cells were assayed for the expression of TMPRSS2 (A) and for the induction of IL18 by qRT‒PCR (B) and Western blotting (C). The results shown in (A, C are from one of three similar experiments. β-actin was used as the loading control.The expression level of IL18 was examined in A549 cells exposed to PM2.5 at 50 μg/ml for 90 days Western blotting The results shown in H) are from one of three similar experiments. β-actin was used as the loading control. The data shown in H) was normalized to β-actin from three independent experiments.
Figure 7A IHC staining of TMPRSS2, IL18, and nuclear AhR in a representative normal lung tissue section. Scale bars, 2.5 mm (low magnification) and 100 μm (high magnification).B IHC staining of TMPRSS2, IL18, and nuclear AhR in lung cancer tissues that displayed high or low expression of TMPRSS2. Scale bars, 2.5 mm (low magnification) and 100 μm (high magnification).
Figure 1(C) Representative image of mESC expressing Nanog-EGFP in +LIF. Bright field, epifluorescence, HILOM, and a max projection image of 2,000 frames are shown.(F) Confocal images of DRAQ5 staining, showing double stranded DNA (top), immunostaining of H3K27ac (middle), and Nanog-EGFP (bottom), along with the progression of differentiation (+2i, +LIF, −LIF, and −LIF 2d).(G) Swarmplots and violinplots of coefficient of variation of DRAQ5 fluorescence in a nucleus of an mESC expressing Nanog-EGFP at the condition of +2i (red, n = 20 cells), +LIF (blue, n = 20), −LIF (green, n = 23), −LIF+TSA (light green, n = 22), −LIF2d (purple, n = 21), and −LIF2d+TSA (pink, n = 19). Double asterisks and single asterisks indicate p-value < 0.05 and 0.05 ≤ p-value < 0.1, respectively, in Student's t-test.
Figure 3(C) Typical example of dwell time histograms of Nanog-EGFP obtained within every 400 frames at frame periods of 50 (green, n = 228 traces), 100 (orange, n = 97), 150 (red, n = 102), 250 (magenta, n = 124), and 450 ms (blue, n = 157) in a nucleus in +LIF.(D) Dwell time histograms of Nanog-EGFP (left) and Oct4-EGFP (right) obtained from all cell data (n = 73 in +2i, 65 in +LIF, and 66 cells in −LIF for Nanog-EGFP, and n = 57 in +2i, 53 in +LIF, and 58 cells in −LIF for Oct4-EGFP) at frame periods of 50, 100, 150, 250, and 450 ms in a nucleus in +2i (red), +LIF (blue), and −LIF conditions (green). Values of koff were obtained by the global fitting approximation.(E) Histograms of dissociation rate of Nanog-EGFP (left) and Oct4-EGFP (right) in an mESC in +2i (top), +LIF (middle), and −LIF (bottom) conditions. Black line shows the fitting result with a normal distribution.(F) Dependence of expression level of Nanog-EGFP (upper) and Oct4-EGFP (lower) on dissociation rate. Considering the distribution of the fluorescent intensity low (blue), middle (magenta), and high (red) were defined as cells showing the log value of the fluorescent intensity of < 1.9, 2.0-2.3, and > 2.4 [a.u.], respectively, for Nanog-EGFP or < 1.9, 2.0-2.2, and < 2.3 [a.u.], respectively, for Oct4-EGFP. Bars and error bars are mean ± SD. For Nanog-EGFP, N = 20 (high), 32 (middle) and 20 (low) for +2i, 26 (high), 18 (middle) and 21 (low) for +LIF, and 20 (high), 26 (middle) and 20 cells (low) for −LIF. For Oct4-EGFP, N = 22 (high), 18 (middle) and 17 (low) for +2i, 17 (high) 16 (middle) and 20 (low) for +LIF, and 19 (high), 19 (middle) and 20 cells (low) for −LIF. Asterisks indicate less than 0.05 of the p-value in Student's t-test.
Figure 4(D, E) Dependence of expression level of Nanog-EGFP (left) and Oct4-EGFP (right) on Rc (D) or D (E). Bars and error bars are mean ± SD. For Nanog-EGFP, N = 20 (high), 32 (middle) and 20 (low) for +2i, 26 (high), 18 (middle) and 21 (low) for +LIF, and 20 (high), 26 (middle) and 20 cells (low) for −LIF. Of Oct4-EGFP, N = 22 (high), 18 (middle) and 17 (low) for +2i, 17 (high) 16 (middle) and 20 (low) for +LIF, and 19 (high), 19 (middle) and 20 cells (low) for −LIF. Asterisks and double asterisks indicate p-values of less than 0.05 and 0.01, respectively, in Student's t-test.
Figure 5(C, D) The average correlation index g(r) of Nanog-EGFP (C) and Oct4-EGFP (D) in +2i (top), +LIF (middle), and −LIF (bottom). Insets are histograms of logarithm value of the intercept g0 (magenta) and the exponential decay constant ξ (orange) of single mESCs. Black lines in insets show the fitting results with a normal distribution. Error bars represent the standard deviation. N = 72 for +2i, 56 for +LIF and 56 cells for −LIF of Nanog-EGFP, and N = 57 for +2i, 53 for +LIF and 58 cells for −LIF of Oct4-EGFP.(E, F) Dependence of expression level of Nanog-EGFP (left) and Oct4-EGFP (right) on log10g0 (E) and log10ξ (F). Error bars represent standard deviations. The definition of low (blue), middle (magenta), and high (red) in E and F were the same as that of Fig. 3F. Bars and error bars are mean ± SD. Of Nanog-EGFP, N = 20 (high), 32 (middle) and 20 (low) for +2i, 26 (high), 18 (middle) and 21 (low) for +LIF, and 20 (high), 26 (middle) and 20 cells (low) for −LIF. Of Oct4-EGFP, N = 22 (high), 18 (middle) and 17 (low) for +2i, 17 (high) 16 (middle) and 20 (low) for +LIF, and 19 (high), 19 (middle) and 20 cells (low) for −LIF. Asterisks and double asterisks indicate p-values of less than 0.05 and 0.01, respectively, in Student's t-test. Exclamation marks and double exclamation marks indicate p-values of less than 0.05 and 0.01, respectively, in Mann Whitney's U-test.