EMBO/soda-nel huggingface dataset:
Activation of serum/glucocorticoid‐induced kinase 1 (SGK1) is important to maintain skeletal muscle homeostasis and prevent atrophy (2012)
Figure 5

Characterization of skeletal muscle of SGK1 transgenic mice.

A.

H&E staining of tibialis anterior sections from 2-month old control and transgenic mice shows no differences (scale bar 90?µm).

B.

Morphometric analyses of minimum Feret's diameter of TA muscle reveal no changes in fiber size distribution or mean fiber size.

C.

Western blots and densitometric analysis of muscle from transgenic and control mice. Downstream targets of the mTOR signalling cascade (p70S6K and 4EBP1) demonstrate significant upregulation of their phosphorylated forms in sgk1tg mice.

D.

Western blot analyses and densitometry of phosphorylated FOXO3a and total FOXO3a of tibialis anterior muscles from control and transgenic mice (n?=?4) show an increase of phosphorylation at S315 and T32.
Activation of serum/glucocorticoid‐induced kinase 1 (SGK1) is important to maintain skeletal muscle homeostasis and prevent atrophy (2012)
Figure 6

SGK1 protects against starvation‐induced muscle atrophy

A.

H&E staining of tibialis anterior sections reveals no changes in muscle architecture (scale bar 90?µm).

B.

Morphometric analysis demonstrates a decrease in muscle fiber size of the wild-type control mice compared with sgk1tg transgenic littermates (p?=?0.0015).

C.

Western blot and densitometry analysis shows that levels of phosphorylated Foxo3a at S315 and T32 are increased in sgk1tg.
Activation of serum/glucocorticoid‐induced kinase 1 (SGK1) is important to maintain skeletal muscle homeostasis and prevent atrophy (2012)
Figure 7

Electroporation of constitutively active Sgk1 protects against immobilization atrophy.

A.

Transfection of constitutively active Sgk1 (CA) into immobilized tibialis anterior muscle (green) reveals increased fiber size diameter when compared to control, EGFP only transfected muscle fibers of immobilized tibialis anterior muscles (100?µm). Laminin ?-1 staining (red) outlines the basement membrane and blue staining marks nuclei (DAPI).

B.

Representation of the minimum Feret's diameter average?±?SD (control and immobilized; n?=?910 per group; 800950 fibers were measured) (*p?=?0.029, *p?=?0.034, *p?=?0.047, p?=?0.43, respectively).

C.

Percentage distribution of the minimum Feret's diameter of tibialis anterior muscle immobilized and transfected with eGFP (GFP), wild-type Sgk1 (WT), kinase dead Sgk1 (KD) and constitutively active Sgk1 (CA) compared to non-immobilized control TA muscle (black dotted line).

D.

Schematic representation of pathways involved in the preservation of skeletal muscle mass during prolonged periods of immobilization. SGK1, in addition to Akt, mediates protein degradation via phosphorylation of Foxo3a, with the subsequent inhibition of proteolysis and autophagy and protein synthesis through the activation of mTOR.
Bif-1 interacts with Beclin 1 through UVRAG and regulates autophagy and tumorigenesis (2007)
figf1

The percentage of dead cells was determined at the indicated timepoints by trypan blue exclusion assay (mean ± s.d.; n = 3). (a) Bif-1+/+ (wild type; WT) and Bif-1−/− (knockout; KO) MEFs were cultured in EBSS for 0, 3, 6 or 12 h. (b) Bif-1−/− MEFs stably transfected with a Bif-1 expression plasmid or control empty vector were incubated in EBSS for 0 or 12 h. Recovery of Bif-1 expression in Bif-1−/− cells was determined by immunoblot analysis (inset). (c) Bif-1+/+ and Bif-1−/− MEFs were cultured in EBSS in the presence of 50 μM z-VAD-fmk or control DMSO for 0, 3, 6 or 12 h. (d) Bax+/+Bak+/+ (WT) and Bax−/−Bak−/− (DKO) MEFs were cultured in EBSS in the presence of 50 μM z-VAD-fmk or control DMSO for 0, 6 or 12 h.
Bif-1 interacts with Beclin 1 through UVRAG and regulates autophagy and tumorigenesis (2007)
figf2

(a, b) Bif-1+/+ and Bif-1−/− MEFs or Bax+/+Bak+/+ and Bax−/−Bak−/− MEFs were infected with Beclin 1 shRNA or control shRNA (SCR shRNA) lentiviruses. Twenty-four hours after infection, the medium was replaced with fresh culture medium and the cells were allowed to recover for an additional 48 h. The cells were then cultured in EBSS for 0, 6 or 12 h, and the percentage of dead cells was determined by trypan blue exclusion assay (mean ± s.d.; n = 3). Knockdown of Beclin 1 expression was confirmed by immunoblot analysis (insets). (c, d) Bif-1+/+ and Bif-1−/− MEFs were pre-treated with 10 mM 3-methyladenine (3-MA), 0.2 μM wortmannin (WM) or control DMSO for 30 min followed by culture in EBSS for the indicated times. The percentage of dead cells was determined by trypan blue exclusion assay (mean ± s.d.; n = 3). Immunoblot analyses were performed to detect LC3 modification and caspase-3 activation. (e) Atg5+/+ and Atg5−/− MEFs were cultured in EBSS in the presence of 50 μM of z-VAD-fmk or control DMSO for 0, 6 or 12 h. The percentage of dead cells was determined by trypan blue exclusion assay (mean ± s.d.; n = 3). (f) Total cell lysates (TCL) were prepared from Atg5+/+ and Atg5−/− MEFs cultured in EBSS for the indicated times and subjected to immunoblot analyses. Uncropped images of the immunoblots in a, b, d and f are shown in the Supplementary Information, Fig. S5.
Bif-1 interacts with Beclin 1 through UVRAG and regulates autophagy and tumorigenesis (2007)
figf3

(a) Bif-1+/+ and Bif-1−/− MEFs were cultured in complete medium (CM) or EBSS for 3 h and analysed by transmission electron microscopy. Arrows indicate autophagosomes. The number of autophagosomes per cross-sectioned cell was counted (mean ± s.d.; n = 42). (b) Bif-1+/+ and Bif-1−/− MEFs were transfected with GFP–LC3, cultured in EBSS or complete medium for 3 h, then analysed by fluorescent microscopy. The number of GFP–LC3 dots per GFP-positive cell was counted (mean ± s.d.; n = 35). (c) Bif-1+/+ and Bif-1−/− MEFs were cultured in EBSS for the indicated times and subjected to immunoblot analysis with anti-LC3, anti-α-tubulin and anti-β-actin antibodies. (d) Bif-1−/− MEFs stably transfected with a Bif-1 expression plasmid or control empty vector were cultured in EBSS for 0, 3 or 6 h and subjected to immunoblot analysis. The immunoblotting results of LC3 in c and d were quantified as the percentage of LC3-II out of total LC3 (LC3-I + LC3-II) and are represented in bar graphs. (e) Stable Bif-1 shRNA-expressing clones or control HeLa cells transfected with GFP–LC3 were cultured in complete medium or EBSS for 2 h and analysed by fluorescent microscopy. The number of GFP–LC3 dots per GFP-positive cell was quantified (mean ± s.d.; n = 66). (f) Bif-1 knockdown HeLa cells were transfected with GFP–LC3 in combination with Bif-1 shRNA-resistant Bif-1–HcRed, Bif-1ΔBAR–HcRed or empty HcRed vector for 20 h. The cells were then cultured in EBSS or complete medium for 2 h and the number of GFP–LC3 dots per HcRed-positive cell was determined using a fluorescence microscope (mean ± s.d.; n = 35). Statistical significance in a, b, e and f was determined by Student's t-test. Uncropped images of the immunoblots in c, d and e are shown in the Supplementary Information, Fig. S5. The scale bar represents 5 μm in the upper panels in a; the scale bar in the lower panel of a is 1 μm; the scale bars in b and e represent 10 μm.
Bif-1 interacts with Beclin 1 through UVRAG and regulates autophagy and tumorigenesis (2007)
figf4

(a) COS7 cells were transfected with Bif-1–HcRed together with GFP–Atg5 or GFP–LC3. Twenty hours after transfection, the cells were cultured in EBSS (middle and bottom panels) or complete medium (top panel) for 2 h and subjected to confocal microscopic analysis. Magnified images are shown as insets. Representative Bif-1–Atg5 or Bif-1–LC-3 double-positive foci are indicated by arrows. (b) COS7 cells transfected with Bif-1–GFP were cultured in EBSS for 2 h and the localization of Bif-1–GFP was examined by immunogold electron microscopy using anti-GFP antibodies. Arrows indicate representative gold particles that are detected on autophagosomal membranes. The scale bars represent 10 μm in the main images and 1 μm in the insets in a, and 1 μm in b.
Bif-1 interacts with Beclin 1 through UVRAG and regulates autophagy and tumorigenesis (2007)
figf5

(a) Bif-1+/+ and Bif-1−/− MEFs were cultured in EBSS for 0 or 6 h and subjected to immunoprecipitation with anti-Bif-1 monoclonal antibodies. The resulting immune complexes and TCL were analysed by immunoblotting with the indicated antibodies. (b) 293T cells were cotransfected with the indicated plasmids for 24 h and subjected to immunoprecipitation with anti-Flag monoclonal antibodies. The resulting immune complexes and TCL were analysed by immunoblotting with anti-Myc or anti-Flag polyclonal antibodies. (c) 293T cells were cotransfected with the indicated plasmids for 22 h, starved in EBSS for 2 h and subjected to immunoprecipitation with anti-Flag monoclonal antibodies. The resulting immune complexes and TCL were analysed by immunoblotting with anti-Myc or anti-Flag polyclonal antibodies. (d) 293T cells were transfected with GFP siRNA or UVRAG siRNA for 24 h, starved in EBSS for 2 h and subjected to immunoprecipitation with anti-Bif-1 monoclonal antibodies. The resulting immune complexes and TCL were analysed by immunoblotting with anti-Bif-1 and Beclin 1 polyclonal antibodies. The reduction of UVRAG protein expression by UVRAG siRNA was confirmed by immunoprecipitation and immunoblot analysis with anti-UVRAG polyclonal antibody. Uncropped images of the immunoblots are shown in the Supplementary Information, Fig. S5.
Bif-1 interacts with Beclin 1 through UVRAG and regulates autophagy and tumorigenesis (2007)
figf6

(a) HeLa cells were transfected with 0.2 μg GFP–LC3 and 0, 0.2 or 0.6 μg of Bif-1SH3–Myc expression plasmids for 24 h. The total amount of plasmid DNA was adjusted at 0.8 μg per transfection with the pcDNA3 vector. The cells were then cultured in EBSS or complete medium for 3 h and the number of GFP–LC3 dots per GFP-positive cell was determined using a fluorescence microscope (mean ± s.d.; n = 40). (b) HeLa cells were transfected with GFP–LC3 in combination with either Bif-1SH3–HcRed, Endophilin A1SH3–HcRed or empty HcRed vector for 20 h. The cells were then cultured in EBSS or complete medium for 2 h and the number of GFP–LC3 dots per HcRed-positive cell was counted using a fluorescence microscope (mean ± s.d.; n = 46). Statistical significance in a and b was determined by Student's t-test and the asterisks indicate P 0.0001. (c) 293T cells were transfected with the indicated plasmids for 24 h and subjected to immunoprecipitation with anti-Flag monoclonal antibodies. The resulting immune complexes and TCL were analysed by immunoblotting with anti-Myc and anti-Flag polyclonal antibodies. Uncropped images of the immunoblots in c are shown in the Supplementary Information, Fig. S5.
Bif-1 interacts with Beclin 1 through UVRAG and regulates autophagy and tumorigenesis (2007)
figf7

(a) Bif-1+/+ and Bif-1−/− MEFs were transfected with Flag–PI(3)KC3, then incubated in complete medium or EBSS for 2 h and subjected to an in vitro PI(3)KC3 kinase assay. The expression of Flag–PI(3)KC3 was confirmed by immunoblot analysis (left panel). A representative autoradiograph is shown (middle). The results were quantified by phosphoimaging with a Typhoon scanner and shown as fold activation (right). The data are presented as means ± s.d. of five independent experiments. (b) Bif-1+/+ and Bif-1−/− MEFs were transfected with p40(phox) PX–EGFP, cultured in complete medium or EBSS for 2 h and analysed using a fluorescence microscope. The number of p40(phox) PX–EGFP-positive vesicles per cell was counted (mean ± s.d.; n = 35). Statistical significance in a and b was determined by Student's t-test. (c) Stable Bif-1 shRNA clones or control HeLa cells were transfected with Flag–PI(3)KC3 alone or in combination with Flag-tagged Beclin 1 and UVRAG for 48 h and subjected to an in vitro PI(3)KC3 lipid kinase assay. The results were quantified and are shown as relative activities (mean ± s.d.; n = 3). (d) 293T cells were cotransfected with Flag–PI(3)KC3 and either wild-type Bif-1, Bif-1ΔSH3 or control empty vector for 24 h and subjected to an in vitro PI(3)KC3 kinase assay (mean ± s.d.; n = 3). (e) Schematic representation of the Bif-1–UVRAG–Beclin 1–PI(3)KC3 protein complex.
Bif-1 interacts with Beclin 1 through UVRAG and regulates autophagy and tumorigenesis (2007)
figf8

(a) Bif-1−/− mice exhibit moderate splenomegaly. The weight of spleens extirpated from Bif-1+/+ (n = 27) and Bif-1−/− (n = 34) mice (6–18-month old) was measured. Statistical significance was determined by Wilcoxon rank test. (b) Hematoxylin and eosin staining of tumour tissues. Tumours were observed in 3 of 21 (14.3%) and 26 of 29 (89.7%) autopsied Bif-1+/+ and Bif-1−/− mice, respectively. The scale bars represent 50 μm.
SIGNR3‐dependent immune regulation by Lactobacillus acidophilus surface layer protein A in colitis (2015)
Figure 1

Lactobacillus acidophilus NCK2187 strain development and characteristics

Agarose gel image illustrating PCR amplicons of ltaS (LBA0447), slpB, and slpX deletions in NCK2187.
SDS–PAGE gel of 5 M LiCl‐purified S‐layer fractions from the parental strains, NCK56 and NCK1909, NCK2030 (LTA+, SlpA+, SlpB−, SlpX−), and NCK2187 (LTA−, SlpA+, SlpB−, SlpX−).
B6 mice were orally gavaged with 109 CFU erythromycin‐resistant NCK56 or NCK2187. Fecal pellets were collected daily and tested for the presence of erythromycin‐resistant strains. n = 3 mice/group. Data are representative of five independent experiments and are shown as mean ± SEM.
Colonic LP cells were co‐cultured with NCK56 or NCK2187 (1:1), and secreted cytokines were measured in the supernatants. Data are shown as mean ± SEM and are representative of two experiments performed in triplicate. *P 0.05, **P 0.01, ***P 0.001.
SIGNR3‐dependent immune regulation by Lactobacillus acidophilus surface layer protein A in colitis (2015)
Figure 2

Lactobacillus acidophilus NCK2187 promotes intestinal regulation in steady state

A B6 mice were orally gavaged with 109 CFU NCK56 (blue) or NCK2187 (green) on days 0, 3, 6, and 9, or left untreated, and immune responses in the colon analyzed at day 14 by flow cytometry.
B, C B6 FoxP3‐GFP mice were treated and evaluated as in (A). Regulatory cytokine production in FoxP3‐GFP+ (green‐dotted bars) versus FoxP3‐GFP− (white bars) cells was measured by intracellular staining and FACS analyses (C).

Data information: For all data shown, n = 5 mice/group. Data represent four individual experiments and are shown as mean ± SEM. *P 0.05, **P 0.01, ****P 0.0001. Black asterisks compare NCK2187 to untreated (PBS) mice, and red asterisks to NCK56‐treated mice.
SIGNR3‐dependent immune regulation by Lactobacillus acidophilus surface layer protein A in colitis (2015)
Figure 3

Lactobacillus acidophilus NCK2187 and its SlpA protect against pathogenic T‐cell‐induced colitis

A B6 Rag1−/− mice were injected with 106 CD4+CD45RBhi T cells and then orally gavaged with NCK56 (red), NCK2187 (green), or SlpA (blue), 1 and 3 days after transfer, and subsequently once a week for four consecutive weeks, or left untreated (magenta). A group of mice was co‐transferred with CD4+CD25+ T cells as a positive control for protection (Tregs; gray). Colitis severity was determined in part by weight loss, diarrhea scores, and FOB. See Supplementary Tables S1, S2 and S3 for statistical analyses results.
B, C Colitis scores based on histopathology and gross morphology of the colons of mice treated as in (A) were also used as measures of disease. Scale bar = 50 μm.
D Circulating levels of proinflammatory cytokines were measured in the sera of the mice transferred and treated as in (A), or sham adoptive transferred (white bars).

Data information: Data represent three individual experiments and are shown as mean ± SEM. n = 5 mice/group. *P 0.05, **P 0.01, ***P 0.001, ****P 0.0001. Black asterisks compare NCK2187 to PBS‐treated adoptively transferred mice, and red asterisks to NCK56‐treated mice.
SIGNR3‐dependent immune regulation by Lactobacillus acidophilus surface layer protein A in colitis (2015)
Figure 4

Lactobacillus acidophilus NCK2187 and its SlpA protect intestinal barrier function and prevent dysbiosis in pathogenic T‐cell‐induced colitis

Colitis was induced in B6 Rag1−/− mice as described in Fig .

A, B Colonic expression of tight junction‐associated genes Cldn3 and Ocln, determined by RT–PCR relative to 18S rRNA (A), as well as passive transepithelial absorption of FITC‐dextran (B), were used as measures of epithelial barrier integrity. Sham adoptive transferred B6 Rag1−/− mice (white bars) were used as baseline controls in some cases. n = 5 mice/group. Data represent three individual experiments and are shown as mean ± SEM. *P 0.05, **P 0.01. Black asterisks compare NCK2187 to PBS‐treated adoptively transferred mice, and red asterisks to NCK56‐treated mice.
C UniFrac analyses were used to calculate distances between the microbial communities of the different samples (week 7), and three‐dimensional scatter plots were generated by using principal coordinate analyses (PCoA). Gray dots = CD4+CD45RBhi T cells + PBS; yellow dots = CD4+CD45RBhi T cells + NCK56; green dots = CD4+CD45RBhi T cells + NCK2187; blue dots = CD4+CD45RBhi T cells + SlpA; red dots = CD4+CD45RBhi T cells + Tregs. Each dot represents the fecal microbiota data of an individual mouse.
D Changes in the relative abundance of different phyla were also analyzed. See Supplementary Table S4 for results of statistical analyses among the different groups. Dark blue = Actinobacteria; green = Bacteroidetes; red = Proteobacteria; yellow = Verrucomicrobia; aqua = Firmicutes; purple = Tenericutes; black = others.
E Comparison of microbial communities at family or order levels. The heat map depicts the relative value in individual mice. n = 5–6 mice/group.
SIGNR3‐dependent immune regulation by Lactobacillus acidophilus surface layer protein A in colitis (2015)
Figure 5

Lactobacillus acidophilus NCK2187 and its SlpA bind to murine SIGNR3 to induce regulatory signals

A B6 mice were orally gavaged with 109 CFU NCK56 or NCK2187, and the colonic gene expression of C‐type lectin receptors were measured by RT–PCR. Each box represents an individual mouse; n = 4. Data represent three individual experiments.
B Binding of SlpA to various hFc fusion proteins was analyzed by flow cytometry. Gray tinted line = SlpA‐coated beads only; orange = SlpA‐coated beads + secondary antibody; green = SlpA‐coated beads + control fusion protein; blue = SlpA‐coated beads + SIGNR1‐hFc; red = SlpA‐coated beads + SIGNR3‐hFc. Binding assay results were confirmed five independent times.
C Binding of SlpA to SIGNR3, but not SIGNR1 expressed by CHO‐S cells. Gray tinted line = untransfected CHO‐S cells; blue = untransfected CHO‐S cells + labeled SlpA; green = SIGNR1‐transfected CHO‐S cells + labeled SlpA; red = SIGNR3‐transfected CHO‐S cells + labeled SlpA. Binding assays in CHO‐S cells were performed three individual times.
D Binding of SlpA to recombinant human DC‐SIGN/CD209‐Fc chimera was analyzed by flow cytometry. Gray tinted line = SlpA‐coated beads only; orange = SlpA‐coated beads + secondary antibody; green = SlpA‐coated beads + control fusion protein; red = SlpA‐coated beads + DC‐SIGN‐Fc. Binding assays were performed and confirmed three times.
E Binding of SlpA to DC‐SIGN expressed by CHO‐S cells. Gray tinted line = untransfected CHO‐S cells; blue = untransfected CHO‐S cells + labeled SlpA; red = DC‐SIGN‐transfected CHO‐S cells + labeled SlpA. Binding assays in CHO‐S cells were performed three individual times.
F, G IL‐1β production by colonic DCs of naïve WT B6 GF or B6 Signr3−/− (KO) mice treated with NCK56 (blue) or NCK2187 (green) four times, or left untreated (black), as determined by flow cytometry. n = 5 mice/group. Data represent four individual experiments and are shown as mean ± SEM. *P 0.05. Black asterisks compare NCK2187 to untreated (PBS) mice, and red asterisks to NCK56‐treated mice.
H Frequency of colonic FoxP3+ Tregs in KO mice treated with NCK56 or NCK2187 was measured by flow cytometry. n = 5 mice/group. Data represent four individual experiments and are shown as mean ± SEM.
SIGNR3‐dependent immune regulation by Lactobacillus acidophilus surface layer protein A in colitis (2015)
Figure 6

Lactobacillus acidophilus NCK2187 and its SlpA do not protect against DSS‐induced colitis in Signr3−/− mice

WT B6 or B6 Signr3−/− (KO) mice were orally gavaged with NCK56, NCK2187, or SlpA on days −3 and −1, and 3% DSS was given in the drinking water. Mice were gavaged with bacteria or purified SlpA every other day for an additional three times and monitored for disease progression.

A Colitis severity was determined in part by weight loss. n = 5 mice/group.
B, C Colitis scores based on histopathology and gross morphology of the colons were also used as measures of disease. Scale bar = 50 μm. n = 5 mice/group. Empty bars = WT; lined bars = KO; white bars = untreated; purple bars = DSS; red bars = DSS + NCK56; green bars = DSS + NCK2187; blue bars = DSS + SlpA.
D Colonoscopies were performed in the different groups with a Multi‐Purpose Rigid™ Telescope attached to a TELE PACK X on day 10.
E Mean relative colonic expression of tight junction‐associated genes in WT mice. n = 5 mice/group.
F Fecal albumin levels in WT mice as a measure of intestinal permeability. n = 5 mice/group.
G UniFrac analyses were used to calculate distances between the microbial communities of the different samples (day 10), and three‐dimensional scatter plots were generated by using PCoA. Light gray: WT + DSS; green: WT + DSS + NCK56; brown: WT + DSS + NCK2187; blue: WT + DSS + SlpA; aqua: KO + DSS; red: KO + DSS + NCK56; yellow: KO + DSS + NCK2187; purple: KO + DSS + SlpA. n = 4–6 mice/group.
H Species richness and microbial diversity in DSS‐treated mice. Top: The Chao richness index was used as a measure of species richness. Bottom: The Shannon diversity index was used to estimate microbial diversity for each group. n = 4–6 mice/group. Color coding is as in (B).

Data information: Data are shown as mean ± SEM and are representative of three different experiments. *P 0.05, **P 0.01, ****P 0.0001.
SIGNR3‐dependent immune regulation by Lactobacillus acidophilus surface layer protein A in colitis (2015)
Figure 7

Lactobacillus acidophilus NCK2187 and its SlpA do not prevent immune infiltration and activation during DSS‐induced colitis in Signr3−/− mice

Signr3+/+ (WT) or Signr3−/− (KO) mice were orally gavaged with NCK56, NCK2187, or SlpA on days −1 and −3, and 3% DSS was given in the drinking water. Mice were gavaged with bacteria or purified SlpA every other day for an additional three times, and immunity was analyzed by flow cytometry at day 10.

A Representative plots indicate the frequency of neutrophils in the colons of untreated or DSS‐treated WT (left) and KO mice (right). Empty bars = WT; lined bars = KO; white bars = untreated; purple bars = DSS; red bars = DSS + NCK56; green bars = DSS + NCK2187; blue bars = DSS + SlpA.
B, C Colonic DCs and macrophages (MΦs) were analyzed by flow cytometry for the production of IL‐1β (B), and colonic FoxP3+ Tregs were evaluated for co‐expression of RORγt+ (C). n = 5 mice/group. Gray tinted line = isotype control; black = untreated; purple = DSS; red = DSS + NCK56; green = DSS + NCK2187; blue = DSS + SlpA.

Data information: Data are shown as mean ± SEM and are representative of three different experiments. *P 0.05, **P 0.01, ***P 0.001, ****P 0.0001.