EMBO/soda-nel huggingface dataset:
Activation of autophagy during cell death requires the engulfment receptor Draper (2010)
figf1

a, Protein extracts from drpr null (w; drpr&Dgr;5/drpr&Dgr;5) pupae at puparium formation (0 h) and wild-type (Canton-S) salivary glands 6 h, 12 h and 14 h after puparium formation, analysed by western blotting with anti-Drpr antibody. b, Samples from control animals (+/w; +/drpr&Dgr;5), n = 12, and drpr null mutants (w; drpr&Dgr;5/drpr&Dgr;5), n = 47, analysed by histology for the presence of salivary gland material (red circles) 24 h after puparium formation. c, Quantification of data from b and d. d, Samples from control animals (+/w; +/UAS-drprIR), n = 11, and those with salivary gland-specific knockdown of drpr (fkh–Gal4/w; UAS-drprIR/+), n = 19, analysed by histology for the presence of salivary gland material (red circles) 24 h after puparium formation. e, Control animals (+/w; +/UAS-drpr-IIR), n = 9, and those with salivary gland-specific knockdown of drpr-I (fkh–Gal4/w; UAS-drpr-IIR/+), n = 20, analysed by histology for the presence of salivary gland material (red circles) 24 h after puparium formation. f, Quantification of data from e. g, Samples from drpr null animals (+/w; +/UAS-Drpr-I; drpr&Dgr;5/drpr&Dgr;5), n = 9, and those with salivary gland-specific expression of Drpr-I (fkh–Gal4/w; UAS-Drpr-I/+; drpr&Dgr;5/drpr&Dgr;5), n = 20, analysed by histology for the presence of salivary gland material (red circles) 24 h after puparium formation. h, Quantification of data from g.
Activation of autophagy during cell death requires the engulfment receptor Draper (2010)
figf2

a, Samples from animals with salivary gland-specific expression of p35 (fkh–Gal4/+; UAS-p35/+), n = 18 (left), drpr null animals (+/w; +/UAS-p35; drpr&Dgr;5/drpr&Dgr;5), n = 10 (middle), and drpr null animals with salivary gland-specific expression of p35 (fkh–Gal4/w; UAS-p35/+; drpr&Dgr;5/drpr&Dgr;5), n = 16 (right), analysed by histology for the presence of salivary gland material 24 h after puparium formation. Red circles, cell fragments; yellow circle, gland fragments. b, Quantification of data from a. c, Salivary glands dissected from animals expressing drprIR specifically in GFP-marked clone cells (heat shock (hs)flp/w; UAS-drprIR/+; act,cd2, FRT > Gal4, UAS-GFP/+) 6 h and 14 h after puparium formation. Stained with GFP antibody (green), to label cells expressing drpr IR, and Lamin antibody (red).
Activation of autophagy during cell death requires the engulfment receptor Draper (2010)
figf3

a, Samples from animals with salivary gland-specific knockdown of Atg12 (fkh–Gal4/w; UAS-Atg12IR/+), n = 21, and those with salivary gland-specific knockdown of both Atg12 and drpr-I (fkh–Gal4/w; UAS-Atg12IR/+; UAS-drpr-IIR/+), n = 19, analysed by histology for the presence of salivary gland material (red circles) 24 h after puparium formation. b, Quantification of data from a. c, GFP–LC3 was expressed in salivary glands of control animals (+/w; UAS-GFP–LC3/+; fkh–Gal4/+) and those with salivary gland-specific knockdown of drpr (w; UAS-drprIR/UAS-GFP–LC3; fkh–Gal4/+). Salivary glands were dissected 6 h and 14 h after puparium formation, imaged for GFP–LC3, and LC3 puncta were quantified using Zeiss Automeasure software. d, Quantification of data from c. Error bars represent s.e.m.; n ≥ 10; p  0.0000001. e, LAMP1–GFP was expressed in control animals (tub-LAMP–GFP/w; +/fkh–Gal4) and those with salivary gland-specific knockdown of drpr (tub-LAMP–GFP/w; UAS-drprIR/+; fkh–Gal4/+). Salivary glands were dissected 14 h after puparium formation, imaged for LAMP–GFP, and LAMP puncta were quantified using Zeiss Automeasure software. f, Quantification of data from e. Error bars represent s.e.m.; n ≥ 10; p  0.05. g, Samples from drpr mutant animals (+/w; +/UAS-Atg16A; drprDgr;5/drprDgr;5), n = 10, and those with salivary gland-specific expression of Atg16A (fkh–Gal4/w; UAS-Atg16A/+; drprDgr;5/drprDgr;5), n = 16, analysed by histology for the presence of salivary gland material (red circles) 24 h after puparium formation. h, Quantification of data from g.
Activation of autophagy during cell death requires the engulfment receptor Draper (2010)
figf4

a, Protein extracts from drpr null (w; drprDgr;5/drprDgr;5) pupae at puparium formation (0 h) and from the fat bodies of wild-type (Canton-S) third-instar larvae were analysed by western blotting with anti-Drpr antibody. Third-instar larvae were either fed or starved for 4 h. b, We either fed Wild-type (Canton-S) third-instar larvae or starved them for 4 h, and dissected fat bodies, stained them with anti-Drpr antibody, and imaged them for Drpr (green). We stained nuclei with 4′,6-diamidino-2-phenylindole (DAPI) (blue). c, We starved third-instar larvae expressing GFP–Atg8a in all cells, and drprIR specifically in dsRed-marked clone cells (hsflp/w; UAS-drprIR/+; hsGFPAtg8a, act,cd2, FRT > Gal4, UAS-dsRed/+), for 4 h. We dissected larval fat bodies and imaged them for GFP–Atg8a (green) and dsRed (red). d, Salivary glands of animals expressing GFP–Atg8a in all cells, and drprIR specifically in dsRed-marked clone cells (hsflp/w; UAS-drprIR/+; hsGFPAtg8a, act,cd2, FRT > Gal4, UAS-dsRed/+), dissected 14 h after puparium formation. Imaged for GFP–Atg8a (green) and dsRed (red). We stained nuclei with Hoechst (blue).
The intramembrane protease SPPL2c promotes male germ cell development by cleaving phospholamban (2018)
Figure 1

FIGURE 1. SPPL2c is a testis-specific ER-resident intramembrane protease.

(A) Predicted topology of murine SPPL2c. Positions of the predicted N-glycosylation site and critical aspartate residues of the active centre (asterisks) are indicated.

(B) Scheme of the two murine SPPL2c isoforms A and B. Epitopes (N-term/C-term) used for generation of antisera are marked. SP, predicted signal peptide.

(C) RT-PCR analysis of SPPL2c expression in different murine tissues. Total RNA from the indicated tissues was either transcribed into cDNA prior to PCR amplification (+RT) or, as negative control, used directly as template (-RT). As indicated in Fig EV1A, a common forward primer in combination with an isoform-specific reverse primer was employed. A fragment of the actin ORF was amplified as control.

(D, E) Western Blot analysis of total lysates from testis isolated from wild type or SPPL2c-/- using antibodies against C- (D) or N-terminal (E) epitopes of SPPL2c. Lysates from HeLa cells transiently expressing the SPPL2c isoforms were analysed in parallel. Full Western Blots are shown in Fig EV1B. *, non-specific band.

(F) SPPL2c was visualised by indirect immunofluorescence in HeLa cells transiently expressing C-terminally Myc-tagged murine SPPL2c isoform A. For detection of SPPL2c, either anti-Myc or the SPPL2c antiserum (C-terminal epitope) were employed as indicated together with anti-KDEL, anti-GM130 or anti-ERGIC53 as indicated in order to label the ER, the cis Golgi apparatus or the ER-Golgi intermediate compartment (ERGIC), respectively. Scale bars, 10 µm.

(G) Lysates from HEK293 cells transiently transfected with murine SPPL2c-myc (isoform A) were treated with the glycosidases PNGase F or Endo H as indicated prior to Western blot analysis with anti-SPPL2c (C-term). Deglycosylation of endogenously expressed LIMP2 was analysed as control.

(H) N-glycosylation of endogenous SPPL2c is similar to the overexpressed protein. Total lysates from testis of wild type or SPPL2c-/- mice were treated and analysed as in (G).

(I) Subcellular fractionation of postnuclear supernatant from murine testis using a self-forming Percoll density gradient. Fractions were collected from bottom to top and subjected to Western blot analysis for SPPL2c. Protein disulfide isomerase (PDI), Cathepsin D and CD44 were detected as organelle marker proteins.
The intramembrane protease SPPL2c promotes male germ cell development by cleaving phospholamban (2018)
Figure 2

FIGURE 2 SPPL2c cleaves selected tail-anchored (TA) proteins with a substrate spectrum distinct from SPP.

(A) HEK293 cells were transiently transfected with murine N-terminally HA-tagged heme oxygenase 1 (HA-mHO-1) alone or in combination with active or inactive (D/A) murine SPP or SPPL2c. Substrate levels were determined by Western blot analysis with anti-HA. For detection of SPP, an antiserum recognizing both endogenous human (h) and overexpressed murine (m) SPP was employed. Murine SPPL2c was visualised using the antiserum generated against the C-terminus of the protein. Actin was detected to control for equal protein loading.

(B) Immunofluorescence analysis of HeLa cells transiently expressing murine HO-1 (HA-mHO-1) alone or together with active or inactive (D/A) SPPL2c-myc. Substrate and proteases were visualised using the HA and Myc epitopes, respectively. Scale bars, 10 µm.

(C) The TA protein Ube2J1 is not proteolysed by co-expressed SPP or SPPL2c in HEK293 cells. Western blot analysis was performed as in (A).

(D) Differential cleavage of CYB5a, RAMP4-2 and RAMP4 by SPPL2c and SPP. Substrates and proteases were co-expressed in HEK293 cells followed by Western blot analysis as described above. Densitometric quantification from at least n=3 independent experiments was performed. Substrate levels were normalised and compared to cells without protease-overexpression. Mean ± SD; unpaired student's t test; ***, p≤0.001; ns, non-significant.

(E) HEK293 cells expressing HA-RAMP4-2 alone or in combination with SPPL2c or SPP were treated with 100 µM (Z-LL)2-ketone (ZLL), 5 µM inhibitor X (InX), 5 µM DAPT or DMSO as control. RAMP4-2 band intensity was quantified from blots of three independent experiments and normalised to cells just over-expressing the substrate. Mean ± SD, unpaired student's t test; ***, p≤0.001, *, p≤0.05; ns, non-significant.

(F) Microsomes isolated from testis of wild type and SPPL2c-/- mice were solubilised in 1% digitonin and proteins were separated by blue native PAGE. After transfer to PVDF membrane, SPPL2c and SPP were detected using the polyclonal antisera introduced above.
The intramembrane protease SPPL2c promotes male germ cell development by cleaving phospholamban (2018)
Figure 3

FIGURE 3. SPPL2c has a critical function in elongated spermatids.

(A) Histochemical visualization of β-galactosidase (β-gal) reporter activity revealed SPPL2c expression within seminiferous tubules. Cryo-sections from PFA-fixed wild type and SPPL2c-/- testis were stained with X-Gal as β-gal substrate. Scale bar, 500 µm.

(B) SPPL2c protein levels were analysed in germ cell populations from testis of wild type mice sorted for their DNA content which was determined by Hoechst 33342 staining as depicted in Fig EV4D. While for 4C, 2C and 1C fractions 10 µg of protein were loaded, for the testis control samples (=total lysates) 20 µg of protein were subjected to electrophoresis. *, non-specific band.

(C) Immunohistochemical detection of SPPL2c was performed on cryosections of Bouin-fixed wild type and SPPL2c-/- testis. The SPPL2c antiserum directed against a C-terminal epitope of the protease and diaminobenzidine as peroxidase substrate were employed. Nuclei were stained with hematoxylin. Scale bar, 100 µm (upper panel) or 50 µm (lower panel).

(D) SPPL2c levels were compared in total lysates prepared from testis of wild type mice sacrificed at the age of 4, 6, 8 or 12 weeks by Western blotting. A testis lysate from an adult SPPL2c-/- mouse was used as negative control.

(E) The weight of both testicles of wild type and SPPL2c-/- mice (n=12 per genotype) was determined and normalised to the body weight. Mean ± SD; unpaired student's t test; *, p≤0.05.

(F) Paraffin sections from Bouin-fixed wild type and SPPL2c-/- testis were stained with hematoxylin & eosin (H&E). Scale bars, 100 µm.

(G) In H&E stained paraffin-sections from Bouin-fixed wild type and SPPL2c-/- testis, the mean number of elongated spermatids per seminiferous tubule was determined. Cells were counted in 20 tubuli in sections from n=6 mice per genotype. Mean ± SD; unpaired student's t test; *, p≤0.05.

(H) SPPL2c is not present in mature spermatozoa. Western blot analysis of total lysates of wild type (+/+) and SPPL2c-/- testis, epididymis and spermatozoa recovered from the cauda epididymidis/vas deferens for SPPL2c.

(I) Mature sperm were isolated from the epididymis of wild type or SPPL2c-deficient mice and analysed by phase-contrast microscopy. Representative pictures of morphologically normal spermatozoa are depicted. Scale bars, 10 µm.

(K) Reduced motility of SPPL2c-/- sperm cells. Spermatozoa were recovered from the cauda epididymidis of wild type and SPPL2c-/- mice (n=3) and subjected to computer-assisted sperm analysis. Average path velocity (VAP), straight line velocity (VSL) and curvilinear velocity (VCL) were determined in micrometer per second. Mean ± SD; unpaired student's t test; **, p≤0.01.

(L) Mean litter sizes for different breeding schemes. The data depicted are based on n=12 (+/+X+/+), n=57 (+/-X+/-), n=11 (-/-X-/-), n=10 (-/-X+/+) and n=11 (+/+X-/-) different breedings, respectively. Mean ± SD; one-way Anova with Newman-Keuls post hoc testing; **, p≤0.01.
The intramembrane protease SPPL2c promotes male germ cell development by cleaving phospholamban (2018)
Figure 4

FIGURE 4. Identification of phospholamban (PLN) as novel substrate of SPPL2c.

(A) Carbonate-washed total membrane fractions from wild type and SPPL2c-/- testis (n=5) were subjected to a label-free quantitative proteome analysis. For each quantified protein, a log2 intensity ratio between SPPL2c-deficient and wild type samples was calculated. Negative values indicate a depletion and positive values an enrichment in SPPL2c-/- samples. In the depicted volcano plot the negative log10 of the p value (y axis) is plotted versus the log2 intensity ratio of SPPL2c-/- versus wild type samples (x axis). All proteins above the significance level of p<0.01 (unpaired Student's t test) are coloured in red. The hyperbolic curve indicates the threshold for a permutation-based FDR correction for multiple hypotheses with p = 0.05 and s0 = 0.1.

(B) Quantification of SPPL2c, C11orf94, Syntaxin 8 (Stx8) and Phospholamban (PLN) in the proteomic dataset. A two-sided Student's t-Test was performed.

(C) HeLa cells were transiently transfected with N-terminally HA-tagged PLN (HA-PLN) and inactive (D/A) SPPL2c-myc The expressed proteins were visualised by indirect immunofluorescence via their HA and Myc epitopes. Scale bars, 10 µm.

(D, E) HEK293 cells were transiently transfected with HA-PLN alone or in combination with active or inactive (D/A) murine SPPL2c Isoform A (D) or Isoform B (E). Substrate processing was analysed by Western blotting with anti-HA. SPPL2c expression was confirmed with the antiserum against the C-terminal epitope. Actin was detected to confirm equal protein loading.

(F) Scheme of mutants generated for analysis of determinants within the PLN transmembrane domain (TMD) required for SPPL2c-mediated intramembrane proteolysis.

(G) Subcellular localisation of the PLN mutants was analysed by indirect immunofluorescence using anti-HA in combination with anti-protein disulphide isomerase A6 (PDIA6) to stain the ER. The 37-41A and 47-51 PLN mutants were not expressed in relevant amounts. Scale bars, 10 µm.

(H) HEK293 cells were transiently transfected with wild type or mutated HA-PLN alone or together with SPPL2c isoform A.

(I) Quantification of n=5 experiments as shown in (H). For each PLN variant, the residual PLN in SPPL2c co-expressing cells is depicted as % of PLN in absence of the protease. Mean ± SD; one-way ANOVA with Dunett's post hoc test. ** p≤0.01, ns = not significant.
The intramembrane protease SPPL2c promotes male germ cell development by cleaving phospholamban (2018)
Figure 5

FIGURE 5. SPPL2c regulates Ca2+ homeostasis in spermatids by cleaving phospholamban.

(A) Accumulation of endogenous PLN in the testis of SPPL2c-/- mice. PLN was immuno-precipitated (IP) from equal protein amounts of total lysates of wild type or SPPL2c-/- testis with the mouse monoclonal antibody. Western blot analysis of the IPs was performed with the rabbit monoclonal PLN antibody. SPPL2c and actin were detected from total lysates directly.

(B) Quantification of normalised PLN/Actin ratios from n=3 biological replicates per genotype. Mean ± SD, unpaired student's t test; **, p≤0.01.

(C) Expression of PLN in murine testis from wild type (wt) SPPL2c-/- (2c-/-) was quantified by qPCR. n=6. Unpaired Student's t-test. ns = not significant.

(D) Immunohistochemical detection of PLN in paraformaldehyde (PFA)-fixed testis from wild type and SPPL2c-/- mice. Cryosections were stained using a rabbit monoclonal PLN antibody or normal rabbit IgG as negative control and diaminobenzidine as peroxidase substrate. Scale bars, 100 µm.

(E) Testis suspensions were stained with 2.5 µM DRAQ5 to determine cellular DNA amount. 1C-co = elongated spermatids, 1C = other haploid cells including round spermatids, 2C = spermatogonia, secondary spermatocytes, Sertoli cells, other somatic cells, 4C = primary spermatocytes, G2 spermatogonia.

(F) Intracellular Ca2+ concentrations were analysed in testis suspensions from either wild type or SPPL2c-deficient mice using the Ca2+-sensitive probe Fluo4-AM. Individual germ cell populations were identified as shown in (E). Bars indicate Median Fluo4-AM fluorescence ± SD of 8 individual samples from 3 mice per genotype normalised to those of wild type samples. Unpaired Student's t-test. *** p≤0.001, ns = not significant.

(G) Median forward scatter (FSC) was controlled in the 1C-co population from the datasets shown in (F). Median FSC ± SD of 8 individual samples from 3 mice per genotype. Unpaired Student's t-test. ns = not significant.

(H, I) Expression of SPPL2c in human testis. (H) Western blot analysis of lysates from HEK293 cells expressing human (h) SPPL2c, human and murine testis using antisera against a C-terminal epitope of murine SPPL2c or human SPPL2c as indicated. (I) Immunohistochemical detection of SPPL2c with the monoclonal 1A1 antibody in paraffin-sections from Bouin's-fixed human testis. In addition, sections were incubated with control immunoglobulins. Arrows indicate elongated spermatids. Scale bars, 30 µm.

EXPANDED VIEW FIGURE LEGENDS
SEUSS Integrates Transcriptional and Epigenetic Control 
of Root Stem Cell Organizer Specification (2020)
Figure 1

Figure 1. Ablation of SEU reduces WOX5 expression and impairs quiescent center (QC) specification.

A Expression patterns of pSEU::SEU-GFP and pWOX5::GFP in the embryos of the indicated genotypes at dermatogen, early globular, and heart stages. White arrows indicate the QC precursor cell, and white dashed lines indicate embryos. Scale bars: 10 µm.

B Quantification of pWOX5::GFP GFP fluorescence in wild-type (WT) and seu-3 mutant embryos. Fluorescence intensity at the early globular stage of WT embryos was set to 1.

C, D Expression pattern of pWOX5::GFP in WT (C) and seu-3 (D) embryos at 5 days after germination (DAG). Scale bars: 20 µm.

E Quantification of GFP fluorescence in the QC of pWOX5::GFP transgenic plants, as shown in (C) and (D). Fluorescence intensity was normalized to the WT.

F RT-qPCR analysis of the relative expression levels of WOX5 in WT and seu-3 roots. Total RNA was extracted from 5 mm root tip sections of seedlings at 5 DAG.

G-J Modified pseudo-Schiff propidium iodide (mPS-PI) staining of stem cell niche areas in the indicated genotypes at 5 DAG. Blue arrows indicate the QC, and red arrows indicate the columella stem cells (CSCs). The numbers denote total number of scored samples, with similar phenotypes showing in (G-J). Scale bars: 20 μm.

K Quantification of the CSC layer in the indicated genotypes at 5 DAG.

L, M Double staining of the QC184 β-glucuronidase (GUS) marker (light blue) and starch granules (dark brown) in WT (L) and seu-3 (M) seedlings at 5 DAG.

N, O Expression pattern of J2341 in WT (N) and seu-3 (O) seedlings at 5 DAG.

P, Q Expression pattern of CS9227 in WT (P) and seu-3 (Q) seedlings at 5 DAG.

Data Information: In (B), (E), (F) and (K), data represent mean ± SD of three independent replicates. n denotes the total number of scored samples. Individual values (black dots) are shown. **P < 0.01, *P < 0.05 (Student's t-test). In (L-Q), white arrows indicate the CSCs. Scale bars: 20 μm.
SEUSS Integrates Transcriptional and Epigenetic Control 
of Root Stem Cell Organizer Specification (2020)
Figure 2

Figure 2. SEU acts in the SHR/SCR pathway to promote WOX5 expression and QC specification.

A-C Expression pattern of pWOX5::GFP in WT (A), scr-3 (B) and plt1-4 plt2-2 (C) at 5 DAG.

D Quantification of GFP fluorescence in the QC of pWOX5::GFP transgenic seedlings, as shown in (A-C). GFP signal intensity in each genotype was normalized relative to that in the WT.

E RT-qPCR analysis of the relative expression levels of WOX5 in WT, scr-3, and plt1-4 plt2-2 roots. Total RNA was extracted from 5 mm root tip sections of seedlings at 5 DAG.

F Primary root length of the indicated genotypes at 5 DAG.

G-J Representative confocal images of the indicated genotypes at 5 DAG. White arrows indicate the QC.

K Quantification of GFP fluorescence in the QC of pSCR::GFP and pSCR::GFP-SCR transgenic seedlings, as shown in (G-J). GFP signal intensity of each genotype was normalized relative to that of the WT.

L-Q Root stem cell niche phenotypes of the indicated genotypes at 5 DAG. White dashed lines indicate the QC region. The numbers denote total number of scored samples, with similar phenotypes showing in (L-Q).

Data information: In (D), (F) and (K), n denotes the total number of scored samples. Individual values (black dots) are shown. Data represent mean ± SD of three independent replicates. In (D) and (F), different lowercase letters indicate significant differences by one-way ANOVA followed by Tukey's multiple comparison test (P < 0.01). In (E) and (K), **P < 0.01 (Student's t-test). Scale bars: 20 µm.
SEUSS Integrates Transcriptional and Epigenetic Control 
of Root Stem Cell Organizer Specification (2020)
Figure 4

Figure 4. SDG4 interacts with SEU and deposits H3K4me3 modification in the WOX5 promoter.

A Y2H assays showing that SEU interacts with SDG4 and SDG25. The yeast transformants were plated on SD/-2 and SD/-4 media to assess protein-protein interactions.

B Schematic diagram of the WOX5 and PCR amplicons (indicated as letters A-I) used for ChIP-qPCR.

C SDG4 physically bind to the WOX5 promoter, as shown by ChIP-qPCR analysis. ACT7, control.

D, E ChIP-qPCR analysis indicating that mutations in SDG4 and SEU genes impair H3K4me3 deposition in the WOX5 promoter. ChIP signal was normalized to the WT. Individual values (black dots) are shown.

F, G Expression pattern of pWOX5::GFP in the indicated genotypes at 5 DAG. Scale bars: 20 µm.

H Quantification of GFP fluorescence in the indicated genotypes expressing pWOX5::GFP. GFP signal intensity in the WT was set to 1. n denotes the total number of scored samples.

I RT-qPCR analysis of the relative expression levels of WOX5 in WT and ashr3-1 roots. Total RNA was extracted from 5 mm root tip sections of seedlings at 5 DAG. Individual values (black dots) are shown.

J-M mPS-PI staining of stem cell niche in the indicated genotypes at 5 DAG. Blue arrows indicate the QC, and red arrows indiate the CSCs. The numbers denote total number of scored samples, with same phenotypes showing in (J-M). Scale bars: 10 μm.

N Quantification of the CSC layer in the indicated genotypes at 5 DAG.

Data information: For (C-E), Chromatin was isolated from 5 mm root tip sections of seedlings at 5 DAG, sonicated, and immunoprecipitated using anti-GFP or anti-H3K4me3 antibodies. The precipitated DNA was used as a template for qPCR analysis. PCR amplicons are shown in (B). In (C), (D), (E), (H),(I) and (N), data represent mean ± SD of three independent replicates. **P < 0.01, *P < 0.05 (Student's t-test).