EMBO/soda-nel huggingface dataset:
A voltage-gated calcium channel regulates lysosomal fusion with endosomes and autophagosomes and is required for neuronal homeostasis. (2015)
Fig 2

Aged <italic>cac</italic> mutant photoreceptor terminals show morphological defects and accumulate late stage AVs.

A. TEM sections of 3- and 27-days-old photoreceptor terminals of flies. Terminals of 27-days-old flies show terminal expansion, loss of capitate projections (CPs) and active zones (AZs) and increased number of mitochondria per terminal. Scale bars, 500 nm. B. Inset from A representing the photoreceptor terminal boxed in white. Yellow arrow indicates double-membraned autophagosome, red indicates amphisome, and blue indicates two fusion-primed AVs. Scale bars, 500 nm. C-E. Quantification of CPs, AZs, and mitochondria per cartridge, respectively. Data are presented as means ± SEM. F. Quantification of the different AVs per cartridge. The mutant terminals accumulate late stage AVs and fusion-primed AVs (ns: not significant; *: p < 0.05; **: p < 0.01; ***: p < 0.001; ****: p < 0.0001). Data are presented as means ± SEM. G. 30-days old fly eye brain complexes stained with neuronal marker Elav (blue) and poly-Ub antibody (red). The right panel is the enlarged images of the boxed regions at the left side. H. The level of p62 is increased in cacJ brain. I. Quantification of H. Data are presented as means ± SEM. (*: p < 0.05).
A voltage-gated calcium channel regulates lysosomal fusion with endosomes and autophagosomes and is required for neuronal homeostasis. (2015)
Fig 3

Autophagy defects observed in <italic>cac</italic> mutants are not due to defects in neurotransmitter release, but due to defects in lysosomal fusion.

TEM sections of 1-day-old and 30-days-old photoreceptor terminals of flies raised in 12 h light/dark conditions. A, B, D, and E. stj and Vha100–1 (V0) mutant terminals accumulate AVs upon aging. G, H. n-Syb mutant photoreceptor terminals show increased SVs, but not a significant accumulation of AVs and fusion-primed AVs. J, K, M and N. Vamp7 and fab1 mutant photoreceptor terminals show the highest increase in AV accumulation. C, F, I, L, O. Quantification of the different AVs for each genotype (**: p < 0.01). Eyes from three animals for each genotype at each age were analyzed and the counted cartridge number were listed as “n = ” in the figure. Data are presented as means ± standard deviation (SD). Scale bars, 1 μm.
A voltage-gated calcium channel regulates lysosomal fusion with endosomes and autophagosomes and is required for neuronal homeostasis. (2015)
Fig 4

<italic>Cacna1a</italic><sup>tg-la</sup> and <italic>Cacna2d2</italic><sup>du-2J</sup> mice have autophagy defects.

A. Calbindin and hematoxylin staining of mice cerebella indicates that Cacna1atg-la mice have progressive PC and granule cell loss upon aging. Scale bar, 200 μm. B, D. Calbindin (green) and 4',6-diamidino-2-phenylindole (DAPI)(blue) staining of mice cerebellum indicates that there are swollen PC axons (arrows) in the granular layers of the Cacna1atg-la mice cerebellum at day 25 (B) and the Cacna2d2du-2J mice at day 50 (D). Scale bar, 20 μm. C, E. The TEM of the axons at the granular layers of the cerebellum showing that many PC axons in Cacna1atg-la and Cacna2d2du-2J mice are swollen and accumulated with numerous mitochondria, autophagosome/MVBs-like structures (red arrows), and various cytoplasmic vesicles or aberrant membrane structures, including stacks of cisternal membranes (blue arrows), and folded ER (yellow arrows). Scale bars for right two panels are 2 μm. Other scale bars are 500 nm. F, G. LC3II and p62 are slightly increased in 15-days-old Cacna1atg-la mice and 50-days-old Cacna2d2du-2J mice. b, d are quantification of a and c. Data are presented as means ± SEM. (*: p < 0.05).
A voltage-gated calcium channel regulates lysosomal fusion with endosomes and autophagosomes and is required for neuronal homeostasis. (2015)
Fig 5

CACNA1A localizes to the lysosomes.

A. CACNA1A co-localizes with LAMP1 in primary cerebellar cultures of both CTL and Cacna1atg-la mutants. Scale bars, 20 μm. B. CACNA1A is present as punctae on the Vacuolin-1 enlarged LAMP1 positive lysosomes in primary cerebellar cultures of both CTL and Cacna1atg-la mutants. Scale bars, 20 μm. C. Quantification of LAMP1 positive organelles that are also CACNA1A positive. Data are presented as means ± SD. (ns: not significant). D. CACNA1A is concentrated in the lysosomal fractions isolated from mice cerebella with iodixanol gradient.
A voltage-gated calcium channel regulates lysosomal fusion with endosomes and autophagosomes and is required for neuronal homeostasis. (2015)
Fig 6

<italic>Cacna1a</italic> mutant neurons display lysosomal fusion defects.

A. LysoTracker Red staining of the primary cerebella neurons from Cacna1atg-la mice and WT controls. The control cells show big bright punctae, while the mutant cells have much dimmer and smaller punctae. B, C and D. Quantification of LysoTracker relative intensity (“n” represents the counted cell number), LysoTracker positive punctae number and size per cell (“n” represents the counted image number). E. Lysosomal marker LAMP1 co-localizes with autophagosome marker LC3 in CTL but not Cacna1atg-la mutant cells. Scale bar, 20 μm. F. Quantification of LC3 and LAMP1 co-localization. Data are presented as means ± SD. (ns: not significant; ***: p < 0.001).
A voltage-gated calcium channel regulates lysosomal fusion with endosomes and autophagosomes and is required for neuronal homeostasis. (2015)
Fig 7

The calcium channel activity of CACNA1A on the lysosome, not the plasma-membrane is required for lysosomal fusion.

A. DQ-BSA labeled late endosomes fuse with LAMP1 labeled lysosomes in CTL but not the mutant cells. Scale bar, 20 μm. B. Quantification of DQ-BSA and LAMP1 co-localization in CTL and Cacna1atg-la cells. Data are presented as means ± SD. (ns: not significant; ***: p < 0.001) C. Bepridil but not ω-agatoxin TK (ω-Aga TK) treatment reduces lysosomal fusion in cultured cerebellar cells. DQ-BSA (Green) labeled late endosomes co-localize with LAMP1 (Red) labeled lysosomes with/without ω-Aga TK (1 µM) or Bepridil (10 µM) treatment. Scale bar, 20 μm. D. Quantification of DQ-BSA and LAMP1 co-localization with ω-Aga TK or Bepridil treatment. Data are presented as means ± SD. (ns: not significant; **: p < 0.001).
Unexpected gene activation following CRISPR-Cas9-mediated genome editing (2021)
Figure 1

Figure 1 - A DSB in the promoter of ABCB1 causes gene upregulation and Taxol-resistance

A) Graphical representation of the ABCB1 genomic region and the location of the gRNA targeting the gene. RPE-1 cells were infected with a Lentivirus carrying one of the gRNAs and after 7 days of puromycin selection one million cells were plate with 8nM of Taxol for Colony Outgrowth Assay. Taxol resistant cells were counted and plotted in the graph. n=3.

B) Crystal violet staining of viability assay on RPE-1 Parental cells and Taxol resistant clones obtained from A. For the clones' nomenclature, sg# represents the sgRNA from where they are derived and C# the clone number.

C) Western Blot showing the levels of the PgP and control (α-TUBB) in RPE-1 parental and Taxol resistant clones.

D) ABCB1 mRNA levels determined by qRT-PCR and normalized to GAPDH expression levels. Each dot represents a technical replicate (n=3).

E) Representative smRNA-FISH images of RPE-1 Parental and clones for the ABCB1 gene and DAPI. The images are projections of 0,5μm sections and a total 5 μm in thickness. Scale bar, 15μm.
Unexpected gene activation following CRISPR-Cas9-mediated genome editing (2021)
Figure 2

Figure 2 - sgRNA integration and transcription activation of ABCB1

A) TLA analysis for ABCB1 contacts in RPE-1 parental and Taxol resistant clone sg6C9 covering the whole genome. Green arrow in sg6C9 shows a de novo interaction found between ABCB1 and a region in chromosome 6.

B) TLA analysis for RPE-1 Parental and sg6C9. Zoom in in the region of chr6 with de novo interaction for sg6C9. Image modified from IGV viewer.

C) Graphical representation of the PCR products to assess vector integration. Two different primer pairs were used to PCR the vector integration: 1) a common EEF1A1 Forward (F) primer with a specific Reverse for each break site (5/11/6/3/12R). Only when EEF1A1 is integrated in cis we will obtain a PCR product. 2) A forward and Reverse primer to amplify each specific break site (5/11/6/3/12F and R). If EEF1A1 is integrated in the break site, the PCR product will be bigger.

D) PCR products using the primers in C(1) over the ABCB1 and EEF1A1 regions in RPE-1 Parental and the different Taxol resistant clones.

E) PCR products using the primers in C(2) over the specific break site in the ABCB1 promoter in RPE-1 Parental and the different Taxol resistant clones.

F) TLA analysis for RPE-1 Parental and sg6C9. Reads are aligned to the lenti-guide vector. The location of the EF1α promoter from the vector is highlighted in red. Image modified from IGV viewer.

G) Percentage of RPE-1 cells targeted with the ABCB1 promoter sgRNAs found with vector integration. Percentages were calculated from data in Fig 1A. Each dot represents a CFA replicate experiment. Horizontal bars represent the mean of lentiviral integratation per sgRNA.
Unexpected gene activation following CRISPR-Cas9-mediated genome editing (2021)
Figure 3

Figure 3 - Chromatin landscape in the ABCB1 promoter region upon LentiGuide vector integration

A) H3K9me3 ChIP-sequencing tracks from the q21.12 arm of chromosome 7 in RPE-1 cells (Parental and Taxol-resistant clones). The ABCB1 gene region is shown in pink. Each colored track shows the H3k9me3 profile of a different Taxol-resistant clone.

B) H3K9me3 ChIP-qPCR in the ABCB1 promoter region. Bar graph show primer pairs amplifying the sgRNA#6 break and spanning this region (+/- base pairs) for each Taxol-resistant clone compared to the parental. Bar plots show the mean of H3K9me3 relative enrichment. Each dot represents a technical replicate (n=3).

C) Graphical representation of the ABCB1 promoter region. The location of the sgRNAs targeting the promoter are shown in blue. The location of the eleven CpG islands analyzed in the methyl array are shown in colored dots.

D) Relative methylation status (1: methylated, 0: non-methylated) of the eleven CpG islands shown in C for RPE-1 parental and the Taxol-resistant clones.
Mycobacterium tuberculosis eis regulates autophagy, inflammation, and cell death through redox-dependent signaling. (2010)
Figure 1

Mtb Eis modulates autophagy in macrophages.

(A) BMDMs were infected with Mtb-WT, Mtb-Δeis, or Mtb-c-eis (MOI = 10) for 4 h (as described in the Materials and Methods), and then incubated for 24 h (left) or the indicated periods of time (right). Cells were fixed, stained with DAPI to visualize nuclei (blue), and immunolabeled with an anti-LC3 antibody. Primary antibody was detected using an Alexa Fluor 488-conjugated goat anti-rabbit IgG (green). Left: representative immunofluorescence images of LC3 punctae; right: quantification of data (LC3-punctated cells were counted manually). ***p<0.001, vs. Mtb-WT-infected condition. Scale bars, 5 µm. (B) BMDMs were infected with Mtb-Δeis in the absence or presence of 3-methyladenine (3-MA; 10 mM) and subjected to confocal analysis as described in Figure 1A. LC3-punctated cells were counted manually. Each condition was assayed in triplicate, and at least 250 cells were counted in each well. ***p<0.001, vs. SC. (C) Immunoblot analyses performed using Abs raised to LC3 or β-actin. Experimental conditions were identical to those outlined in panel A. Gel images representative of three experiments are shown. (D) Electron micrographs of Mtb-Δeis-infected BMDMs under low (left) and high (right) magnification show the accumulation of autophagic vesicles (black arrow, initial autophagic vacuoles; white arrow, degradative autophagic vacuoles). Scale bars: 2 µm (left), 0.5 µm (right). (E) Immunoblot analyses performed using Abs raised to LC3 or β-actin. BMDMs were infected with Mtb-Δeis in the presence or absence of 3-MA (10 mM) or bafilomycin A1 (Baf-A1; 100 nM). Gel images representative of three experiments are shown. The ratio of the intensities of the LC3-II/LC3-I and β-actin bands is indicated below each lane (C and E). UI, uninfected; SC, solvent control (0.1% distilled water (B), 0.1% DMSO (E)).
Mycobacterium tuberculosis eis regulates autophagy, inflammation, and cell death through redox-dependent signaling. (2010)
Figure 2

Mtb-<italic>Δeis</italic> infection increases production of proinflammatory cytokines and ROS by BMDMs.

(A and B) BMDMs were infected with Mtb-WT, Mtb-Δeis or Mtb-c-eis at different MOIs (0.1, 1 and 10) for 18 h (A) or for the indicated periods of time (B; MOI = 10). Supernatants were assessed by ELISA for levels of TNF-α and IL-6. Data (A and B) are presented as the mean±SD of five experiments. (C and D) BMDMs were stimulated with Mtb-WT, Mtb-Δeis, or Mtb-c-eis for 30 min. Cells were then incubated with 10 µM DHE or 5 µM DCFH-DA for 15 min, washed thoroughly, and immediately analyzed for superoxide or H2O2 production by flow cytometry (C, Left). Cells were labeled with MitoSOX for 30 min and analyzed for mitochondrial ROS levels by flow cytometry (D, top). Quantitative analysis of ROS generation (C, right; D, bottom). *p<0.05, **p<0.01, ***p<0.001, vs. Mtb-WT-infected condition. UI, uninfected.
Mycobacterium tuberculosis eis regulates autophagy, inflammation, and cell death through redox-dependent signaling. (2010)
Figure 3

Increased ROS generation plays a critical role in autophagy and proinflammatory cytokine production in Mtb-<italic>Δeis</italic>-infected macrophages.

(A–C) BMDMs were infected with Mtb-Δeis (MOI = 10) for 18 h in the presence or absence of DPI (10 µM), NAC (20 mM), catalase (Cat, 1 mU/mL), or tiron (5 mM). (A) Representative immunofluorescence images (top); percentage of endogenous LC3-punctated cells (bottom). (B) Immunoblot analyses of BMDMs with antibodies raised to LC3 or β-actin. Gel images are representative of three experiments. The ratio of the intensities of the LC3-II/LC3-I and β-actin bands is indicated below each lane. (C) Experimental conditions were identical to those outlined in Figure 3A. Supernatants collected 18 h after infection were assessed for cytokine levels by ELISA. Data represent the mean±SD of five experiments. (D–F) BMDMs from WT and NOX2 KO mice were infected with Mtb-WT, Mtb-Δeis, or Mtb-c-eis for 18 h. (D) Numbers of LC3-punctated cells (counted manually) are shown (at least 250 cells were counted in each well). (E) Immunoblot analyses performed using Abs raised to LC3 or β-actin. BMDMs from WT and NOX2 KO mice were infected with Mtb-Δeis for 18 h. Gel images representative of three experiments are shown. (F) Supernatants collected 18 h after infection were assessed for cytokine levels by ELISA. Data are presented as the mean±SD of at least three separate experiments, each performed in triplicate. ***p<0.001, vs. SC (A and C); WT mice (D and F). UI, uninfected; SC, solvent control (0.1% DMSO).
Mycobacterium tuberculosis eis regulates autophagy, inflammation, and cell death through redox-dependent signaling. (2010)
Figure 4

Macrophages infected with Mtb-<italic>Δeis</italic> show reduced cell viability and increased caspase-independent cell death.

(A) BMDMs were infected with Mtb-WT, Mtb-Δeis, or Mtb-c-eis (MOI = 10) for the indicated periods of time, washed to remove unbound mycobacteria, and then incubated in complete DMEM at 37°C in 5% CO2. Cell viability was assessed by PI staining and then examined by fluorescence microscopy. (B and C) Experimental conditions were identical to those outlined in panel A. BMDMs were infected with the three strains of mycobacteria for 36 h. (B) Apoptosis was assessed using a TUNEL/apoptosis detection kit, according to the manufacturer's protocol. Cells were then examined under a laser-scanning confocal microscope (LSM 510; Zeiss). Percentages of TUNEL-positive, PI-positive, and TUNEL-/PI-double-positive cells were calculated. Data are representative of three separate experiments. (C) Morphological changes in BMDMs infected with Mtb-WT, Mtb-Δeis, or Mtb-c-eis at MOIs of 1 and 10. Representative images are shown. Scale bars: 50 µm (low magnification), 20 µm (high magnification). (D) Macrophages were infected with Mtb-WT, Mtb-Δeis, or Mtb-c-eis in the presence or absence of zVAD-fmk (20 µM) or 3-MA (10 mM). Staurosporine (STS; 500 nM) was used as a positive control. After 36 h, cells were stained with PI and then examined by fluorescence microscopy. Data are presented as the mean±SD of at least three separate experiments, each performed in duplicate. *p<0.05, ***p<0.001, vs. Mtb-WT-infected condition (A and B); Mtb-Δeis–infected condition without inhibitors (D). UI, uninfected.
Mycobacterium tuberculosis eis regulates autophagy, inflammation, and cell death through redox-dependent signaling. (2010)
Figure 5

Infection with Mtb-<italic>Δeis</italic> induces cell death through JNK-dependent signaling.

(A and B) BMDMs were infected with Mtb-WT, Mtb-Δeis, or Mtb-c-eis (MOI = 1) for the indicated periods of time, and then subjected to Western blot analysis using Abs raised to p-ERK1/2, p-p38, p-JNK, and β-actin. Data shown are representative of three independent experiments that all yielded similar results (A). Expression of phospho-MAPK/β-actin protein in cytoplasmic extracts of BMDMs was quantified densitometrically (B). (C and D) BMDMs were pretreated with U0126 (5, 10, 20 µM), SB203580 (SB; 1, 5, 10 µM), or SP600125 (SP; 5, 10, 20 µM) for 45 min, and then infected with Mtb-Δeis for 30 min (C) or 36 h (D). (C) Cells were then incubated with DHE for 15 min, washed rapidly and thoroughly, and analyzed immediately for superoxide levels by flow cytometry. Quantitative DHE fluorescence data represent the mean±SD of four experiments. (D) Cell death after 36 h was assessed by PI staining and then examined by fluorescence microscopy. (E) Raw264.7 cells were transfected with siRNA specific for JNK1 (siJNK) or a non-specific control siRNA (siNS). At 24 h after transfection, cells were infected with Mtb-Δeis for 36 h. Cell death was then assessed by PI staining, and then examined by flow cytometry. Transfection efficiency was assessed by RT-PCR (inset). Data represent the mean±SD of five random fields and are representative of three independent experiments (D and E). *p<0.05, **p<0.01, ***p<0.001, vs. Mtb-WT-infected condition (B); SC (C and D); siNS (E). UI, uninfected; SC, solvent control (0.1% DMSO).
Mycobacterium tuberculosis eis regulates autophagy, inflammation, and cell death through redox-dependent signaling. (2010)
Figure 6

<italic>In vivo</italic> analysis of autophagic vesicles, inflammation, and cell death in infected mice with Mtb-<italic>Δeis</italic>.

C57BL/6 mice were challenged, by aerosol, with 10–30 CFU Mtb-WT, Mtb-Δeis, or Mtb-c-eis and sacrificed 4 weeks post-infection. (A) High- and low-magnification electron micrographs of lung tissue sections from mice infected with Mtb-Δeis show accumulation of autophagic vesicles (black arrow, bacteria in autophagic vacuoles; white arrow, degradative autophagic vacuoles). Scale bars: 2 µm (left upper), 0.5 µm (right). Numbers of autophagic vacuoles per cell in each TEM section (left lower) (mean±SD; n = 50). (B) Quantitative RT-PCR analysis of lung tissue from Mtb-WT-, Mtb-Δeis-, and Mtb-c-eis-infected mice. Total RNA was extracted from paraffin-embedded lung tissue sections, as described in the Materials and Methods. (C) To assess in vivo cell death, bronchoalveolar lavage fluid cells from Mtb-WT-, Mtb-Δeis-, and Mtb-c-eis-infected mice were subjected to PI staining, and analyzed by flow cytometry. Data are presented as the mean±SEM (n = 4). (D) Numbers of CFUs in lung and spleen 4 weeks after infection with Mtb-WT, Mtb-Δeis, or Mtb-c-eis. Data are presented as log10 CFU±SEM (n = 4). (E) BMDMs were infected with Mtb-WT, Mtb-Δeis, or Mtb-c-eis and then analyzed by CFU assay. CFU data represent the mean±SD of four individual experiments. **p<0.01, ***p<0.001, vs. Mtb-WT-infected condition. UI, uninfected.
Mycobacterium tuberculosis eis regulates autophagy, inflammation, and cell death through redox-dependent signaling. (2010)
Figure 7

Eis modulates ROS release and proinflammatory cytokine production through its <italic>N</italic>-acetyltransferase domain.

(A) Intracellular superoxide production was analyzed by flow cytometric analysis. BMDMs were infected with Mtb-Δeis (MOI = 10) in the presence or absence of recombinant Eis protein (rEis; 5, 10, 20 µg/mL) or 30 kDa Mtb antigen (30 k; 5, 10, 20 µg/mL). Upper, representative flow cytometric analysis; lower, quantitation of superoxide generation. (B) THP-1 cells were transfected with mock, Eis-WT, or Eis-ΔAT constructs, and infected with Mtb-Δeis for 30 min. Cells were stained with DHE (for superoxide) or DCFH-DA (for H2O2) and subjected to flow cytometric analysis. Inset, transfection efficiency. (C and D) Experimental conditions were identical to those outlined in panels A and B, respectively. Supernatants were collected 18 h after infection and assessed by ELISA for levels of TNF-α and IL-6. Data are presented as the mean±SD of five experiments. (E and F) THP-1 cells transfected with mock, Eis-WT, or Eis-ΔAT constructs were pretreated with SP600125 (SP; 20 µM) or SB203580 (SB; 5 µM) for 45 min before infection with Mtb-Δeis for 30 min (E) or 18 h (F). E, Intracellular superoxide production was analyzed by flow cytometric analysis. F, ELISA analysis for TNF-α and IL-6 levels. Data are presented as the mean±SD of three experiments. **p<0.01, ***p<0.001, vs. SC (A, C, E, and F); mock control (B and D). UI, uninfected; SC, solvent control (0.1% DMSO).
Rabl2 GTP hydrolysis licenses BBSome-mediated export to fine-tune ciliary signaling (2020)
Figure 1

Figure 1. Murine Rabl2 is dispensable for ciliogenesis.

A. Upregulation of Rabl2 during multiciliogenesis. Cultured mTECs were induced to undergo multicilia formation by growing at an air-liquid interface (ALI) for the indicated time and subjected to immunoblotting. Ift52, Ift139, Arl6, and acetylated tubulin (Ac-tub) served as ciliary markers. Gapdh served as the loading control.

B. Rabl2 levels in different tissues from 8-week-old mice.

C. Rabl2 knockout strategy and representative genotyping PCR results using the indicated primers (P1-P4). FRT, flippase recognition target; LacZ, β-galactosidase gene; Neo, neomycin-resistant gene; IRES, internal ribosome entry site; LoxP, Cre recombinase recognition site.

D. Complete depletion of Rabl2 in tissues (from 8-week-old mice) upon its gene knockout.

E-H. Normal ciliary formation in representative bright-field (E), scanning electron (F, G, H), or immunofluorescent (F, G, H) micrographs of the indicated tissues (from 8-week-old mice) or cells. Cultured mEPCs were fixed at day 7 post serum starvation. Ac-tub served as ciliary marker. DAPI labeled nuclear DNA. Typical cilia or flagella are indicated by arrows, in which yellow arrows denote primary cilia.

I, J. Normal ciliary formation of Rabl2-/- MEFs. MEFs derived from E12.5 mouse embryos were serum-starved for 48 h to induce primary cilia (I). Statistical results (J) were from three independent experiments. Ciliogenesis of at least 200 cells was scored in each experiment and condition. Ciliary lengths of 80 cells were measured in each experiment and condition and the results of the same genotypes were pooled together.

K, L. Rabl2 deficiency did not repress ciliary localization of IFT-B. Ciliated MEFs were immunostained for Ift81 and Ift57, two IFT-B subunits (K; see Fig 4F for diagram of IFT-B). Relative intensity of ciliary Ift81 (L), presented in arbitrary unit, was obtained by normalizing to that of corresponding Ac-tub. Four MEF strains prepared respectively from two embryos per genotype were used for quantification. 50 cilia were quantified from each MEF strain and results of the same genotypes were pooled together.

Data information: Statistical results are presented as mean ± s.d. Student's t-test: ns, no significance; **P < 0.01.