EMBO/sd-character-level-ner huggingface dataset:
A pooled, genome-wide CRISPR screen for candidate genes involved in HIV-1 latency.Figure 1A. The outline of the genome-wide CRISPR screen strategy. C11 cells were infected with a lentiviral library containing Cas9 proteins and sgRNAs that target 19050 human genes. After fourteen days of puromycin selection, genomic DNA was extracted from GFP-positive cells after two-rounds of sorting. The candidate genes were then identified by next generation sequencing. B. Flow cytometry of cells infected with the lentiCRISPR v2.0 library where the expression of GFP indicates latency reactivation. Target genes were enriched through two-round sorting. Continuously cultured C11 latent cells infected with the lentiCRISPRv2.0 served as control. Blue dots and the encircled area represent the GFP-positive cells for flow cytometry analysis. C. Validation of sgRNAs in unsorted and sorted C11 cells by PCR after lentiCRISPR v2.0 library infection. D. Before next generation sequencing, the GFP expression in two-round sorted C11 cells was confirmed by fluorescence microscopy. Scale bar, 100 μm. E. Fold change (Log2) of the abundance of target genes in sorted C11 cells. Enriched sgRNA genes are highlighted.
Figure 5(A) Survival percentage of 3-4 dpf zebrafish embryos (n ≥156) upon infection with S. pneumoniae T4 alone or its isogenic PLY mutant, T4Δply. Injection with E3 growth medium served as mock control. *** denotes P < 0.0005 and **** denotes P < 0.0001 by Mantel Cox test. Exact P values are shown in Appendix Table S4. (B) Zebrafish survival percentage upon infection with T4 alone or together with peptide P2 or CP2 or P2-conjugated CaP NPs (P2-NPs). *** denotes P < 0.0005 and **** denotes P < 0.0001 by Mantel Cox test. Exact P values are shown in Appendix Table S4. (C) Survival of mice (n=10) upon intranasal infection with 2x106 CFU of S. pneumoniae T4 together with peptide P2 or CP2 or P2-NPs over 3 days post infection. Infected mice were checked twice daily in the morning at 9 am (early check) and in evening at 7 pm (late check) for clinical symptoms. Unloaded NPs and the isogenic PLY mutant strain (T4Δply) served as negative controls. * denotes P < 0.05 and ** denotes P < 0.005 by Mantel Cox test. Exact P values are shown in Appendix Table S4. Bacterial load in the lungs of infected mice (n=10) were measured post sacrifice. Data represent mean ± s.e.m. * denotes P < 0.05, ** denotes P < 0.01 and **** denotes P <0.0001 by one-way ANOVA with Bonferroni's post hoc test for multiple comparisons. n.s. denotes not significant. Exact P values are shown in Appendix Table S4. levels of pro-inflammatory cytokines TNF-α in the lungs of infected mice (n=10) were measured post sacrifice. Data represent mean ± s.e.m. * denotes P < 0.05, ** denotes P < 0.01 and **** denotes P <0.0001 by one-way ANOVA with Bonferroni's post hoc test for multiple comparisons. n.s. denotes not significant. Exact P values are shown in Appendix Table S4. levels of pro-inflammatory cytokines IL-12 in the lungs of infected mice (n=10) were measured post sacrifice. Data represent mean ± s.e.m. * denotes P < 0.05, ** denotes P < 0.01 and **** denotes P <0.0001 by one-way ANOVA with Bonferroni's post hoc test for multiple comparisons. n.s. denotes not significant. Exact P values are shown in Appendix Table S4.
a) Representative physical pictures of 18-month-old WT and Atg5 Tg mice fed with a regular diet.f3(a) Representative physical pictures of 18-month-old WT and Atg5 Tg mice fed with a regular diet. (b) Body weight chart of ageing-induced obesity in WT and Atg5 Tg mice. The mice were fed with a regular diet for 24 months (n=180--250) and their body weights were monitored monthly. The bars represent the mean±s.d. ***P0.0001 versus control; Student's t-test. . (c) Average daily oral food intake normalized to body weight, measured on regular diet. The food intake of mice (n=18-24) was measured for 2 weeks at 3-, 6-, 12- and 18-month-old age. d) Quantification of gonadal fat pads from 18-month-old mice (n=11). ***P0.0001 versus control; Student's t-test. (e,f) Quantification of visceral (e) and subcutaneous (f) fat pads of 18-month-old mice (n=6). ***P0.0001 versus control; Student's t-test. (g) Enhanced glucose tolerance of Atg5 Tg mice. WT and Atg5 Tg mice were starved overnight and then given with an i.p injection of glucose (1 mg g−1 body weight). The results are the mean±s.d. of 12-week-old WT and Atg5 Tg mice (n=12). *P0.01, **P0.001, ***P0.0001 versus control; Student's t-test. (h) Enhanced insulin sensitivity of Atg5 Tg mice. WT and Atg5 Tg mice were starved for 6 h and then i.p injected with 0.75 IU soluble insulin. *P0.01, **P0.001 versus control; Student's t-test. (i) Plasma levels of triglycerol (TG) in WT and Atg5 Tg mice. The mice were fasted for 3 h before measurement. Young mice (4-week old, n=8); old mice (18-month old, n=8). ***P0.0001 versus control; Student's t-test (j) Plasma leptin levels in WT and Atg5 Tg mice (n=8-12). ***P0.0001 versus control; Student's t-test. ( (k) GSH/GSSG ratio in the liver (n=4 males, three times trial). **P0.001, ***P0.0001 versus control; Student's t-test. (l) Respiration rates of mitochondria in WT and Atg5 Tg MEFs (n=2). (m) Wire-hang endurance test of Atg5 Tg and WT mice (n=10). All the bars represent the mean±s.d. ***P0.0001 versus control; Student's t-test.
Autophagosome Levels in Bim-Depleted and -Overexpressing Cells.Figure 2(A) Bim−/− and Bim+/+ MEFs were cultured in 6-well dishes. Cells were treated with vehicle or Bafilomycin A1 (Baf) for 4 hr. Blots were probed with the indicated antibodies. LC3-II/tubulin in wild-type control MEFs is set as 1. The relative value of LC3-II/tubulin in Bim-knockout MEFs is shown (n = 5). Data are mean ± SD. ∗∗∗p < 0.001. (B) HeLa cells were transfected with control siRNA and Bim siRNA. After 48 hr, cells were treated with vehicle or Baf for 4 hr. Blots were probed with the indicated antibodies and analyzed as described in (A). ∗∗∗p < 0.001. (C) Bim−/− and Bim+/+ MEFs were transfected with GFP-LC3. The percentages of cells with GFP-LC3 vesicles were assessed. Data are shown as mean ± SD. ∗∗p < 0.01. (D) HeLa cells were transfected with control plasmid (pMKO)/GFP-LC3 or pMKO-BimshRNA/GFP-LC3 (to ensure that GFP-LC3-positive cells also contain BimshRNA or control plasmid, the ratio of pMKO or pMKO-BimshRNA/GFP-LC3 is 3:1). The percentages of cells with GFP-LC3 vesicles were assessed. Data are shown as mean ± SD. ∗∗∗p < 0.001. (E) HeLa cells were transfected with control plasmid (pMKO) or pMKO-BimshRNA. Blots were probed with the indicated antibodies. Data are shown as mean ± SD. ∗∗∗p < 0.001. (F) HeLa cells were transfected with control plasmid (pMKO)/GFP-PX or pMKO-BimshRNA/GFP-PX (3:1). The percentages of cells with GFP-vesicles were assessed. Data are shown as mean ± SD. ∗∗p < 0.01. (G) DFCP1-GFP cells were transfected with control siRNA or Bim siRNA. The numbers of GFP puncta were assessed. Data are shown as mean ± SD.∗p < 0.05.Images represent DFCP1-GFP vesicles in cells transfected with control siRNA and Bim siRNA. Arrows mark DFCP1-GFP puncta. (H) HeLa cells were treated with control siRNA or Bim siRNA. Blots were probed with the indicated antibodies. Data are shown as mean ± SD. ∗∗p < 0.01. (I) Bim(EL)/control siRNA, Bim(EL)/Bim siRNA, Bim(EL) EE/control siRNA, Bim(EL) EE/Bim siRNA, vector/control siRNA, and vector/Bim siRNA were transfected into HeLa cells. Blots were probed with the indicated antibodies. Data are shown as mean ± SD. ∗∗p < 0.01. (J) HeLa cells were transfected with control plasmid or Bim(EL) EE. Blots were probed with the indicated antibodies. Data are shown as mean ± SD. ∗∗p < 0.01. (K) Bax/Bak DKO MEFs were transfected with control plasmid /GFP-LC3, wild-type Bim(EL)/GFP-LC3 (3:1), or Bim (EL) EE/GFP-LC3. The percentages of cells with GFP-LC3 vesicles were assessed. Data are shown as mean ± SD. ∗∗p < 0.01. (L) Two independent control vector (pcDNA3) or Bim(EL) EE stably expressing HeLa cell clones were transfected with GFP-LC3 respectively. The percentages of cells with GFP-LC3 vesicles were assessed. Data are shown as mean ± SD. ∗∗∗p < 0.001. (M) HeLa cells were transfected with control plasmid/GFP-PX or Bim(EL) EE/GFP-PX (3:1). The percentages of cells with GFP-vesicles were assessed. Data are shown as mean ± SD. ∗∗p < 0.01. Fluorescent microscope images show GFP-PX vesicles.
The ADAP-SKAP55 module is dependent on NFATc1, Ca2+, and Fyn to control PD1 expression.Figure 3A-C WT, ADAP−/−, or SKAP55−/− OT-I CD8+ CTLs were stimulated with OVA257-264-pulsed or unpulsed EL4 cells to detect the levels of NFATc1 expression, activation and nuclear entry by immunoblotting (A) or immunostaining (B). Alternatively, nuclear extracts from these CTLs were incubated with the N1 DNA probes containing NFAT-binding sites for an EMSA. The unlabeled competitor probe (Comp. N1) and the mutant probe that contains the same sequence except for carrying a mutated NFAT-binding site (Comp. mutN1) were included as controls (C). D WT, ADAP−/−, or SKAP55−/− OT-I CD8+ CTLs were pretreated with DMSO or 5 μM CsA, then stimulated with OVA257-264-pulsed EL-4 cells for 4 h to examine surface PD-1 expression (mean of triplicates ± SD). E WT, ADAP−/−, or SKAP55−/− OT-I CTLs were pretreated with the DMSO control, 10 μM PP2 or 75 μM 2APB, then stimulated with OVA257-264-pulsed EL-4 cells for 4 h to examine surface PD-1 expression (mean of triplicates ± SD). F GFP, ADAP, or SKAP55 was overexpressed with pGL3-NFAT Luciferase reporter plasmid into Jurkat cells. Cells were stimulated with anti-CD3 and anti-CD28 in the presence or absence of PP2 or 2APB for 6 h, followed by measuring luciferase readings (mean of triplicates ± SD).
a) Wild-type (WT), atg17Δ, atg7Δ and atg19Δ strains containing RPL25–GFP were grown to mid log phase, starved in SD-N for 48 hours and examined under a fluorescence microscope (left panel).figf1(a) Wild-type (WT), atg17Δ, atg7Δ and atg19Δ strains containing RPL25-GFP were grown to mid log phase, starved in SD-N for 48 hours and examined under a fluorescence microscope (left panel). The arrows indicate vacuolar accumulation of the GFP signal. CVT, cytoplasm-to-vacuole-targeting pathway. After the times indicated, at least 100 cells per sample were scored for the presence of Rpl25p-GFP in the vacuole (right panel). Scale bar, 5 μm. (b) Wild-type and atg7Δ strains containing RPL25-GFP or GFP-ATG8 were grown and starved as in a. Samples were taken at indicated time points and GFP cleavage was analysed by western blotting. (c) The levels of endogenous Rps3p (top) and Rpl3p (middle) were analysed by immunblotting in wild-type and atg7Δ cells. Ponceau staining (bottom) controlled for equal loading. Uncropped images of blots are shown in Supplementary Information, Fig. S6. Starv, starvation. (d) Wild-type and pep4Δ cells expressing RPL25-GFP were analysed as in b; 0.1 mM of PMSF was added to pep4Δ cells at the time of starvation to block all vacuolar proteases completely. The LI-COR imaging system was used to quantify the ratio between cleaved GFP and full-length protein as described in Methods. The last time point of wild-type cells containing RPL25-GFP was set to 1 arbitrary unit (a.u.). One of two independent experiments is shown. (e) Wild-type cells containing either RPL25-GFP or control GFP (HOG1-GFP) were analysed as in d. The average of four independent experiments is shown, and the standard deviation was less than 10% (Supplementary Information, Table S3).
Figure 4 Figure 4. Restored p53 activity by nutlin3a impairs MGC formation and inos dysregulation A Representative images of unstimulated BMMΦ from wt and p53-/- mice at day 6 with hemacolor staining (left), LP stimulation for 6 days and quantification for MGC formation by hemacolor staining (right). Depicted are means ± SEM of 3 independent experiments; scale bar, 50µm. B BMMΦ were stimulated with LP for indicated times and either lysed with RIPA-buffer for western blot analysis of p53 levels or used to quantify transcript levels p21 relative to gapdh by qRT-PCR as fold change to unstimulated. Depicted are mean ± SEM from 3 independent experiments. C BMMΦ stimulated with LP from (B) were used for p53 ChIP at indicated timepoints and quantified by qRT-PCR for amplification of p53-associated p21 promoter region as fold change to unstimulated. Depicted are means ± SEM from 3 independent experiments. D Representative images of BMMΦ stimulated with LP, fixed and stained with γ-tubulin (grey) for centrosomes, α/β-tubulin (green) for cytoplasm and DAPI (blue), and quantification of centrosome number per cell. Depicted are means ± SEM from 3 independent experiments with each >100cells; scale bar, 20µm. E BMMΦ were stimulated with LP for 6 days and supplemented with 10µM nutlin3a for indicated times and MGC formation was quantified by hemacolor staining. Depicted are means ± SEM from 3 independent experiments. F BMMΦ were stimulated with LP and 10µM nutlin3a and quantified for inos transcript levels by qRT-PCR at indicated times as fold change to unstimulated. Depicted are means ± SEM from 4 independent experiments. *p < 0.05, **p < 0.01 (two-tailed unpaired t-test).
a) Survival rate of wild-type mice (WT) and indicated knockout mice after intranasal infection with 750 plaque forming units (PFU) of the PR8 virus.figf1(a) Survival rate of wild-type mice (WT) and indicated knockout mice after intranasal infection with 750 plaque forming units (PFU) of the PR8 virus. (b) Hematoxylin and eosin (H&amp;amp;E)-stained lung sections from mice infected with PR8 virus on day 2. Arrows indicate cellular infiltrates and necrotic debris occluding the airways. Original magnification, ×40. (c) Disease severity ('lung score') and extent of cell death (necrosis score) on day 2 after infection based on analysis of H&amp;amp;E-stained lung sections. (d) Necrosis score on day 7, estimated visually from H&amp;amp;E-stained lung sections, and flow cytometry analysis for annexin V-stained lung cell populations. (e) Immunohistochemistry staining of neutrophils occluding the airways of PR8-infected mice. Original magnification, ×20. (f,g) Flow cytometry analysis of neutrophil, dendritic cell (DC) and macrophage cell numbers on days 2 and 7. (h) Pulmonary viral titers on days 2 and 7 after infection in Ripk2−/− and WT mice. *P 0.05, **P 0.01, ***P 0.001 (log-rank test (a) and unpaired two-sided Student's t-test (c,d,f,g). Data are pooled from three experiments (a,f-h; mean ± s.e.m. in f-h; n = 10-12 mice total) or are representative of three experiments (b-e; mean ± s.e.m.; n = 3-5 mice per experiment).
a) Upon starvation, de-ubiquitination by the Ubp3p/Bre5p ubiquitin protease may trigger the engulfment of ribosomal subunits by autophagic membranes, followed by their degradation in the vacuole.figf5(a) Upon starvation, de-ubiquitination by the Ubp3p/Bre5p ubiquitin protease may trigger the engulfment of ribosomal subunits by autophagic membranes, followed by their degradation in the vacuole. (b) Alternatively, ubiquitination of ribosomal subunits or other factors by an unknown E3 ligase may trigger the engulfment of 60S subunits, and a subsequent de-ubiquitination step promoted by Ubp3p/Bre5p may be required for the maturation and/or fusion of the autophagosome with the vacuole. Ub, ubiquitin.
Electron micrographs showing ultrastructures of cotyledon cells of germinatedV.Figure 4.Electron micrographs showing ultrastructures of cotyledon cells of germinatedV. mungo seeds (A-G), immunogold localization of α-amylase in cotyledon cells (H), and ultrastructure of cells of detached cotyledons (I). Toluidine blue (TB) staining of sections from cotyledons of day 3 V. mungo seedlings. Conversion from TB-stained cells (region I) to TB-stainless cells (region III) was accompanied with that of the PSV to LV. (B) SGs and PSVs in TB-stained cells (A, region I). The PSV was filled with storage proteins. Arrowheads indicate border between SGs and the cytoplasm. (C) SGs and PSVs in cotyledons at region II in A. Electron density of PSVs became low. SGs were surrounded with membranous structure (arrows). LED areas were found between SGs and the cytoplasm. (D) SG and LV in TB-stainless cells (region III in A). The PSV was converted to the LV in the cells. The areas around SG with LED were enlarged. (E) Ultrastructure of TB- stainless cells. SGs wrapped with a LED area contacted with LVs (arrowheads). Vesicles with similar density to LVs were observed. (F) LED membranes around the SGs fused with the LV membranes (arrow). (G) SGs were observed in LVs. The shape of SGs were largely different from those in B-E. (H) An Immunogold image representing degradation of SGs by α-amylase localized in LVs. Gold particles from anti-α-amylase antibody were densely detected in the peripheral region of SGs, which is inserted into the inside of the LV. (I) PSVs were not converted to LVs, and SGs were not taken up into PSVs in the cells of detached cotyledons. Neither low density areas around SGs nor vesicles with similar density to LV was observed in the cells. LV, lytic vacuole; Mt, mitochondrion; PSV, protein storage vacuole; SG, starch granule. Bars: (A) 50 μm; (E and G) 2 μm; (D, F, and I) 1 μm; (B, C, and H) 200 nm.
The VcdRP dual-function RNA regulator inhibits CTX productionA.Figure 1A. Violin plot of V. cholerae ΔhapR cells expressing the indicated sRNAs (x-axis) plotted as relative fold-repression (y-axis) of secreted CTX protein fractions in comparison with a control strain (pCMW) grown under AKI conditions. Indicated in grey is the only known repressor of CTX to date (VqmR) and indicated in pink is the yet uncharacterized sRNA, Vcr082 (VcdRP). B. Gene synteny analysis of the genomic loci encoding vcdRP (formerly known as vcr082) in various Vibrio strains. Homologous genes are depicted using the same colors. C. Alignment of vcdRP sequences, including the promoter regions from various Vibrio species: V. cholerae (Vch), V. furnissi (Vfu), V. vulnificus (Vvu), V. harveyi (Vha), V. parahaemolyticus (Vpa), V. anguillarum (Van) and V. splendidus (Vsp). The -35 box, -10 box, transcriptional start site (TSS; arrow), the start and stop positions of VcdP (blue box), the highly conserved base-pairing region (four consecutive cytosines) and rho-independent terminator (brackets) are indicated. D. Northern blot analysis of V. cholerae wild-type strain examined for the expression of VcdRP monitored over bacterial growth in the indicated media. The solid triangle represents the band corresponding to the full length primary vcdRP transcript, whereas the open triangles correspond to the different processed isoforms. Probing with 5S rRNA confirmed equal loading. E. Sequence conservation of the 29 amino acid small protein, VcdP, in different Vibrio species. F. Western blot analysis of the production of chromosomally-encoded VcdP::SPA, corresponding to the same growth conditions as the Northern blot in (D). The SPA epitope contains the 3×FLAG and the calmodulin binding peptide sequences separated by a TEV protease cleavage site (Zeghouf et al, 2004). Therefore, anti-Flag antibody was used to examine the levels of VcdP. RNAP served as a loading control.
Figure 1(A) Summary of HMCES recovery upon TAP-MS analyses performed with 293T cells stably expressing SFB-PCNA. Cells were treated with CPT: camptothecin (1 μg/ml for 6 h); HU: hydroxyurea (2 mM for 16 h), UV: ultraviolet light irradiation (10 mJ/cm2 harvested after 6 h post-irradiation) or Thymidine (100 mM for 6 h). Recovered peptide-spectrum matches (PSMs) for PCNA and HMCES are indicated for each condition. (B) Schematic representation of HMCES domain organization and deletion mutants used in this study. (C) Immunoprecipitation (IP) of SFB-tagged full-length (FL) HMCES and indicated HMCES deletion mutants was conducted to decipher the region responsible for PCNA interaction. (D) GST-PCNA pulldown assay was used to evaluate the interaction between PCNA and full-length HMCES or indicated HMCES deletion mutants. (E) aniPOND-Western blotting analysis revealed the presence of HMCES on replication fork. Cells were pulsed with EdU for 10 mins, and click-reaction was performed. In thymidine chase samples, cells were incubated with thymidine for another 30 mins prior to click-reaction. (F) Analysis of HMCES level throughout the cell cycle. Cells were synchronized by double thymidine block and released for the indicated time points. CYCLIN E and CYCLIN B1 were used as markers for cell cycle phases.
Decomposing the sources of noise shows time has a small contribution to overall mRNA count variance.Figure 4(A) Noise decomposition of model M4, estimated with 100 simulations of the data set using the posterior mean parameter values combined with the measured cell areas. The variable η2 represents the variance/mean2. (B) Simulations from model M4 to show the percentage of variance attributed to time as a function of the number of cells in each sample. (C) Simulation of the 3-dimensional mean mRNA trajectory of Bmal1, Nr1d1 and Cry1. Each point on the cycle represents a different circadian time. (D) Simulation of the 3-dimensional probability distribution of Bmal1, Nr1d1 and Cry1, averaged over time. The surface represents the area of highest probability density that integrates to 80% of the total probability.
Figure 8A model for the increased level of body fat storage by intestinal microbial alteration in the castrated cattle.
Fig.Figure 3A, UV-Vis spectra of the photosensory domain of A. nidulans FphA (PGP) after co-expression together with bacterial HO (BphO) or A. alternata HoxA, HoxB or HoxA plus HoxB overnight at 20°C in the dark. PGP was purified via the Strep-tag system and concentrated before recording the spectra. Inset, left lane: Molecular mass markers stained with Coomassie Blue. Middle lanes: Zinc-induced red fluorescence (labeled zinc, Zn2+) of equal amounts of PGP expressed in E. coli along with bacterial BphO (O) or A. alternata HoxA plus HoxB (A+B). right lanes: Coomassie Blue staining (C.B.) of the polyacrylamide gel used for the Zn2+ fluorescence. Red/far-red light induced difference spectrum of purified PGP expressed with HoxA and HoxB or with HoxA alone after large-scale batch fermentation and purification. Red/far-red light induced difference spectrum of purified PGP expressed with HoxB alone after large-scale batch fermentation and purification. E, Overlay of normalized (at 705 nm) difference spectra plus a difference spectrum of the photosensory domain in the presence of the bacterial HO BphO
Figure 4A Schematic overview of protocols used to derive naïve-like hESC lines by 2a2iL from human blastocysts. Two protocols were used to generate naïve hESC lines. In protocol 1, isolated ICMs were cultured in hESCs medium that contained basic fibroblast growth factor (bFGF) for 4 days followed by 2a2iL-supplemented medium until day 10. Dome-shaped colonies representative of the naïve state formed after single-cell dissociation. In protocol 2, ICMs were exposed to 2a2iL-supplemented medium at the beginning of seeding. B Morphology of ICM-outgrowths by protocols 1 and 2 during the first 4 days after seeding. nRH11-14 (naïve Royan H11, 12, 13, and 14): naïve hESC lines produced from human embryos using 2a2iL condition. Scale bar: 100 µm. C Comparison of the efficiency of naïve-like hESCs derivation from human blastocysts by two protocols. D Representative example of naïve-like hESCs (nRH13) at passage 33. Dome-shaped colonies were stained for ALP activity and karyotyped. Scale bars: 200 and 100 µm in the inset. E Expression of the common pluripotency markers OCT4, SOX2, and TRA-1-81, and naïve-related pluripotency markers NANOG, KLF17, and TFCP2L1 in nRH13. Nuclei were stained with 4′, 6-diamidino-2-phenylindole (DAPI). Scale bar: 100 µm. F qRT-PCR analysis for pluripotency markers in nRH13 in comparison to a primed hESC line, pRH6. *P<0.05, **P<0.01, ***P<0.001 (t-test). Data presented as mean ± SD (n=3). G Expression of lineage markers after embryoid body (EB) formation and spontaneous differentiation in nRH13 cells. *P<0.05, ***P<0.001 (t-test). Data presented as mean ± SD (n=3).