EMBO/soda-nel huggingface dataset:
A non-canonical function of Plk4 in centriolar satellite integrity and ciliogenesis through PCM1 phosphorylation (2015)
Figure 3

Figure 3. Plk4-mediated phosphorylation of PCM1 at S372 is critical for the proper localisation of centriolar satellites, its dimer formation and interaction with other satellite components

A, B, C, D. siRNA-treated U2OS cells (PCM1 for A and B; Plk4 for C and D) were transfected with plasmids producing various EGFP-PCM1 constructs (WT*, S372A*, S372D* or S372E*), fixed and stained with the indicated antibodies (arrowheads point to the centrosome) (A and C). Quantification data are shown in B and D. >200 cells were counted and classified into three categories: normal (around and away from the centrosome), aggregated or dispersed. Data represent the mean + SD (n=3). Statistical analysis was performed using two-tailed unpaired student’s t-tests. ***p<0.001, **p<0.01, *p<0.05, n.s. (not significant). Note that the percentage of cells displaying “dispersed” is analysed. Scale bars, 5 μm (A and C).

E. PCM1 siRNA-treated U2OS cells were transfected with two types of plasmids (EGFP- and mCherry-tagged) that produce PCM1-WT* and PCM1-S372A*. Immunoprecipitation and subsequent immunoblotting were performed with the indicated antibodies. Quantification data is shown on the right. Statistical analysis was performed using two-tailed unpaired student’s t-tests. *p<0.05, **p<0.01, n.s. not significant.

F. U2OS cells were treated with PCM1 siRNA and transfected with empty vectors (EV) or EGFP plasmids that produce PCM1-WT* or PCM1-S372A*. Immunoprecipitation was performed with an anti-GFP antibody, followed by immunoblotting with the indicated antibodies.
A non-canonical function of Plk4 in centriolar satellite integrity and ciliogenesis through PCM1 phosphorylation (2015)
Figure 4

Figure 4. Plk4-mediated phosphorylation of PCM1 at S372 is essential for ciliogenesis

A. Immunofluorescence microscopy was performed using the indicated antibodies in hTERT-RPE-1 cells treated with control, PCM1 or Plk4 siRNA. Regions around the basal body (insets) are enlarged on the right hand side. Scale bars, 5 μm (left) and 1 μm (right).

B. Quantification. At least 150 cells were counted (n=3). Statistical analysis was performed using two-tailed unpaired student’s t-tests. ***p<0.001, ****p<0.0001.

C. hTERT-RPE-1 cells treated with PCM1 siRNA were transfected by an empty vector (EV) or plasmids producing PCM1-WT*, -S327A* or -S372E*, and immunofluorescence microscopy performed. Enlarged images of the regions around the basal body are shown in insets. Scale bars, 5 μm and 1 μm (inset).

D. Quantification. At least 200 cells were counted (n=3). Statistical analysis was performed using two-tailed unpaired student’s t-tests. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, n.s. not significant.

E. Model. Plk4 plays a decisive role in not only centriole duplication but also centriolar satellite integrity and primary cilium formation. The latter two roles, at least in part, are executed via PCM1 phosphorylation. Plk4 directly or indirectly phosphorylates PCM1 at S372 (I), which promotes its own dimerisation/oligomerisation (II) and interaction with other components of centriolar satellites (III). These processes ensure PCM1 acts as a structural platform for centriolar satellites. Processes II and III are not necessarily sequential steps. It is shown that centriolar satellites and PCM1 play a critical role in centriole duplication and assembly [25, 49]. Under conditions of serum starvation or developmental cues, centriolar satellites deliver various factors from the cytoplasm to the basal body, thereby helping formation of cilia [26, 30, 37, 50, 51].
53BP1 Ablation Rescues Genomic Instability In Mice Expressing ‘RING-less’ BRCA1 (2016)
Figure 1

Figure 1. Targeting of Brca1 exon 2 leads to production of a stable, N-terminal-truncated protein isoform.

A. Structure of Brca1ex2 allele. * Note that exon 4 was annotated in error in original descriptions of the gene structure and is not drawn. RT-PCR shows a novel product from the Brca1ex2 allele, corresponding to splicing of exon 1 directly to exon 3.

B. Western blot to detect BRCA1 and BARD1 in MEFs expressing WT and mutant Brca1.

C. Immunofluorescent (IF) detection of BRCA1 and RAD51 at IR-induced nuclear foci in cells expressing WT and mutant forms of Brca1. Scale bar: 10 ing

D. Quantification of IF, showing proportion of cells with RAD51 foci that also had >5 BRCA1 foci. N=2.

E. Quantification of IF, showing proportion of RAD51 foci that colocalized with BRCA1 foci. N=2.

F. Immunofluorescent detection of BARD1 at IR-induced nuclear foci in MEFs expressing WT and mutant forms of Brca1. Scale bar: 10 μ S
53BP1 Ablation Rescues Genomic Instability In Mice Expressing ‘RING-less’ BRCA1 (2016)
Figure 2

Figure 2. Conditional deletion of Brca1 exon 2 causes genomic instability despite normal recruitment of RAD51 to DNA double-strand breaks.

A. Western blot showing abundance of BRCA1 and BARD1 protein in Brca1Δ2/Δ2;Trp53bp1-/- mouse B cells.

B. Analysis of chromosome aberrations in mice with targeted deletion of Brca1 exon 2 and Trp53bp1. Left, metaphase spreads were prepared from B cells treated with olaparib. Arrows show examples of chromosome aberrations. Right, quantification of chromosome breaks (CSB), chromatid-type breaks (CTD), radial chromosomes, and other abnormalities in mouse B cells. Error bars indicate SD, N=3

C. RAD51 foci in B cells after 10 Gy IR exposure. Chart shows mean percentage of cells with >5 foci, N=3. Error bars indicate SD, statistical analysis by two-tailed Student t test.

D. Percentage of cells showing >5 RAD51 foci after 4hrs treatment with 10 μM olaparib (OLA) or 10 μM camptothecin (CPT). N=2.

E. Average number of γ-H2AX foci per cell in cells that were not treated (NT) or exposed to 2 Gy ionizing radiation. N=2.

F. Sister chromatid exchanges in cells that were not treated (NT) or exposed to 2 μM olaparib (OLA) for 16 hrs. Black arrows in images show SCEs in olaparib-treated cells. Scale bar: 5 μm. N=2.
53BP1 Ablation Rescues Genomic Instability In Mice Expressing ‘RING-less’ BRCA1 (2016)
Figure 4

Figure 4. Lifespan and tumor predisposition of mice expressing RING-less BRCA1.

A. Kaplan-Meier survival curve for Brca1ex2/+;Trp53bp1-/- and Brca1ex2/ex2;Trp53bp1-/- mice. N=20 animals in each group. Statistical analysis by Mantel-Cox log-rank test.

B. Kaplan-Meier survival curve for Brca1ex2/+;Trp53bp1-/-;Trp53+/- and Brca1ex2/ex2;Trp53bp1-/-;Trp53+/- mice. N=20 animals in each group. Statistical analysis by Mantel-Cox log-rank test.

C. Percentage of Brca1ex2/+;Trp53bp1-/- and Brca1ex2/ex2;Trp53bp1-/- mice showing signs of abnormal tissue morphology at death. N=46 animals inspected for Brca1ex2/+;Trp53bp+/+, N=54 animals inspected for Brca1ex2/ex2/Trp53bp1-/-.

D. Tissues affected by abnormal growth from N=46 Brca1ex2/+/Trp53bp1-/- and N=54 Brca1ex2/ex2/Trp53bp1-/- mice.
53BP1 Ablation Rescues Genomic Instability In Mice Expressing ‘RING-less’ BRCA1 (2016)
Figure 5

Figure 5. Arrest in spermatogenesis in mice expressing RING-less BRCA1.

A. H&E-stained sections of seminiferous tubules from testes of WT and Brca1ex2/ex2/Trp53bp1-/- male mice. Scale bar: 100 μm.

B. Immunofluorescence on meiotic chromosomes from WT and Brca1ex2/ex2;Trp53bp1-/- spermatocytes. Spreads were stained for SYCP3 to indicate chromosome axes and extent of synapsis and were stained for SYCP3 to indicate chromosome axex body. Scale bar: 5 apsi

C. Quantification of the number of γ-H2AX foci observed at the pachytene stage in the indicated genotypes. P value is Mann-Whitney, two-tailed.

D. G2M checkpoint analysis in mouse B cells after IR treatment. Left, mitotic cells were identified by flow cytometry as having 4c DNA content (based on propidium iodide staining) and pSer10-H3+. Right, quantification of flow cytometry data, showing mitotic cells after IR as percentage of that seen in untreated cells. N≥2.
Npas4 regulates medium spiny neuron physiology and gates cocaine-induced hyperlocomotion (2021)
Figure 1

Figure 1: Npas4 is sparsely expressed in the nucleus accumbens of saline injected control animals and is upregulated by cocaine injection.

a, Left: Time course of habituation and treatments with cocaine (15 mg/kg) or saline solutions. Right: striatal sub-divisions where Npas4 expression was analysed by immunohistochemistry (NAc: nucleus accumbens; dStr: dorsal striatum).

b, Representative confocal images of NeuN (green) and Npas4 (red) immunostaining performed 1h after the last injection of cocaine or saline (scale bar: 50 µm).

c, Quantifications of Npas4-positive cells (fold change normalized to the 30min saline control group) in the NAc and dStr. Mean + SEM; n = 4 mice per group. 10 images (5 per hemisphere) per mouse for the NAc and 10 images (5 per hemisphere) for the dStr. p** < 0.01, unpaired Student's t-test.
Npas4 regulates medium spiny neuron physiology and gates cocaine-induced hyperlocomotion (2021)
Figure 2

Figure 2. Differential induction of Npas4 in mouse cultured medium spiny neurons and human iPSC-derived organoids through distinct stimuli.

a, DARPP-32 (D32) immunostaining shows that striatal neuron cultures consist mainly of D32-positive medium spiny neurons (MSNs). 40x magnification, scale bar = 50 µm. MAP2 marks neurons, Hoechst (Hoe) stains DNA. n = 3 cultures from independent mouse litters.

b, 100x magnification images show intracellular distribution of D32 in D32-positive neurons. Scale bar = 10 µm.

c, MSN cultures at DIV11 were treated for 2h with either 10 µM of the D1-agonist SKF-38393 (SKF) alone or with SKF and 10 µM of the selective D1-antagonist SCH-23390 (SCH). qPCR was performed to determine the relative mRNA levels of the immediate early genes cFos and Arc. Mean + SEM; one-way ANOVA with Holm-Sidak post-hoc correction; p** < 0.01, p**** < 0.0001; n = 3 independent cultures as biological replicates.

d, MSNs were depolarized with potassium chloride (HiK, 55 mM) or treated with the D1-agonist SKF-38393 (10 µM) for 1h, 2h or 4h. Subsequently, qPCR was performed to assess immediate early gene mRNA levels. Mean + SEM; two-way ANOVA with Bonferroni post-hoc test; indicated p-values for induction over control for each treatment; p* < 0.05, p** < 0.01, p***<0.001; n = 4 independent cultures as biological replicates.

e, To assess differences in mRNA load induced by HiK vs SKF, the values from the experiments in panel d) were summed up over time for each treatment and the sums for each treatment were divided by each other to yield the ratio IHiK/ISKF. Values for this ratio were compared through one-way ANOVA with Tukey's post-hoc test; mean + SEM; p*<0.05, p*** < 0.001; n = 4 independent cultures as biological replicates.

f, immunohistochemistry analysis shows that cells in human iPSC-derived forebrain organoids express the forebrain neuron marker FOXG1 (green) and the neuron differentiation marker MAP2 (red). Hoe = Hoechst. Scale bar = 30µm.

g, Differential induction of Npas4 through HiK and not forskolin in hiPSC-derived forebrain organoids. Organoids were treated with HiK (55 mM) or the PKA activator forskolin plus TTX (Fsk, 10 µM; TTX 1 µM) for 1h before qPCR analysis was performed to assess mRNA levels of Npas4 and Arc. Mean + SEM; n = 7 organoids in control condition, n = 5 organoids for HiK, n = 5 organoids for Fsk/TTX. One-way ANOVA with Tukey's post-hoc test, ns = not significant, p* < 0.05, p***<0.001.
Npas4 regulates medium spiny neuron physiology and gates cocaine-induced hyperlocomotion (2021)
Figure 4

Figure 4. Npas4 overexpression affects a large mRNA set including transcripts encoding NMDA receptors and subunits for Ca2+ channels.

a-c, hippocampal neurons were infected with rAAVs expressing GFP (control) or Npas4 (Npas4 overexpression) on DIV4. On DIV11, RNA was harvested and subjected to microarray transcript analysis. a, overexpression of Npas4 leads to ≥ 1.5-fold upregulation of the levels of 1091 sequences and to a ≥ 1.5-fold downregulation of the levels of 1133 sequences, indicating that Npas4 regulates a widespread transcriptional response. b, gene ontology analysis (GO) for biological processes via the Panther tool for up- and downregulated genes from panel a). Shown is a selection of relevant processes. Upregulated genes are enriched for cell-signaling processes (i.e., TGF-beta signaling, hormone responses), behaviour (i.e., locomotion) and developmental processes (i.e., anatomical structure development). Downregulated genes are enriched for synapse regulation (i.e., regulation of synapse assembly), dendritic organization (i.e. dendrite morphogenesis) and cognitive processes (i.e., learning, cognition, locomotion). c, selection of genes from the Npas4 regulon with relevance to synaptic transmission and dendrite morphology, both of which are processes implicated in habitual learning.

d, Npas4 overexpression leads to basal upregulation of genes encoding for proteins targeted to glutamatergic synapse function and Ca2+ channels in MSNs. MSNs were infected with rAAVs expressing mCherry-NLS control or Npas4 on DIV4 or DIV5 and expression of the indicated genes was analysed by qPCR on DIV11 or DIV12. Shown are mRNA levels normalized to mCherry-control. The dashed line represents normalized control levels. pCAG = CAG promoter, myc = myc-tag, flag = flag-tag. Mean + SEM; unpaired, two-tailed t-test for each gene, p*<0.05, p**<0.01, p***<0.001. n = 5 independent cultures as biological replicates.

e, Npas4 controls activity-dependent upregulation of genes for glutamatergic synapse formation and Ca2+ channels. MSNs were infected with rAAVs expressing shRNA-scrambled (control) or shNpas4 (Npas4 knockdown) on DIV5. On DIV 12, cultures were stimulated with HiK (55 mM) for 1h or 6h and mRNA levels were analysed by qPCR. mRNA level = fold change in mRNA compared to shscrambled unstimulated control. Mean + SEM; two-way ANOVA with Bonferroni post-hoc test, p* < 0.05, p***<0.001. n = 5 independent cultures as biological replicates.
Npas4 regulates medium spiny neuron physiology and gates cocaine-induced hyperlocomotion (2021)
Figure 5

Figure 5: Npas4 regulates dendritic spine density in D1R-MSNs under basal conditions and after repeated cocaine exposure.

a, Time course of AAV injections, habituation and treatments to assess the impact of Npas4 knock-down on MSN neuronal morphology under basal conditions and after repeated cocaine injections.

b, Representative images of AAV-shscr-GFP- and AAV-shNpas4-GFP-infected cells together with the sparse labelling of D1R-MSNs achieved through a co-injection of AAV-Flex-tdTomato and AAV-PPTA-Cre (to label D1 MSNs) or AAV-ENK-Cre (to label D2 MSNs) in a 1/10,000 ratio. Scale bar = 100 µm.

c, Representative confocal images of staining for Npas4 expression (red) in the NAc of mice infected with AAV shscr-GFP or AAV-shNpas4-GFP 1h after cocaine treatment. Scale bar = 20 µm. Note the presence of Npas4-positive neurons infected with the AAV shscr-GFP (arrowheads) and not in cells infected with the AAV-shNpas4.

d, Quantifications of GFP-positive and Npas4-positive cells 1h post cocaine treatment. Data were normalized so that the number of GFP- and Npas4-positive cells was set to 100%. 20 fields of view were analysed from n = 3 mice in the case of AAV-shscr-GFP and 30 fields of view from n = 3 mice for AAV-shNpas4-GFP. Mean + SEM; unpaired Student's t-test; p* < 0.05, n = 3 mice per group.

e, Confocal images of tdTomato-labelled D1-MSNs expressing shscr-GFP or shNpas4-GFP (left panels; scale bar: 20 µm). 3D volume-renderings of MSN dendrites from the NAc shell (right panels; scale bar: 2 µm).

f, Quantification of spine density in D1-MSNs, defined as the number of spines normalized to 10µm of dendrite for saline control and after cocaine injection. Mean + SEM; two-way ANOVA, Bonferroni post-hoc test, p* < 0.05; p** < 0.01; p*** < 0.001. n=40 neurons from 4 mice in each experimental group.

g, Confocal images of tdTomato-labelled D2-MSNs expressing shscr-GFP or shNpas4-GFP (left panels; scale bar: 20 µm). 3D volume-renderings of MSN dendrites from the NAc shell (right panels; scale bar: 2 µm).

h, Quantification of spine density in D2-MSNs, defined as the number of spines normalized to 10µm of dendrite for saline control and after cocaine injection. Mean + SEM; two-way ANOVA, Bonferroni post-hoc test, all non-significant. n=40 neurons from 4 mice in each experimental group.
Npas4 regulates medium spiny neuron physiology and gates cocaine-induced hyperlocomotion (2021)
Figure 6

Figure 6. Npas4 regulates MSN membrane excitability and corticostriatal transmission.

a-d, Scatter plot of passive and active membrane properties of MSNs in ex vivo slice preparations from shRNA-AAV-infected mice, plotted for MSNs as D1-positive (D1+), D1-negative (D1-) and those two populations pooled (all). a, rheobase; mean ± SEM. b, firing rate estimated at twice the value of the rheobase; mean ± SEM. c, input/output gain values recorded in shscr- or shNpas4-injected mice, mean ± SEM. The increased rheobase and lower input-output gain indicate a lower excitability for MSNs in the Npas4-knockdown group. d, facilitation at corticostriatal synapses examined in MSNs with paired-pulse stimulation at 20 and 4 Hz. Representative current traces and scatter plots of paired-pulse experiments illustrate a potent facilitation in MSNs in mice injected with scr-shRNA and a significant decrease of the facilitation in mice injected with shNpas4, with a stronger effect in D1-negative MSNs. Mean ± SEM.

Data information: For n-numbers, please see Table EV1. Mann-Whitney-Test, p*<0.05, p**<0.01.
Npas4 regulates medium spiny neuron physiology and gates cocaine-induced hyperlocomotion (2021)
Figure 7

Figure 7. Npas4 gates cocaine-induced hyperlocomotion.

a, Time course of AAV injections, habituation and treatments to assess the impact of Npas4 knock-down on cocaine-induced hyperlocomotion.

b, cocaine-induced hyperlocomotion is attenuated by Npas4-knockdown while locomotor behaviour after saline injection is unaffected on all days. Mean + SEM; two-way ANOVA followed by Bonferroni post hoc test. p* < 0.05, p** < 0.01, ns = not significant. n = 12 - 16 animals per condition.
TRAF3IP3 mediates the recruitment of TRAF3 to MAVS for antiviral innate immunity (2019)
Figure 1

Figure 1. TRAF3IP3 associates with active MAVS-Region III.

A Diagram for three constructs expressing MAVS-Region III C-terminally tagged with GCN4-derived peptides, whose sequences are presented in bold.

B Recombinant MAVS-Region III-dimer (lanes 1-4), trimer (lanes 5-8) or tetramer (lanes 9-12) were subjected to IRF3 dimerization assay respectively in vitro. Increasing doses of recombinant MAVS-Region III were used and shown in Fig EV1C. Immunoblotting analysis was performed following Native-PAGE for the reaction mixture.

C pcDNA3-based constructs expressing MAVS-Region III-dimer, trimer and tetramer were transfected into HEK293T cells respectively. ISG54 induction were measured thirty-six hours after transfection by quantitative PCR (qPCR). Expression levels of MAVS-Region III were shown in Fig EV1D.

D Recombinant MAVS-Region III-dimer or tetramer were immunoprecipitated with anti-FLAG M2 beads with or without a preincubation with S100 from HEK293T cells following an experimental procedure outlined in Fig EV1G. The IP products were resolved in SDS-PAGE and a specific protein band pulled-down with MAVS-Region III-tetramer was indicated by an arrow. TRAF3IP3 was identified in the specific band by mass spectrometry.

E Protein samples as described in (D) were subjected to immunoblotting analysis. The TRAF3IP3 band was indicated by an arrow and the asterisk indicated nonspecific bands

F pcDNA3-FLAG-TRAF3IP3 (full-length and truncated forms) and pcDNA3-HA-MAVS were transfected into HEK293T cells, which were harvested thirty-six hours post transfection. Co-IP experiments were performed with anti-HA antibody, followed by immunoblotting. A diagram depicting various TRAF3IP3 deletions used in the co-IP experiments was shown in Fig EV1H.

G Human cells, including THP1, HeLa, HEK293, HEK293T as well as Traf3ip3-/- HEK293T, were lysed and subjected to immunoblotting to examine TRAF3IP3 expression.

Data information: These data are presented as the mean values based on three independent experiments, and error bars indicate standard deviation. P values were determined by two-tailed Student's t-test. ***P<0.001.
TRAF3IP3 mediates the recruitment of TRAF3 to MAVS for antiviral innate immunity (2019)
Figure 2

Figure 2. TRAF3IP3 regulates interferon production in response to RNA virus infection.

A Three pairs of small interfering RNA against TRAF3IP3 were transfected into HEK293T cells respectively and immunoblotting were then employed to examine the knockdown efficiency. A pair of scrambled RNA oligoes was transfected and signified as con.

B Three pairs of si-TRAF3IP3 together with IFNβ-luciferase reporter were transfected into HEK 293T cells. Twenty-four hours after transfection, the cells were infected with (SeV) or without (mock) Sendai virus, which were harvested six hours post infection to measure the luciferase activity.

C Si-TRAF3IP3 (oligo #1) were transfected into HEK 293 cells, which were then treated with mock, VSV, SeV, poly(I:C) or HSV-1. After various treatments, IFNB inductions were measured by qPCR.

D Immunoblotting to detect TRAF3IP3 in wild type and Traf3ip3-/- HEK293T cells.

E Wild type and Traf3ip3-/- HEK293T cells were treated with or without VSV, SeV or poly (I:C) as indicated for twelve hours. IFNB induction was measured by qPCR.

F Wild type and Traf3ip3-/- HEK293T cells were infected with GFP-VSV. Fluorescent images were taken to examine GFP-VSV proliferation at eight hours post infection (left). Scale bar represents 10 micrometers. GFP-VSV titers were quantitated by plaque assay (right).

G Various expression constructs were transfected into WT and Traf3ip3-/- HEK293T cells. Thirty-six hours after transfection, IFNB induction was measured by qPCR. Protein expression levels were shown in Fig EV2A.

H Mavs-/- HEK293T cells were transfected with empty vector, pcDNA-FLAG-MAVS, pcDNA-FLAG-MAVS-(Region III only), or pcDNA-FLAG-MAVS-(ΔRegion III), together with pcDNA3-FLAG-TRAF3IP3 or not as indicated. Twenty-four hours after transfection, cells were infected with SeV for twelve hours. IFNB induction was measured by qPCR. Protein expression levels were shown in Fig EV2B.

Data information: All data are presented as the mean values based on three independent experiments, and error bars indicate s.d. P values were determined by unpaired two-tailed Student's t-test. *P<0.05 and **P<0.01. NS indicates no statistically significant difference.
TRAF3IP3 mediates the recruitment of TRAF3 to MAVS for antiviral innate immunity (2019)
Figure 3

Figure 3. TRAF3IP3 is specifically required for TBK1-IRF3 activation.

A The experiment was performed as described in Fig 2C. Instead of IFNB, ISG54 and IL6 inductions were measured by qPCR.

B HEK293 cells were transfected with si-TRAF3IP3 oligos and then treated with various stimuli. Following subcellular fractionation, S5 fractions were obtained and subjected to native PAGE for detecting IRF3 dimer by immunoblotting. The whole cell lysates were also subjected to SDS-PAGE for anti- IRF3 and tubulin immunoblotting.

C The experiment was performed as described in Fig 2E. ISG54 and IL6 induction was measured by qPCR.

D Wild type and Traf3ip3-/- HEK293T cells were infected with or without VSV. At indicated time points, Cells were collected and analyzed by immunoblotting. Following subcellular fractionation, S5 fractions were subjected to native PAGE to examine IRF3 dimer, and P5 fractions contained mitochondria were subjected to SDD-AGE to examine MAVS aggregation. The mitochondrial outer membrane protein Prohibitin was immunoblotted as an internal control.

Data information: …All data are presented as the mean values based on three independent experiments, and error bars indicate s.d. P values were determined by unpaired two-tailed Student's t-test. **P<0.01. NS indicates no statistically significant difference.