EMBO/soda-nel huggingface dataset:
Ribonucleotide reductase inhibitors suppress SAMHD1 ara-CTPase activity enhancing ara-C efficacy (2019)
Figure 4

Figure 4 | dCTPαS-activated SAMHD1 is a poor ara-CTPase

Intracellular dNTP measurements using a primer-extension assay in SAMHD1+/+ THP-1 cells treated for 4 or 24 h with either 50 µM HU (middle panel) or 2.5 nM 3-AP (right panel), ratios of dCTP-to-dATP were calculated. Bars indicate mean values of three independent experiments, error bars indicate s.e.m. Statistical analyses were done using unpaired two-tailed t-tests: **, P < 0.01. For absolute values of dNTP pool measurements, see Appendix Fig S11.

Intracellular ara-CTP (upper panel) and dCTP:dATP ratio (lower panel) in the indicated cell lines following the indicated treatment determined using HPLC-MS/MS. SAMHD1-/- THP-1 cells were treated with 500 nM ara-C and SAMHD1+/+ THP-1 cells were treated with either solvent, 500 nM ara-C or a combination of 500 nM ara-C and an RNRi (HU, 50 µM; dF-dC, 10 nM; 3-AP, 150 nM) for 24 h prior. Values relative to mean ara-CTP amounts in ara-C treated SAMHD1+/+ THP-1 cells shown (indicated by dashed line). Circles, columns and error bars correspond to individual values, means and s.e.m. of at least three experiments performed independently. Analyses were performed using unpaired two-tailed t-tests. *, P < 0.05; **, P < 0.01.

Quantificaiton of dCK phosphorylation at serine-74 (S74) with respect to total dCK in SAMHD1+/+ THP-1 cells treated with either solvent, 500 nM ara-C or a combination of 500 nM ara-C and an RNRi (HU, 50 µM; dF-dC, 10 nM; 3-AP, 150 nM) for 24 h. Circles and squares, columns and error bars correspond to individual measurements, means and s.e.m. of one representative out of two independent experiments performed in triplicates. Statistical analyses were performed using unpaired two-tailed t-test. *, P < 0.05; **, P < 0.01.

Measurement of released inorganic triphosphate (PPPi) from hydrolysis of ara-CTP (200 µM) by recombinant SAMHD1 (0.35 µM) in the presence of GTP (200 µM) and a titration of different non-hydrolysable dNTP analogues (dNTPαS) in the enzyme-coupled malachite green assay. Error bars indicate s.e.m. of two independent experiments performed in triplicate and quadruplet.

Tables and their legends

Table 1 | Hazards ratios (HR) for mRNA levels of SAMHD1 and RRM1, RRM2, and RRM2B (all log-transformed using the natural logarithm) in univariable regression as well as hazard ratios for SAMHD1 in multivariable regression models in ara-C-treated AML patients.

TCGA cohort

EFSa,h,i

completec

EFS

18 monthsd

OSb,h,i

complete

OS

18 months

TARGET cohort

12 monthsd

12 months

a Event-free survival. b Overall survival. c Complete follow-up period. d Follow-up censored after the first 18 or 12 months after diagnosis. e Ribonucleoside-diphosphate reductase (large) subunit M1. f Ribonucleoside-diphosphate reductase (small) subunit M2. g Ribonucleoside-diphosphate reductase (small) subunit M2B. h adjusted for age, sex, and cytogenetic risk group. i Shown are hazard ratios, 95% confidence intervals and p-values calculated with Wald test. Bold text indicates p-values < 0.05.

Expanded View Figure Legends
Temporal Activation of LRH-1 and RAR-γ in Human Pluripotent Stem Cells Induces a Functional Naïve-Like State (2020)
Figure 1

Figure 1. 2a2iL induces conversion of primed hPSCs into a naïve-like state.

A Determination of a time window for 2a2iL treatment needed to induce a naïve-like pluripotency state in primed hPSCs. One day treatment with 2a followed by the addition of 2a2iL for up to 14 days. Scale bars: 200 µm.

B Schematic illustration that outlines induction of the naïve-like state from primed hPSCs. Primed hPSCs were first cultured as single cells in conventional hPSC medium in the presence of ROCK inhibitor (ROCKi) and Y27632 (day 0). The medium was replaced by serum-free medium plus 2a2iL on day 1 without ROCKi. Compact dome-shaped colonies began to appear on day 3. The resultant naïve-like cells were passaged as single cells in the absence of ROCKi on day 5 in 2iL medium. 2a2iL-hPSCs were passaged every 3 to 4 days in 2iL, and kept typical naïve-like morphology for over 50 passages.

C Characteristics of 2a2iL-RH6 cells represented by dome-shaped colonies, ALP activity, and a normal male karyotype (46, XY). 2a2iL-RH6 expressed the common pluripotency markers OCT4, NANOG, SOX2, and TRA-1-81, but not SSEA1. Nuclei were stained with 4′, 6-diamidino-2-phenylindole (DAPI) and Propidium iodide (PI). Scale bars: 200, 100, and 50 µm in the inset.
Temporal Activation of LRH-1 and RAR-γ in Human Pluripotent Stem Cells Induces a Functional Naïve-Like State (2020)
Figure 2

Figure 2. 2a2iL-hPSCs show characteristics of naïve pluripotency.

A Doubling time in pRH6 versus 2a2iL-RH6. **P<0.01 (t-test). Data presented as mean ± SD (n=3).

B Cloning efficiency in the presence (+) and absence (−) of ROCK inhibitor (ROCKi) in pRH6 versus 2a2iL-RH6. ROCKi (−)*P<0.05, ROCKi (+) *P<0.01 (t-test), data presented as mean ± SD (n=3).

C Firefly luciferase expression of constructs that contain distal and proximal enhancers of OCT4 after transfection of pRh6 and 2a2iL-RH6. The ratio of firefly luciferase expression to Renilla luciferase expression (expressed from co-transfected plasmid) measured 48 h after transfection showed proximal enhancer activity in pRH6 and distal enhancer activation in 2a2iL-RH6. OCT4 PE-Luc ***P<0.001, OCT4 DE-Luc *P<0.05 (t-test). Data presented as mean ± SD (n=3).

D qRT-PCR analysis of pluripotency-related genes in pRH6 and 2a2iL-RH6. *P<0.05, **P<0.01, ***P<0.001 (t-test). Data presented as mean ± SD (n=3).

E Immunofluorescence staining for several naïve specific markers in 2a2iL-RH6. Scale bar: 100 µm.

F Western blot analysis of naïve-related pluripotency markers in pRH6 and 2a2iL-RH6. **P<0.01, ***P<0.001 (t-test). Data presented as mean ± SD (n=3).

G Immunofluorescence staining against pRH5 and 2a2iL-RH5 (46, XX) for H3K27me3. Nuclei were stained with 4′, 6-diamidino-2-phenylindole (DAPI). Reactivation of inactive X-chromosome in female 2a2iL-RH5 was confirmed by the lack of H3K27me3 nuclear foci. Scale bar: 50 µm.
Temporal Activation of LRH-1 and RAR-γ in Human Pluripotent Stem Cells Induces a Functional Naïve-Like State (2020)
Figure 3

Figure 3. Functional pluripotency assessment of 2a2iL-hPSCs.

A Phase contrast images of embryoid bodies (EBs) from 2a2iL-RH6 cells that differentiated into neurons. Scale bars: 200 and 100 µm in the inset.

B qRT-PCR analysis of 20 day-EBs from pRH6 and 2a2iL-RH6 for markers of different embryonic germ layer derivatives. *P<0.05, **P<0.01, ***P<0.001 (t-test). Data presented as mean ± SD (n=3).

C Immunofluorescence images of differentiated 2a2iL-RH6 cells that depict expression of markers for neuron-like cells (TUJ-1), cardiac-muscle-like cells (CTNT), and hepatocyte-like cells (SOX17). Nuclei were stained with 4′, 6-diamidino-2-phenylindole (DAPI). Scale bar: 100 µm.

D 2a2iL-RH6 cells form teratomas that consist of three embryonic germ layer-derived tissues. (i) Rosette structures (ectoderm), (ii) cartilage (mesoderm), and (iii) primitive gut (endoderm; scale bar 50 µm).

E From left to right: Phase contrast and GFP representation in 2a2iL-RH6-eGFP; presence of injected 2a2iL-RH6-eGFP in the mouse blastocyst and integration into the inner cell mass (ICM) after 3, 24 and 48 h incubation periods. Scale bars: 200, 100, and 50 µm in the inset.

F Fluorescence images of tissue-sections from human-mouse chimeras in E10.5 showed cells positive for GFP (top) and human nuclear antigen (HNA) (bottom) in different parts of the embryos, including the head (i), trunk (ii), and tail (iii) with the higher magnification in the right panels. Scale bars: 200 and 100 µm in the inset.

G Genomic PCR analysis for a human specific mitochondrial sequence (391 bp) in human-mouse chimeras.

H Immunofluorescence staining against NANOG shows lack of expression in E10.5 human-mouse chimera tissue sections. Scale bars: 200 and 100 µm in the inset.

I Immunofluorescence staining against TUJ1, BRACHYURY (BRA), and SOX17 in E10.5 human-mouse chimera tissue sections represent differentiation-derivatives of 2a2iL-RH6. Scale bars: 200 and 100 µm in the inset.
Temporal Activation of LRH-1 and RAR-γ in Human Pluripotent Stem Cells Induces a Functional Naïve-Like State (2020)
Figure 4

Figure 4. 2a2iL allows derivation of naïve-like hESCs from human blastocysts

A 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).
STING agonism turns human T cells into interferon-producing cells but impedes their functionality (2023)
Figure 1

Figure 1. cGAMP treatment in addition with TCR stimulation leads to antiviral cytokine release in primary human T cells

CD4 and CD8 T cells were stimulated with cGAMP (20 μg/ml) and αCD3/CD28 beads as indicated. Cell lysates after 4 h of stimulation were analyzed by immunoblotting. Blots depict one representative donor out of three.

CD4 T cells were stimulated with cGAMP (20 μg/ml) and αCD3/CD28; and human IFNβ (top) and IP10 levels (bottom) in the supernatant were analyzed by ELISA at the indicated time points. Data panels depict mean ± SD of biological duplicates from three independent donors.

Statistics from (B) across all three donors, indicate significance by a repeated measures two-way ANOVA with Dunnett correction for multiple testing: ****p < 0.0001; **p < 0.01; ns, not significant.

CD4 T cells were pre-treated with cycloheximide (2 μg/ml) for 2 h, then treated with cGAMP (20 μg/ml) and αCD3/CD28 (3/28) for 4 h. Lysates were analyzed by immunoblotting. One representative donor out of two is shown.

CD4 T cells were pre-treated with cycloheximide (2 μg/ml) for 2 h, then treated with cGAMP (20 μg/ml) and αCD3/CD28 (3/28) for 4 h, then analyzed by quantitative PCR for IFNB1 and OASL expression. Data are depicted as mean + SEM of 3 independent donors (for cGAMP and αCD3/CD28 treated cells without CHX only 2 data points were obtained). Statistics were determined by a two-way ANOVA on log-transformed data using Šidák correction for multiple testing: ***p < 0.001; **p < 0.01; *p < 0.05; ns, not significant.
STING agonism turns human T cells into interferon-producing cells but impedes their functionality (2023)
Figure 2

Figure 2. Primary human T cells display canonical STING signaling

Pooled CD4 T cell WT and STING1-/- clones were stimulated in triplicates with cGAMP (20 μg/ml) and αCD3/CD28 for 4 h and analyzed by RNA sequencing. Principal component analysis based on the top 5000 highly variable genes (HVGs).

Heatmap showing interferon stimulated genes (ISGs) among the top 100 HVGs according to (Samarajiwa et al, 2009) clustered hierarchically across all conditions and replicates. Color coding corresponds to normalized and log2 transformed read counts. Genes differentially expressed between cGAMP-treated and cGAMP + αCD3/CD28-treated conditions are annotated.

CRISPR/Cas9 targeted CD4 T cells for indicated genes were treated with cGAMP (20μg/ml) and αCD3/CD28 beads for 48 h. Human IFNβ levels were analyzed by ELISA. Data panels depict mean of biological duplicates from three independent donors.

Statistics from (C) across all three donors, indicate significance by a repeated measures two-way ANOVA with Dunnett correction for multiple testing: ****p < 0.0001; ***p < 0.001; *p < 0.05; ns, not significant.

CD4 T cells were stimulated with the indicated stimuli for 4 h. Lysates were analyzed by immunoblotting. One representative donor out of three is shown.

CRISPR/Cas9 targeted CD4 T cells for the indicated genes were treated with the indicated stimuli for 48 h. Human IFNβ levels were analyzed by ELISA. Data are depicted as mean + SEM of 3 independent donors. Statistics indicate significance by two-way ANOVA with Dunnett correction for multiple testing: ****p < 0.0001; ns, not significant.
STING agonism turns human T cells into interferon-producing cells but impedes their functionality (2023)
Figure 3

Figure 3. Critical role for NF-kB signaling in STING-driven antiviral immunity

Simplified model of signaling downstream of TCR engagement.

CD4 T cells were pre-treated with Dasatinib (100, 10, 1 and 0.1 nM), Tacrolimus (1000, 100, 10 and 1 nM), TPCA-1 (4, 2, 1 and 0.5 μM) and Torin1 (50, 5, 0.5 and 0.05 nM) for 30 min, then treated with cGAMP (20 μg/ml) and αCD3/CD28 for 16 h. Ηuman IFNβ levels in the supernatant were analyzed by ELISA. Data panels depict mean of biological duplicates from three independent donors.

CD4 T cells were treated with cGAMP (20 μg/ml) and indicated additional stimuli for 16 h. Human IFNβ levels were analyzed by ELISA. Data panels depict mean of biological duplicates from three independent donors (for cGAMP and PHA treated cells of Donor C only one data point was obtained).

CD4 T cells were treated with cGAMP and indicated stimuli together with increasing concentrations of TPCA-1 for 4 h. Cell lysates were analyzed by immunoblotting. One representative donor out of two is shown.

CD4 T cells were stimulated with cGAMP (20 μg/ml) and additional stimuli as indicated. Cell lysates after 4 h of stimulation were analyzed by immunoblotting. One representative donor out of two is shown.

CD4 T cells were stimulated with cGAMP (20 μg/ml) and additional stimuli as indicated. Cell lysates after 4 h of stimulation were analyzed by immunoblotting. One representative donor out of two is shown.
STING agonism turns human T cells into interferon-producing cells but impedes their functionality (2023)
Figure 4

Figure 4. STING activation induces cell death in primary human T cells.

Freshly isolated CD4 T cells were treated with cGAMP, αCD3/CD28 beads and ABT737/S63845 (A/S) for 48 h and analyzed by flow cytometry. Treatment induced cell death was assessed by Annexin V and propidium iodide staining. Data are depicted as mean + SEM of 3 independent donors. Statistics indicate significance by two-way ANOVA with Dunnett correction for multiple testing: ***p < 0.001; *p < 0.05; ns, not significant.

Freshly isolated CD4 (left) and CD8 (right) T cells were treated as indicated for 48 h and analyzed by flow cytometry. Treatment induced cell death was assessed by Annexin V and propidium iodide staining. Data are depicted as mean + SEM of 4 (CD4) or 3 (CD8) independent donors. Statistics indicate significance by two-way ANOVA with Šidák correction for multiple testing: **p < 0.01; ns, not significant.

CD4 T cells were treated with indicated concentrations of cGAMP for 18 h, and ABT737/S63845 for 2 h. Lysates and supernatant precipitations were analyzed by immunoblotting. One representative donor out of two is shown.

CRISPR/Cas9 targeted CD4 T cells for indicated genes were treated with the indicated stimuli for 48 h and analyzed by flow cytometry. Each panel shows propidium iodide and Annexin V intensity for one representative donor out of three. Digits indicate the percentage of the parental population (single events without subcellular debris).

Summary from (D), whereas data are depicted as mean + SEM of 3 independent donors. Statistics indicate significance by two-way ANOVA with Dunnett correction for multiple testing: **p < 0.01; ns, not significant.
STING agonism turns human T cells into interferon-producing cells but impedes their functionality (2023)
Figure 5

Figure 5. STING activation impairs proliferation in primary human T cells.

CellTrace Violet (CTV) stained, freshly isolated CD4 and CD8 T cells were activated with αCD3/CD28 beads, stimulated with cGAMP for 96 h and analyzed by flow cytometry. Each panel shows profiles from activated (black), cGAMP-treated (green) and resting (light gray) single and live cells from one representative donor.

Summary from (A) across four (CD4) or six (CD8) independent donors. Division indices for each condition were calculated using proliferation modeling. Plot shows log2 transformed fold changes (log2FC) of division indices of treated samples compared to those of activated samples without any additional treatment. Individual data points ± SEM are shown. Statistics indicate significance by one-sample t-test: ***p < 0.001; **p < 0.01.

CRISPR/Cas9 targeted CD4 T cells for indicated genes were treated with cGAMP for 96 h, analyzed by flow cytometry and subjected to the same analysis as in (A). Two representative donors out of three are shown.

Summary of (C) across 3 independent donors, analyzed as in (B). Data are depicted as mean ± SEM of 3 independent donors. Statistics indicate significance by one-way ANOVA with Dunnett correction for multiple testing: *p < 0.05; ns, not significant.

Freshly isolated CD4 T cells were treated with cGAMP (40 μg/ml) and αCD3/CD28 (3/28) for 48 h and analyzed by Seahorse XF mito stress test. Data are depicted as mean ± SEM of 3 independent donors. Statistics indicate significance by two-way ANOVA with Šidák correction for multiple testing: **p < 0.01; *p < 0.05; ns, not significant.

Cells treated as in (E) were subjected to the Seahorse XF glycolysis stress test. Data are depicted as mean ± SEM of 3 independent donors. Statistics indicate significance by two-way ANOVA with Šidák correction for multiple testing: **p < 0.01; *p < 0.05; ns, not significant.

Expanded View Figure legends
BRD4-PRC2 Represses Transcription of T-helper 2-Specific Negative Regulators during T-cell Differentiation (2023)
Figure 1

Fig. 1. Brd4 represses a key gene transcriptional program during Th2 cell differentiation.

(A) Volcano plot of RNA-seq data showing differential expression of mRNA transcripts in mouse Th2 cells treated with or without JQ1 (250nM). Data are representative of two biological replicates. (B) Global ChIP-seq analysis of Brd4 occupancy at the genomic regions of mouse Th2 cells treated with or without JQ1 (250nM). Data are representative of three biological replicates.

(C) Identification of direct targets of BRD4-activated and -repressed genes in Th2 cells.

(D) Brd4 ChIP-seq intensity associated with BRD4-activated and -repressed genes in Th2 cells, treated with or without JQ1.

(E) Gene ontology (GO) analysis of direct targets of BRD4-activated and -repressed genes in Th2 cells.

(F) ChIP-seq tracks Brd4 on select genes (Foxp3, Nr4a2, Med1, Taf10, Ar, and Fbxw7) in Th2 cells, treated with or without JQ1.

(G) Heatmap of RNA-seq analysis showing up- and down-regulation of select genes in Th2 cells, treated with or without JQ1.

Data information: Mouse naïve CD4+ T cells were cultured in Th2 polarization condition and treated with or without inhibitors on Day 0 and were differentiated for 6 days before analysis, unless otherwise specified.
BRD4-PRC2 Represses Transcription of T-helper 2-Specific Negative Regulators during T-cell Differentiation (2023)
Figure 2

Fig. 2. Foxp3 inhibits Gata3 gene transcription while Fbxw7 promotes Gata3 protein degradation. (A) ATAC-seq tracks of accessible cis-regulatory regions of Foxp3, Fbxw7, Gata3, and Il4-13 in mouse naïve, Th1, Th2, Th17 and Treg cells.

(B) qPCR analysis of Il4, Il5, Il13, Gata3, Foxp3 and Fbxw7 in mouse Th2 cells infected with sh-Ctrl, sh-Foxp3, and sh-Fbxw7 lenti-virus.

(C) Immunoprecipitation of Flag-tagged Fbxw7 with anti-Flag affinity gel, followed by Western blotting of Myc to detect Myc-Gata3 in HEK293T cells transfected with Myc-Gata3 and/or Flag-Fbxw7 plasmids.

(D) Flow cytometric (left) and statistical analysis (right) of IL-4 and Gata3 in mouse Th2 cells infected with sh-ctrl and sh-Fbxw7.

(E) Flow cytometric analysis of IL-4 and Gata3 in mouse naïve CD4+ T cells cultured in Th2 polarization were treated with JQ1 (500nM) at Day 0, and then treated with DMSO or MG132 (20μM) for 6hrs before harvest.

(F) Western blotting of Gata3 in mouse Th2 cells treated with or without JQ1 (500nM), and then treated with cycloheximide (CHX; 25μg/ml) for 0h, 0.5h, 1h and 2h before harvest to stop protein synthesis.

(G) Co-IP to immunoprecipitated Gata3, followed by Western blotting of ubiquitin in mouse naïve CD4+ T cells cultured in Th2 polarization treated with JQ1 (500nM) at Day 0, and then treated with DMSO or MG132 (20μM) for 6hrs before harvest.

Data information: Mouse naïve CD4+ T cells were cultured in Th2 polarization condition and treated with or without inhibitors on Day 0 and were differentiated for 6 days before analysis, unless otherwise specified. All Western blotting data are representative of three independent experiments. All data represent mean±SD and average of three independent experiments. Data are analyzed by Paired t test. *p<0.05; **p<0.01; and ***p< 0.001.
BRD4-PRC2 Represses Transcription of T-helper 2-Specific Negative Regulators during T-cell Differentiation (2023)
Figure 3

Fig. 3. Brd4 recruits PRC2 subunits to repress Th2 negative regulators through H3K27me3.

(A) ChIP-seq tracks of Gata3 and Brd4 co-occupancy on the gene loci of Il4, Il5, Il13, Foxp3 and Fbxw7 in mouse Th2 cells.

(B) ChIP-qPCR analysis of the binding of Brd4 on the gene loci of Il4, Il5, Il13, Foxp3 and Fbxw7 in mouse Th2 cells, retrovirally transduced with vectors expressing shRNA targeting YY1 and Gata3.

(C) ChIP-qPCR analysis of histone modifications on the gene loci of Foxp3 and Fbxw7 in mouse Th2 cells treated with or without JQ1 (250nM).

(D) Immunoprecipitation of Brd4 in mouse Th2 cells, followed by Western blotting of Ezh2, Suz12, EED and G9a.

(E) ChIP-qPCR analysis of Brd4, Ezh2, Suz12 and EED binding on the gene loci of Foxp3 and Fbxw7 in mouse Th2 cells versus Th0 cells cultured for 3 days.

(F) ChIP-qPCR analysis of Brd4, Ezh2, Suz12 and EED binding on the gene loci of Il4, Il5, Foxp3 and Fbxw7 in mouse Th2 cells treat with or without JQ1 (250nM).

Data information: Mouse naïve CD4+ T cells were cultured in Th2 polarization condition and treated with or without inhibitors on Day 0 and were differentiated for 6 days before analysis, unless otherwise specified. All Western blotting data are representative of three independent experiments. All ChIP-qPCR data represent mean±SD and are representative of three independent experiments. Data are analyzed by Paired t test. *p<0.05; **p<0.01; and ***p< 0.001.