Journal: JCI Insight

Article Title: Virus-induced RGMa expression drives neurodegeneration in HTLV-1–associated myelopathy

doi: 10.1172/jci.insight.184530

Figure Lengend Snippet: ( A ) The concentration of NF-L (pg/mL) in the supernatant when NB-1 cells were cocultured with HAM-PBMCs ( n = 7) or HD-PBMCs ( n = 7). ( B ) The concentration of NF-L (pg/mL) in the supernatant when NB-1 cells were cocultured with HAM-PBMCs ( n = 9) and with mogamulizumab (antiCCR4) in a dose-dependent manner for 72 hours. ( C ) The comparison of RGMA mRNA gene expression levels using DNA microarray among normal CD4 + T cells (HD CD4 + : n = 4), HAM patient–derived CD4 + T cells (HAM CD4 + : n = 4), ACs ( n = 2), and smoldering/chronic-type-ATL patient–derived ( n = 3) HTLV-1–infected CD4 + T cells (Non-HAM infected CD4 + T cells: n = 5), and acute-type-ATL patient–derived HTLV-1–infected CD4 + T cells (Acute ATL infected cells: n = 3). ( D ) The comparison of the expression levels of the genes associated with the inhibition of neuroregeneration ( OMG , MAG , RTN4 , and WNT5A ) between HD CD4 + ( n = 4) and HAM CD4 + T cells ( n = 4). ( E ) The enrichment levels of H3K27me3 –2916 bp upstream from the TSS of the RGMA gene locus in HD CD4 + ( n = 3), HAM CD4 + ( n = 4), and acute-ATL infected cells ( n = 4). Data are shown as mean ± SD. ** P < 0.01; *** P < 0.001 by unpaired t test ( A and D ) or 1-way ANOVA with Dunnett’s multiple-comparison test ( B , C , and E ). NF-L, neurofilament light chain.

Article Snippet: In the experiment analyzing Tax , HBZ , and RGMA gene expression levels in cultured HAM-PBMCs, mRNA was purified using the Magnetic mRNA isolation kit (New England BioLabs).

Techniques: Concentration Assay, Comparison, Gene Expression, Microarray, Derivative Assay, Infection, Expressing, Inhibition

Journal: JCI Insight

Article Title: Virus-induced RGMa expression drives neurodegeneration in HTLV-1–associated myelopathy

doi: 10.1172/jci.insight.184530

Figure Lengend Snippet: ( A ) The validation of RGMA mRNA gene expression levels using qRT-PCR in HD CD4 + ( n = 6) and HAM CD4 + T cells ( n = 6). ( B ) Expression of RGMa protein in CD3 + CD4 + CCR4 + T cells from HAM-PBMCs. Representative dot plots of CCR4 and normal goat IgG (upper) or RGMa expression (bottom) in CD3 + CD4 + gated cells from HD-PBMCs (left) or HAM-PBMCs (right) cultured for 2 days. ( C ) Graph shows the percentage of RGMa protein–expressing cells in CCR4 – cells or CCR4 + cells in CD3 + CD4 + gated cells from HAM-PBMCs ( n = 8) cultured for 2 days, compared with the isotype control, normal goat IgG. ( D ) Graph shows the percentage of RGMa protein–expressing cells among CD3 + CD4 + CCR4 + gated cells from HD-PBMCs ( n = 5) or HAM-PBMCs ( n = 8) cultured for 2 days. Data are shown as mean ± SD. * P < 0.05; ** P < 0.01 by unpaired t test ( A and D ) or 1-way ANOVA with Dunnett’s multiple-comparison test.

Article Snippet: In the experiment analyzing Tax , HBZ , and RGMA gene expression levels in cultured HAM-PBMCs, mRNA was purified using the Magnetic mRNA isolation kit (New England BioLabs).

Techniques: Biomarker Discovery, Gene Expression, Quantitative RT-PCR, Expressing, Cell Culture, Control, Comparison

Journal: JCI Insight

Article Title: Virus-induced RGMa expression drives neurodegeneration in HTLV-1–associated myelopathy

doi: 10.1172/jci.insight.184530

Figure Lengend Snippet: ( A ) Tax (left), HBZ (middle), and RGMA (right) gene expression levels in cultured HAM-PBMCs ( n = 7) in a time-dependent manner. RPL19 was used as an internal control. ( B ) Tax-dependent RGMA mRNA gene induction in Jurkat cells, which were infected with lentivirus carrying the Tax gene. Top: Tax expression in the Jurkat cells was confirmed by Western blotting. β-Actin was measured as an internal control. Bottom: The induction levels of the RGMA gene were evaluated by qRT-PCR in a time-dependent manner ( n = 3). ( C ) Tax -dependent RGMA mRNA gene induction in JPX9 cells treated with 20 μM CdCl 2 in a time-dependent manner. Tax mRNA (upper) and RGMA mRNA (bottom) were measured by qRT-PCR ( n = 3). GAPDH was measured as an internal control. ( D ) Tax-dependent RGMa protein induction in JPX9 cells treated with 20 μM CdCl 2 for 3 days. Dot plots of Tax and normal goat IgG (upper) or RGMa expression (bottom) in JPX9 cells. JPX9(-), untreated JPX9 cells; 20 μM CdCl 2 JPX9, CdCl 2 -supplemented JPX9 cells. Data are shown as mean ± SD. * P < 0.05; ** P < 0.01; *** P < 0.001 by 1-way ANOVA with Dunnett’s multiple-comparison test ( A ), 2-sided Student’s t test ( B ), or an unpaired t test ( C ). Experiments were performed in triplicate ( B and C ).

Article Snippet: In the experiment analyzing Tax , HBZ , and RGMA gene expression levels in cultured HAM-PBMCs, mRNA was purified using the Magnetic mRNA isolation kit (New England BioLabs).

Techniques: Gene Expression, Cell Culture, Control, Infection, Expressing, Western Blot, Quantitative RT-PCR, Comparison

Journal: Journal of Biological Chemistry

Article Title: Expression Profiles of Arabidopsis thaliana in Mineral Deficiencies Reveal Novel Transporters Involved in Metal Homeostasis

doi: 10.1074/jbc.m309338200

Figure Lengend Snippet: FIG. 6. Functional characterization of AtOPT3. A, hybridization of an 600-bp AtOPT3 cDNA probe, corresponding to the 3-terminal exon, to RNA extracted from Arabidopsis roots of plants grown in control conditions (Ctl), copper (-Cu), manganese (-Mn), zinc (-Zn), and iron (-Fe) deficiency. The ethidium bromide-stained RNA gel is shown for quantification. B, growth of ctr1 expressing AtOPT3 on YPG-Ura plates supplemented with 10 M CuSO4 compared with the mutant transformed with the vector alone. C, growth of smf1 expressing AtOPT3 on manganese-limited medium, with and without 1 mM EGTA compared with the growth of the mutant transformed with the vector alone.

Article Snippet: Genome-wide Analysis Provides Insight into Metal Transport—We have used Affymetrix Arabidopsis DNA chips containing 8,300 genes (which cover about one-third of the ge- FIG. 7.

Techniques: Functional Assay, Hybridization, Control, Staining, Expressing, Mutagenesis, Transformation Assay, Plasmid Preparation

Journal: Cells

Article Title: Cap-Specific m 6 Am Methyltransferase PCIF1/CAPAM Regulates mRNA Stability of RAB23 and CNOT6 through the m 6 A Methyltransferase Activity

doi: 10.3390/cells13201689

Figure Lengend Snippet: Exploration of PCIF1 target genes by genome-wide gene expression analysis. ( A ) RT-qPCR analysis of total RNAs isolated from HeLa cells treated with control siRNA (siNC) or two distinct PCIF1-targeted siRNAs (siPCIF1 #1 and #2). ( B ) Immunoblotting analysis of total protein extracts from HeLa cells treated with control siRNA (siNC) and two distinct PCIF1-targeted siRNAs (siPCIF1 #1 and #2) with anti-PCIF1 and anti-b-actin antibodies. ( C , D ) Venn diagrams showing the overlap between the two indicated siRNA-mediated downregulated ( C ) and upregulated ( D ) genes identified by the gene expression profile analyzed by DNA microarray using a Human Genome U133 Plus 2.0 Array (Affymetrix). The condition for selecting differentially expressed genes are as follows: (1) cut-off condition: expression > 100, (2) fold change: >2, (3) p -value < 0.05.

Article Snippet: An amount of 1.5 μg of anti-m 6 A antibody (Abcam Limited, Cambridge, UK, ab151230) or normal rabbit IgG (Medical & Biological Laboratories Co.) was incubated with 30 μL of Dynabeads Protein G (Thermo Fisher Scientific) at room temperature for 1 h. Total RNA (50 μg) was added to the antibody-Dynabead complexes and incubated at 4 °C for 4 h. After washing three times, the bound RNA was purified from the beads using SepasolTM-RNA I Super G. The first strand of cDNA was synthesized from m 6 A-RNA using ReverTra AceTM qPCR RT Master Mix with gDNA Remover (Toyobo Co., Osaka, Japan) with random hexamer primers according to the manufacturer’s instructions. cDNA was quantified using GeneAce SYBRTM qPCR Mix II (Nippon Gene Co., Tokyo, Japan) and a Mx3000P real-time PCR system (Agilent).

Techniques: Genome Wide, Gene Expression, Quantitative RT-PCR, Isolation, Control, Western Blot, Microarray, Expressing

Journal: Cells

Article Title: Cap-Specific m 6 Am Methyltransferase PCIF1/CAPAM Regulates mRNA Stability of RAB23 and CNOT6 through the m 6 A Methyltransferase Activity

doi: 10.3390/cells13201689

Figure Lengend Snippet: Expression of RAB23 and CNOT6 is regulated by PCIF1 at both the mRNA and protein levels. ( A – C ) RT-qPCR analysis of total RNAs isolated from HeLa cells treated with control siRNA (siNC) and two distinct PCIF1-targeted siRNAs (siPCIF1 #1 and #3), using the specific primer set detecting PCIF1 ( A ), RAB23 ( B ), and CNOT6 ( C ) expression. ( D , E ) Immunoblotting analysis of total protein extracts from HeLa cells treated with control siRNA (siNC) and two distinct PCIF1-targeted siRNAs (siPCIF1 #1 and #3) with the indicated antibodies. Signal intensities obtained from immunoblotting were quantified using ImageJ software version 1.52. The y-axis represents the fold change relative to the levels in HeLa cells treated with control siRNA. Data are expressed as the mean ± standard deviation of three independent experiments. Asterisks represent statistically significant differences between the control siRNA treatment and the indicated PCIF1-targeted siRNA (Student’s t -test, * p < 0.05, ** p < 0.01, *** p < 0.001).

Article Snippet: An amount of 1.5 μg of anti-m 6 A antibody (Abcam Limited, Cambridge, UK, ab151230) or normal rabbit IgG (Medical & Biological Laboratories Co.) was incubated with 30 μL of Dynabeads Protein G (Thermo Fisher Scientific) at room temperature for 1 h. Total RNA (50 μg) was added to the antibody-Dynabead complexes and incubated at 4 °C for 4 h. After washing three times, the bound RNA was purified from the beads using SepasolTM-RNA I Super G. The first strand of cDNA was synthesized from m 6 A-RNA using ReverTra AceTM qPCR RT Master Mix with gDNA Remover (Toyobo Co., Osaka, Japan) with random hexamer primers according to the manufacturer’s instructions. cDNA was quantified using GeneAce SYBRTM qPCR Mix II (Nippon Gene Co., Tokyo, Japan) and a Mx3000P real-time PCR system (Agilent).

Techniques: Expressing, Quantitative RT-PCR, Isolation, Control, Western Blot, Software, Standard Deviation

Journal: Cells

Article Title: Cap-Specific m 6 Am Methyltransferase PCIF1/CAPAM Regulates mRNA Stability of RAB23 and CNOT6 through the m 6 A Methyltransferase Activity

doi: 10.3390/cells13201689

Figure Lengend Snippet: Ectopic expression of siRNA-resistant PCIF1 restores the normal levels of target mRNA expression. ( A , B ) RT-qPCR analysis of total RNAs isolated from HeLa cells transfected with a control empty vector (Vec) or a vector expressing siRNA-resistant PCIF1 (PCIF1siR) under treatment with control siRNA (siNC) or PCIF1-targeted siRNAs (siPCIF1 #3), using the specific primer set detecting RAB23 ( A ) and CNOT6 ( B ) expression. The y -axis represents the fold change relative to the levels in HeLa cells treated with control siRNA. Data are expressed as the mean ± standard deviation of three independent experiments. Asterisks represent statistically significant differences between indicated pairs (Student’s t -test, *** p < 0.001).

Article Snippet: An amount of 1.5 μg of anti-m 6 A antibody (Abcam Limited, Cambridge, UK, ab151230) or normal rabbit IgG (Medical & Biological Laboratories Co.) was incubated with 30 μL of Dynabeads Protein G (Thermo Fisher Scientific) at room temperature for 1 h. Total RNA (50 μg) was added to the antibody-Dynabead complexes and incubated at 4 °C for 4 h. After washing three times, the bound RNA was purified from the beads using SepasolTM-RNA I Super G. The first strand of cDNA was synthesized from m 6 A-RNA using ReverTra AceTM qPCR RT Master Mix with gDNA Remover (Toyobo Co., Osaka, Japan) with random hexamer primers according to the manufacturer’s instructions. cDNA was quantified using GeneAce SYBRTM qPCR Mix II (Nippon Gene Co., Tokyo, Japan) and a Mx3000P real-time PCR system (Agilent).

Techniques: Expressing, Quantitative RT-PCR, Isolation, Transfection, Control, Plasmid Preparation, Standard Deviation

Journal: Cells

Article Title: Cap-Specific m 6 Am Methyltransferase PCIF1/CAPAM Regulates mRNA Stability of RAB23 and CNOT6 through the m 6 A Methyltransferase Activity

doi: 10.3390/cells13201689

Figure Lengend Snippet: PCIF1 does not regulate expression of its target genes at the transcriptional level. ( A , E ) Schematic illustrations of the RAB23 ( A ) and CNOT6 ( E ) genes. The transcription start sites are indicated by arrows, the exons are shown as black boxes, and the polyadenylation signals are indicated by arrowheads. The positions of the PCR primer set for RT-qPCR amplification of the precursor and mature mRNAs are indicated by arrows. ( B – D , F – H ) RT-qPCR analysis of total RNAs isolated from HeLa cells treated with control siRNA (siNC) and two distinct PCIF1-targeted siRNAs (siPCIF1 #1 and #3) using the indicated primer sets. ( I , J ) ChIP analyses of the RAB23 ( I ) and CNOT6 ( J ) gene promoters (position 2 in ) using antibodies against Pol II in HeLa cells treated with control siRNA (siNC) and two distinct PCIF1 targeted siRNAs (siPCIF1 #1 and #3). Normal rabbit IgG was used as the negative control. Data are expressed as the mean ± standard deviation of three independent experiments. Asterisks represent statistically significant differences between the control siRNA treatment and the indicated PCIF1 targeted siRNA (Student’s t -test, n.s. p > 0.05, *** p < 0.001).

Article Snippet: An amount of 1.5 μg of anti-m 6 A antibody (Abcam Limited, Cambridge, UK, ab151230) or normal rabbit IgG (Medical & Biological Laboratories Co.) was incubated with 30 μL of Dynabeads Protein G (Thermo Fisher Scientific) at room temperature for 1 h. Total RNA (50 μg) was added to the antibody-Dynabead complexes and incubated at 4 °C for 4 h. After washing three times, the bound RNA was purified from the beads using SepasolTM-RNA I Super G. The first strand of cDNA was synthesized from m 6 A-RNA using ReverTra AceTM qPCR RT Master Mix with gDNA Remover (Toyobo Co., Osaka, Japan) with random hexamer primers according to the manufacturer’s instructions. cDNA was quantified using GeneAce SYBRTM qPCR Mix II (Nippon Gene Co., Tokyo, Japan) and a Mx3000P real-time PCR system (Agilent).

Techniques: Expressing, Quantitative RT-PCR, Amplification, Isolation, Control, Negative Control, Standard Deviation

Journal: Cells

Article Title: Cap-Specific m 6 Am Methyltransferase PCIF1/CAPAM Regulates mRNA Stability of RAB23 and CNOT6 through the m 6 A Methyltransferase Activity

doi: 10.3390/cells13201689

Figure Lengend Snippet: PCIF1 regulates the stability of target gene mRNAs in opposite ways. After HeLa cells were treated with a negative control siRNA (siNC) and two PCIF1-targeted siRNAs (siPCIF1 #1 and #3) for 72 h, actinomycin D was added to inhibit transcription. Cells were harvested at 0, 2, 4, 8, and 12 h after treatment, and total RNA was isolated. The amount of residual mRNA was analyzed by RT-qPCR at each time point, using the specific primer set detecting PCIF1 ( A ), CRAB23 ( B ), and CNOT6 ( C ) mRNAs. The relative value was calculated using the expression level of β-actin mRNA (ACTB) as a normalizer. The relative values at each time point were calculated relative to time zero. Asterisks represent statistically significant differences between the control siRNA treatment and the indicated PCIF1-targeted siRNA (Student’s t -test, * p < 0.05, *** p < 0.001).

Article Snippet: An amount of 1.5 μg of anti-m 6 A antibody (Abcam Limited, Cambridge, UK, ab151230) or normal rabbit IgG (Medical & Biological Laboratories Co.) was incubated with 30 μL of Dynabeads Protein G (Thermo Fisher Scientific) at room temperature for 1 h. Total RNA (50 μg) was added to the antibody-Dynabead complexes and incubated at 4 °C for 4 h. After washing three times, the bound RNA was purified from the beads using SepasolTM-RNA I Super G. The first strand of cDNA was synthesized from m 6 A-RNA using ReverTra AceTM qPCR RT Master Mix with gDNA Remover (Toyobo Co., Osaka, Japan) with random hexamer primers according to the manufacturer’s instructions. cDNA was quantified using GeneAce SYBRTM qPCR Mix II (Nippon Gene Co., Tokyo, Japan) and a Mx3000P real-time PCR system (Agilent).

Techniques: Negative Control, Isolation, Quantitative RT-PCR, Expressing, Control

Journal: Cells

Article Title: Cap-Specific m 6 Am Methyltransferase PCIF1/CAPAM Regulates mRNA Stability of RAB23 and CNOT6 through the m 6 A Methyltransferase Activity

doi: 10.3390/cells13201689

Figure Lengend Snippet: Ectopic expression of siRNA-resistant wild-type but not methyltransferase-deficient mutant PCIF1 restored normal levels of target mRNA expression. ( A ) Immunoblotting analysis of total protein extracts from HeLa cells transfected with a control empty vector (Vec), a vector expressing siRNA-resistant wild-type PCIF1 (siR_wt), or methyltransferase-deficient mutant PCIF1 (siR_mut) under treatment with control siRNA (siNC) or PCIF1-targeted siRNAs (siPCIF1: siPCIF1 #3 was used for PCIF1 suppression) with the indicated antibodies. ( B , C ) RT-qPCR analysis of total RNAs isolated from HeLa cells treated as in ( A ), using the specific primer set detecting RAB23 ( B ) and CNOT6 ( C ) expression. Data are expressed as the mean ± standard deviation of three independent experiments. Asterisks represent statistically significant differences between the indicated pairs (Student’s t -test, n.s. p > 0.05, * p < 0.05, ** p < 0.01, *** p < 0.001).

Article Snippet: An amount of 1.5 μg of anti-m 6 A antibody (Abcam Limited, Cambridge, UK, ab151230) or normal rabbit IgG (Medical & Biological Laboratories Co.) was incubated with 30 μL of Dynabeads Protein G (Thermo Fisher Scientific) at room temperature for 1 h. Total RNA (50 μg) was added to the antibody-Dynabead complexes and incubated at 4 °C for 4 h. After washing three times, the bound RNA was purified from the beads using SepasolTM-RNA I Super G. The first strand of cDNA was synthesized from m 6 A-RNA using ReverTra AceTM qPCR RT Master Mix with gDNA Remover (Toyobo Co., Osaka, Japan) with random hexamer primers according to the manufacturer’s instructions. cDNA was quantified using GeneAce SYBRTM qPCR Mix II (Nippon Gene Co., Tokyo, Japan) and a Mx3000P real-time PCR system (Agilent).

Techniques: Expressing, Mutagenesis, Western Blot, Transfection, Control, Plasmid Preparation, Quantitative RT-PCR, Isolation, Standard Deviation

Journal: Cells

Article Title: Cap-Specific m 6 Am Methyltransferase PCIF1/CAPAM Regulates mRNA Stability of RAB23 and CNOT6 through the m 6 A Methyltransferase Activity

doi: 10.3390/cells13201689

Figure Lengend Snippet: PCIF1 suppression resulted in a significant decrease in m 6 A levels of both RAB23 and CNOT6 mRNAs. MeRIP-qPCR analysis was performed using HeLa cells treated with control siRNA (siNC) or PCIF1-targeted siRNA (siPCIF1: siPCIF1 #3 was used for PCIF1 suppression). RT-qPCR analysis of RNAs purified from the immunoprecipitates of HeLa cell extracts using the anti-m 6 A antibody, using the specific primer set detecting RAB23 ( A ), CNOT6 ( B ), and ACTB ( C ) mRNAs. The y -axis represents the fold change relative to the RNA levels in the immunoprecipitate by anti-m 6 A antibody from HeLa cells treated with control siRNA (siNC). Normal rabbit IgG (IgG) was used as a negative control. Data are expressed as the mean ± standard deviation of three independent experiments. Asterisks represent statistically significant differences between the indicated pairs (Student’s t -test, n.s. p > 0.05, *** p < 0.001).

Article Snippet: An amount of 1.5 μg of anti-m 6 A antibody (Abcam Limited, Cambridge, UK, ab151230) or normal rabbit IgG (Medical & Biological Laboratories Co.) was incubated with 30 μL of Dynabeads Protein G (Thermo Fisher Scientific) at room temperature for 1 h. Total RNA (50 μg) was added to the antibody-Dynabead complexes and incubated at 4 °C for 4 h. After washing three times, the bound RNA was purified from the beads using SepasolTM-RNA I Super G. The first strand of cDNA was synthesized from m 6 A-RNA using ReverTra AceTM qPCR RT Master Mix with gDNA Remover (Toyobo Co., Osaka, Japan) with random hexamer primers according to the manufacturer’s instructions. cDNA was quantified using GeneAce SYBRTM qPCR Mix II (Nippon Gene Co., Tokyo, Japan) and a Mx3000P real-time PCR system (Agilent).

Techniques: Control, Quantitative RT-PCR, Purification, Negative Control, Standard Deviation

Journal: Cell reports

Article Title: Variation of Human Neural Stem Cells Generating Organizer States In Vitro before Committing to Cortical Excitatory or Inhibitory Neuronal Fates

doi: 10.1016/j.celrep.2020.107599

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: The following primary antibodies were used at the concentration indicated by manufacturer’s protocol: CaM Kinase II alpha (6G9) (NB100–1983), LMX1A (NBP1–81303) Novusbio; SYNAPSIN (106 001), HOMER (160 003) Synaptic System; EGFR (Ab231), FGFR1 phosphoY654 (Ab59194), TBR1 (Ab31940), REELIN (Ab18570), CYCLIN D1 (Ab10540), FGFR2 (Ab10648), BMPR1A (Ab38560) Abcam; HES1 (11988), p-SMAD1/5 (9516), CYCLIN D1 (2926), pERK1/2 (4370), FGFR1 (9740) Cell Signaling Technology; PAX6 (PRB-278P) BioLegend; NESTIN (MAB1259), OTX2 (AF1979), PDGFR alpha (AF1062; AF307), SOX2 (AF2018; MAB2018), SOX21 (AF3538), TuJ1 (MAB1195), EGFR (AF1280), O4 (MAB 1326) R&D Systems; GFAP (Z 0334) DAKO; HES5 (sc-13859), CUX1 (sc-13024), TLE4 (sc-9125), FGFR3 (sc-9007), LHX2 (sc-19344) Santa Cruz Biotechnology; FOXP2 (ABE73), TBR1 (AB2261), REELIN (MAB5366) Millipore; FOXG1 (M227) Takara/Clontech; anti GAD65/67 was kindly gifted by Dr. Christian Geis, Hans Berger Department of Neurology, Jena University Hospital, Germany ( ).

Techniques: Virus, Plasmid Preparation, Recombinant, Transfection, Antibody Labeling, In Vitro, Microarray, Gene Expression, Derivative Assay, Software

Journal: PLoS ONE

Article Title: Expression of MicroRNA-15b and the Glycosyltransferase GCNT3 Correlates with Antitumor Efficacy of Rosemary Diterpenes in Colon and Pancreatic Cancer

doi: 10.1371/journal.pone.0098556

Figure Lengend Snippet: ( A ) Venn diagram representing overlap of the genes significantly modulated (p<0.05 and fold change (FC) >±50%) by RE-2 at 30, 60 and 100 µg/mL after 48 h treatment in SW620 cells according to microarray data. ( B ) Up-regulation of GCNT3 gene expression in SW620 colon cancer cells by five RE's with different composition (RE-1, RE-2, RE-3, RE-4 and RE-5) at the same concentration (90 µg/mL) during 48 h. Bars represent the mean ± SEM of two independent experiments, each performed with biological triplicates and technical duplicates. ANOVA with Bonferroni's post hoc test was applied to test statistically significant differences among RE's. ( C ) Up-regulation of GCNT3 gene expression in SW620 colon cancer cells by 48 h treatment with RE-4 at 60 µg/mL and carnosic acid, carnosol, and the combination of both compounds at the concentrations present in this RE. Bars represent the mean ± SEM of two independent experiments, each performed with biological triplicates and technical duplicates. Student's t test was applied to determine the statistically significant differences between treated and control cells. ( D ) Down-regulation of miR-15b gene expression in SW620 colon cancer cells by 48 h treatment with RE-4 at 60 µg/mL and carnosic acid, carnosol, and the combination of both compounds at the concentrations present in this RE. Student's t test was used to test statistically significant differences between treated and control cells. ( E ) Modulation of miR-15b and miR-939 (as a negative control) in plasma of colon cancer xenograft nude mice after orally intake of RE-5 (1 mg/mL in drinking water), in comparison with the control group of mice. Bars represent mean ± SEM (n = 8). Student's t test was used to assess statistically significant differences between treated and control groups. * p≤0.05, ** p≤0.01, *** p≤0.001. RE: supercritical rosemary extract, Cc+Cl: Carnosic acid and carnosol.

Article Snippet: For gene expression analysis of GCNT3, we used the specific Taqman gene expression assay Hs01921181_s1 (Applied Biosystems). qRT-PCR was performed in the 7900HT Real-Time PCR System (Applied Biosystems), according to the manufacturer's protocol.

Techniques: Microarray, Gene Expression, Concentration Assay, Control, Negative Control, Clinical Proteomics, Comparison

Journal: PLoS ONE

Article Title: Expression of MicroRNA-15b and the Glycosyltransferase GCNT3 Correlates with Antitumor Efficacy of Rosemary Diterpenes in Colon and Pancreatic Cancer

doi: 10.1371/journal.pone.0098556

Figure Lengend Snippet: Modulation of gene and miRNA expression by RE-2 in colon cancer cells.

Article Snippet: For gene expression analysis of GCNT3, we used the specific Taqman gene expression assay Hs01921181_s1 (Applied Biosystems). qRT-PCR was performed in the 7900HT Real-Time PCR System (Applied Biosystems), according to the manufacturer's protocol.

Techniques: Expressing

Journal: Molecular psychiatry

Article Title: Ovarian steroids regulate gene expression related to DNA repair and neurodegenerative diseases in serotonin neurons of macaques

doi: 10.1038/mp.2014.178

Figure Lengend Snippet: Gene names, symbol, AB assay ID, context sequence, and NCBI gene reference information of genes included on the Taqman qRT-PCR card. The microarray signal intensity of a representative probe set is also shown.

Article Snippet: 10. PARP2* , poly(ADP-ribosyl)transferase-like 2 , capable of catalyzing a poly(ADP-ribosyl)ation reaction , XM_001088314.2 , Rh02857777_m1 , 13 , 172 , 39.

Techniques: Sequencing, Microarray, Activity Assay, Transduction, Translocation Assay, Ubiquitin Proteomics, Conjugation Assay, Activation Assay, Membrane

Journal: Nature communications

Article Title: TGF-β3-expressing CD4+CD25(-)LAG3+ regulatory T cells control humoral immune responses.

doi: 10.1038/ncomms7329

Figure Lengend Snippet: Figure 2 | LAG3 þ Treg regulate B-cell functions through Fas. (a–d) Treatment of MRL/lpr mice with adoptive transfer of T-cell subsets. Ten-week-old MRL/lpr mice were injected i.v. with LAG3 þ Treg (n ¼ 9), CD4 þCD25 LAG3 T cells (LAG3 T; n ¼ 9), CD4 þCD25 þ Treg (CD25 þ Treg; n ¼ 9) or CD4 þCD25 CD45RBhigh T cells (naive T; n ¼ 8) from MRL/ þ mice (1 105 cells each). The mice of LAG3 þ Treg x3 group (n ¼ 8) were injected with LAG3 þ Treg (1 105 cells) at 10 weeks of age followed by a twice weekly injection of the same amount of LAG3 þ Treg. The control group received PBS (n ¼ 13). (a) Proteinuria progression. *Po0.05 versus control group (Mann–Whitney U-test). (b) Quantification of serum anti-ds DNA antibodies. *Po0.05 (Bonferroni post-test). (c) Haematoxylin and eosine (H&E) staining (upper panels) and IgG immunofluorescent staining (lower panels) of kidney sections. Scale bars, 50 mm. (d) Glomerular scores. *Po0.05 (Mann–Whitney U-test). (e) LAG3 þ Treg-mediated suppression of in vitro NP-specific antibody responses. B cells and Th cells purified from NP-OVA/alum-pre-immunized B6 mice and OT-II mice, respectively, were incubated with or without LAG3 þ Treg from non-immunized OT-II mice in the presence or absence of anti-FasL blocking antibody, and supernatants were analysed for anti-NP-BSA antibodies using ELISA. See also Supplementary Fig. 1e (n ¼ 6 per group). *Po0.05 (Bonferroni post-test). (f) NP-specific antibody responses of Rag1KO mice injected with B6 B cells and OT-II Th cells with or without LAG3 þ Treg from WT, B6/lpr or B6/gld mice, as outlined in Fig. 1e (n ¼ 6 per group). Anti- FasL blocking antibody (200 mg per mouse) was injected i.v. weekly. *Po0.05 (Bonferroni post-test). (g–i) Flow cytometry plots (g) and quantification of splenic CD4 þCD25 CXCR5 þPD-1 þ TFH (h) and B220 þGL-7 þFas þ GCB (i) from the same mice as in f. Statistical significances in h,i were analysed by Bonferroni post-test (*Po0.05). The experiments in e,f were repeated three times. The means±s.d. are indicated.

Article Snippet: B cells undergoing apoptosis on day 3 and total IgG production in the culture supernatants on day 7 were determined using the Annexin V Apoptosis Detection Kit (BD Pharmingen) and a mouse IgG ELISA Quantitation Set (Bethyl Laboratories), respectively, according to the manufacturer’s protocol.

Techniques: Adoptive Transfer Assay, Injection, Control, MANN-WHITNEY, Staining, In Vitro, Incubation, Blocking Assay, Enzyme-linked Immunosorbent Assay, Flow Cytometry

Journal: Nature communications

Article Title: TGF-β3-expressing CD4+CD25(-)LAG3+ regulatory T cells control humoral immune responses.

doi: 10.1038/ncomms7329

Figure Lengend Snippet: Figure 3 | LAG3 þ Treg suppress B-cell activation through TGF-b3. (a) Microarray comparisons of the gene expression profiles between B6 CD25 þ Treg and B6 LAG3 þ Treg. Normalized expression values from B6 CD4 þCD25 CD45RBhigh naive Tcells are depicted according to the colour scale shown. (b) Tgfb3 mRNA expression in sorted T-cell subsets taken from the spleens of B6 mice (n ¼ 3 per group). (c–e) TGF-b1, 2 and 3 protein levels in the culture supernatants of the indicated T-cell subsets from B6 mice determined using ELISA. Cells were seeded at 1 105 cells per well (n ¼ 4 per group). (f) CFSE- labelled B cells were stimulated with or without anti-IgM mAb in the presence or absence of rTGF-b3 (n ¼ 3 per group). (g) Viability of anti-IgM-stimulated B cells in the presence or absence of rTGF-b3 (1 ng ml 1) was assessed by 7-AAD (n ¼ 3 per group). (h) The effects of TGF-b3 on total IgG production in the culture supernatants of anti-CD40/IL-4-stimulated B cells, determined as in Fig. 1d (n ¼ 3 per group). *Po0.05 (unpaired two-tailed Student’s t-test). (i–k) STAT6 (i), Syk (j) and NF-kB p65 (k) phosphorylation in stimulated B cells with or without rTGF-b3, calculated as the ratio of phosphorylated to total protein levels (n ¼ 3 per group). See also Supplementary Fig. 10. *Po0.05 (unpaired two-tailed Student’s t-test). (l) TGF-b3 protein levels in the culture supernatants of freshly isolated B6 LAG3 þ Treg or, naive B6 CD4 þ Tcells cultured under Th0, Th1, Th2 or Th17 conditions determined using ELISA. Cells were seeded at 3 105 cells per well (n ¼ 3 per group). The means±s.d. are indicated.

Article Snippet: B cells undergoing apoptosis on day 3 and total IgG production in the culture supernatants on day 7 were determined using the Annexin V Apoptosis Detection Kit (BD Pharmingen) and a mouse IgG ELISA Quantitation Set (Bethyl Laboratories), respectively, according to the manufacturer’s protocol.

Techniques: Activation Assay, Microarray, Gene Expression, Expressing, Enzyme-linked Immunosorbent Assay, Two Tailed Test, Phospho-proteomics, Isolation, Cell Culture

Journal: Nature communications

Article Title: TGF-β3-expressing CD4+CD25(-)LAG3+ regulatory T cells control humoral immune responses.

doi: 10.1038/ncomms7329

Figure Lengend Snippet: Figure 5 | PD-1 expression on B cells is important for the suppressive activity of LAG3 þ Treg. (a) Resistance to TGF-b3-mediated B-cell suppression in PD-1-deficient (PD-1KO) mice. CFSE-labelled B cells from B6, PD-1KO, B6/lpr or B6/gld mice were stimulated in vitro for 72 h with anti-IgM and anti-CD40 in the presence or absence of rTGF-b3 (1 ng ml 1). Histograms are gated on B220 þ B cells. (b) Bcl-xL and Bcl-2a1 mRNA levels in anti-IgM-stimulated B cells from WT or PD-1KO mice in the presence or absence of rTGF-b3 (1 ng ml 1; n ¼ 3 per group). *Po0.05 (unpaired two-tailed Student’s t-test). (c) Blockade of LAG3 þ Treg-mediated suppression of in vitro NP-specific antibody responses by anti-PD-L1 blocking mAb. B cells and Th cells purified from NP-OVA/alum-pre-immunized B6 mice and OT-II mice, respectively, were incubated with or without LAG3 þ Treg from non-immunized OT-II mice in the presence or absence of anti-PD-L1 blocking mAb, and supernatants were analysed for anti-NP-BSA antibodies by ELISA (n ¼ 6 per group). *Po0.05 (Bonferroni post-test). (d) NP-specific antibody responses of Rag1KO mice injected with B6 B cells and OT-II Th cells from B6 or PD-1KO mice with or without LAG3 þ Treg from B6 mice. Anti-PD-L1 blocking antibody (200 mg per mouse) was injected i.v. every 3 days. Anti-NP-BSA antibody levels were determined as in Fig. 1e (n ¼ 6 per group). *Po0.05 (Bonferroni post-test). (e) PD-1 expression on B cells. B cells from B6 mice were stimulated in vitro for 72 h with or without anti-IgM and anti-CD40 antibodies in the presence or absence of rTGF-b3 (1 ng ml 1). Histograms are gated on B220 þ B cells. Data are representative of three independent experiments. The means±s.d. are indicated.

Article Snippet: B cells undergoing apoptosis on day 3 and total IgG production in the culture supernatants on day 7 were determined using the Annexin V Apoptosis Detection Kit (BD Pharmingen) and a mouse IgG ELISA Quantitation Set (Bethyl Laboratories), respectively, according to the manufacturer’s protocol.

Techniques: Expressing, Activity Assay, In Vitro, Two Tailed Test, Blocking Assay, Incubation, Enzyme-linked Immunosorbent Assay, Injection

Journal: Nature communications

Article Title: TGF-β3-expressing CD4+CD25(-)LAG3+ regulatory T cells control humoral immune responses.

doi: 10.1038/ncomms7329

Figure Lengend Snippet: Figure 6 | IL-27 induces TGF-b3-producing Egr2 þ Treg from naive T cells. (a) IL-27-mediated induction of Egr2 and LAG-3 on CD4 þ T cells. Freshly isolated naive CD4 þ T cells were stimulated with anti-CD3/CD28 mAb in the presence or absence of IL-27. Cells were stained for Egr2 and LAG-3 expression on day 5. (b) Quantitative RT–PCR analysis of Tgfb3 mRNA expression in naive WT CD4 þ Tcells activated as in a, assessed on day 3. *Po0.05 (unpaired two-tailed Student’s t-test). (c) ELISA for TGF-b3 in culture supernatants of activated naive WT, Egr2 CKO, STAT1 KO, or Stat3fl/flCD4-Cre þ

Article Snippet: B cells undergoing apoptosis on day 3 and total IgG production in the culture supernatants on day 7 were determined using the Annexin V Apoptosis Detection Kit (BD Pharmingen) and a mouse IgG ELISA Quantitation Set (Bethyl Laboratories), respectively, according to the manufacturer’s protocol.

Techniques: Isolation, Staining, Expressing, Quantitative RT-PCR, Two Tailed Test, Enzyme-linked Immunosorbent Assay