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    Thermo Fisher superscript iii firststrand synthesis kit
    p53 modulates transcription of MnSOD as well as other target genes HepG2 cells were transfected with control siRNA or p53 siRNA along with MnSOD promoter (−555 to+24)-driven luciferase reporter vectors. After 24 h of co-transfection, cells were split into separate dishes and grown for another 24 h. A , cells were collected for luciferase activity as a measure of MnSOD gene transcription. B .” C , HepG2 cells were transfected with p53 expression vector (pcDNA3.1/p53, 4µg) or empty-vector (pcDNA3.1, equivalent amount), and total RNA was purified. The mRNA level of Bax gene under p53 overexpression conditions was determined by <t>RT-PCR.</t> A transcriptional off-target, the β-actin mRNA, was examined as an internal control. Statistical analyses were performed in <t>three</t> independent experiments **, p
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    p53 modulates transcription of MnSOD as well as other target genes HepG2 cells were transfected with control siRNA or p53 siRNA along with MnSOD promoter (−555 to+24)-driven luciferase reporter vectors. After 24 h of co-transfection, cells were split into separate dishes and grown for another 24 h. A , cells were collected for luciferase activity as a measure of MnSOD gene transcription. B .” C , HepG2 cells were transfected with p53 expression vector (pcDNA3.1/p53, 4µg) or empty-vector (pcDNA3.1, equivalent amount), and total RNA was purified. The mRNA level of Bax gene under p53 overexpression conditions was determined by RT-PCR. A transcriptional off-target, the β-actin mRNA, was examined as an internal control. Statistical analyses were performed in three independent experiments **, p

    Journal: The Journal of biological chemistry

    Article Title: Specificity Protein 1-dependent p53-mediated Suppression of Human Manganese Superoxide Dismutase Gene Expression *

    doi: 10.1074/jbc.M601083200

    Figure Lengend Snippet: p53 modulates transcription of MnSOD as well as other target genes HepG2 cells were transfected with control siRNA or p53 siRNA along with MnSOD promoter (−555 to+24)-driven luciferase reporter vectors. After 24 h of co-transfection, cells were split into separate dishes and grown for another 24 h. A , cells were collected for luciferase activity as a measure of MnSOD gene transcription. B .” C , HepG2 cells were transfected with p53 expression vector (pcDNA3.1/p53, 4µg) or empty-vector (pcDNA3.1, equivalent amount), and total RNA was purified. The mRNA level of Bax gene under p53 overexpression conditions was determined by RT-PCR. A transcriptional off-target, the β-actin mRNA, was examined as an internal control. Statistical analyses were performed in three independent experiments **, p

    Article Snippet: For reverse transcription cDNA was generated using 2 µg of total RNA, oligo(dT) primer, and SuperScript™III first strand reverse transcriptase according to the manufacturer’s instructions (SuperScript™III first strand synthesis for RT-PCR, Invitrogen) in a total volume of 20µl.

    Techniques: Transfection, Luciferase, Cotransfection, Activity Assay, Expressing, Plasmid Preparation, Purification, Over Expression, Reverse Transcription Polymerase Chain Reaction

    Induction of the GSR system under starvation. B . abortus 2308 wt (black bars) and the isogenic lovhk :: km (dark grey bars) and ∆lovR (light grey bars) mutant strains were grown in TSB medium up to logarithmic phase. First, an aliquot was drawn (time 0 h), then the rest of the culture was washed and resuspended in modified MM1 minimal medium, and aliquots were drawn at 0.5 h, 1 h and 2 h. Expression of phyR gene was analyzed by qRT-PCR. The if-1 housekeeping gene was used as a reference. Data represent the average of three independent experiments and are reported as fold induction relative to wt at time 0 h in TSB ± standard error. p -values between each strain and wt (at each time point) were determined by one-way ANOVA and post-hoc Tukey’s multiple comparisons test (* = p

    Journal: PLoS ONE

    Article Title: LOV Histidine Kinase Modulates the General Stress Response System and Affects the virB Operon Expression in Brucella abortus

    doi: 10.1371/journal.pone.0124058

    Figure Lengend Snippet: Induction of the GSR system under starvation. B . abortus 2308 wt (black bars) and the isogenic lovhk :: km (dark grey bars) and ∆lovR (light grey bars) mutant strains were grown in TSB medium up to logarithmic phase. First, an aliquot was drawn (time 0 h), then the rest of the culture was washed and resuspended in modified MM1 minimal medium, and aliquots were drawn at 0.5 h, 1 h and 2 h. Expression of phyR gene was analyzed by qRT-PCR. The if-1 housekeeping gene was used as a reference. Data represent the average of three independent experiments and are reported as fold induction relative to wt at time 0 h in TSB ± standard error. p -values between each strain and wt (at each time point) were determined by one-way ANOVA and post-hoc Tukey’s multiple comparisons test (* = p

    Article Snippet: Real Time quantitative RT-PCR assays Reverse transcription was performed with SuperScript III First Strand Synthesis System (Invitrogen) following the manufacturer’s instructions using random decamer primers (Invitrogen) and RNasin ribonuclease inhibitor (Promega).

    Techniques: Mutagenesis, Modification, Expressing, Quantitative RT-PCR

    LOVHK modulates the GSR system in B . abortus . B . abortus 2308 wt and in the isogenic lovhk :: km , ∆lovR , ∆phyR and lovhk :: km /pMR_ lovhk strains were cultured in TSB up to logarithmic phase, and expression of lovhk , lovR , dps and rpoH1 genes was analyzed by qRT-PCR. The if-1 housekeeping gene was used as a reference. Data represent the average of three independent experiments, and are reported as fold induction relative to wt ± standard error. p -values between each strain and wt were determined by one-way ANOVA and post-hoc Tukey’s multiple comparisons test (** = p

    Journal: PLoS ONE

    Article Title: LOV Histidine Kinase Modulates the General Stress Response System and Affects the virB Operon Expression in Brucella abortus

    doi: 10.1371/journal.pone.0124058

    Figure Lengend Snippet: LOVHK modulates the GSR system in B . abortus . B . abortus 2308 wt and in the isogenic lovhk :: km , ∆lovR , ∆phyR and lovhk :: km /pMR_ lovhk strains were cultured in TSB up to logarithmic phase, and expression of lovhk , lovR , dps and rpoH1 genes was analyzed by qRT-PCR. The if-1 housekeeping gene was used as a reference. Data represent the average of three independent experiments, and are reported as fold induction relative to wt ± standard error. p -values between each strain and wt were determined by one-way ANOVA and post-hoc Tukey’s multiple comparisons test (** = p

    Article Snippet: Real Time quantitative RT-PCR assays Reverse transcription was performed with SuperScript III First Strand Synthesis System (Invitrogen) following the manufacturer’s instructions using random decamer primers (Invitrogen) and RNasin ribonuclease inhibitor (Promega).

    Techniques: Cell Culture, Expressing, Quantitative RT-PCR

    PhyR expression is decreased in the lovhk mutant. A. B . abortus 2308 wt and the isogenic lovhk :: km , ∆lovR and lovhk :: km /pMR_ lovhk strains were cultured in TSB up to logarithmic phase, and expression of the phyR gene was analyzed by qRT-PCR. The if-1 housekeeping gene was used as a reference. Data represent the average of three independent experiments, and are reported as fold induction relative to wt ± standard error. p -values between each strain and wt were determined by one-way ANOVA and post-hoc Tukey’s multiple comparisons test (* = p

    Journal: PLoS ONE

    Article Title: LOV Histidine Kinase Modulates the General Stress Response System and Affects the virB Operon Expression in Brucella abortus

    doi: 10.1371/journal.pone.0124058

    Figure Lengend Snippet: PhyR expression is decreased in the lovhk mutant. A. B . abortus 2308 wt and the isogenic lovhk :: km , ∆lovR and lovhk :: km /pMR_ lovhk strains were cultured in TSB up to logarithmic phase, and expression of the phyR gene was analyzed by qRT-PCR. The if-1 housekeeping gene was used as a reference. Data represent the average of three independent experiments, and are reported as fold induction relative to wt ± standard error. p -values between each strain and wt were determined by one-way ANOVA and post-hoc Tukey’s multiple comparisons test (* = p

    Article Snippet: Real Time quantitative RT-PCR assays Reverse transcription was performed with SuperScript III First Strand Synthesis System (Invitrogen) following the manufacturer’s instructions using random decamer primers (Invitrogen) and RNasin ribonuclease inhibitor (Promega).

    Techniques: Expressing, Mutagenesis, Cell Culture, Quantitative RT-PCR

    HbCIPK2 interacted with HbFd1. (A) Examination of the interaction between HbCIPK2 and HbFd1 in yeast. The interactions were verified by the yeast growing on selective medium (SC/-Leu-Trp-His-Ura with 35 mM 3-AT) and conducting ß-Gal assays. Bait vector with HbCIPK2 or prey vector with HbFd1 was transformed with empty vector as negative control, and the plasmids pPC97-Fos and pPC86-Jun provided by Invitrogen system were used as positive control. (B) Co-BiFC assay to verify the interaction of HbCIPK2 and HbFd1 in Arabidopsis protoplasts. Construct AtCBF1::RFP was used as reference to co-localize the interaction of HbFd1 with HbCIPK2, and transformants expressing SPYNE/HbFd1::SPYCE and SPYNE::HbCIPK2/SPYCE were used as negative controls. (C) Co-IP assay to show the interaction between HbCIPK2 and mature HbFd1 in cell line HEK293, co-expressing HbCIPK2-Myc and HbFd1△cTP-Flag. Cell proteins before (Input) and after (IP) immunoprecipitation were separated in SDS-PAGE gels, transferred onto the nitrocellulose membranes, and analyzed by protein gel blotting with antibodies as indicated. All assays repeated three times.

    Journal: PLoS ONE

    Article Title: Identification and Expression Analysis of a Novel HbCIPK2-Interacting Ferredoxin from Halophyte H. brevisubulatum

    doi: 10.1371/journal.pone.0144132

    Figure Lengend Snippet: HbCIPK2 interacted with HbFd1. (A) Examination of the interaction between HbCIPK2 and HbFd1 in yeast. The interactions were verified by the yeast growing on selective medium (SC/-Leu-Trp-His-Ura with 35 mM 3-AT) and conducting ß-Gal assays. Bait vector with HbCIPK2 or prey vector with HbFd1 was transformed with empty vector as negative control, and the plasmids pPC97-Fos and pPC86-Jun provided by Invitrogen system were used as positive control. (B) Co-BiFC assay to verify the interaction of HbCIPK2 and HbFd1 in Arabidopsis protoplasts. Construct AtCBF1::RFP was used as reference to co-localize the interaction of HbFd1 with HbCIPK2, and transformants expressing SPYNE/HbFd1::SPYCE and SPYNE::HbCIPK2/SPYCE were used as negative controls. (C) Co-IP assay to show the interaction between HbCIPK2 and mature HbFd1 in cell line HEK293, co-expressing HbCIPK2-Myc and HbFd1△cTP-Flag. Cell proteins before (Input) and after (IP) immunoprecipitation were separated in SDS-PAGE gels, transferred onto the nitrocellulose membranes, and analyzed by protein gel blotting with antibodies as indicated. All assays repeated three times.

    Article Snippet: Total RNA from H . brevisubulatum seedling treated with various stresses (350 mM NaCl, 350 mM mannitol and 10% PEG6000 stressed for 6 hrs, and 4°C for 12 hrs, respectively) upon the emergence of the third shoot was extracted using RNeasy Plant Mini kit (QIAGEN, Stockach, Germany) and first strand cDNA was synthesized using the SuperScript III First-Strand (Invitrogen).

    Techniques: Plasmid Preparation, Transformation Assay, Negative Control, Positive Control, Bimolecular Fluorescence Complementation Assay, Construct, Expressing, Co-Immunoprecipitation Assay, Immunoprecipitation, SDS Page

    γ-T3 downregulates Ang-1 expression in prostate cancer cells. PC-3 cells were treated with 10 μg/mL γ-T3 for 72 h under a serum-free condition. ( A ) qRT-PCR and ( B ) ELISA analyses were performed to examine the effects of γ-T3 on Ang-1 expression in PC-3. ( C ) Addition of exogenous Ang-1 (600 ng/mL) partially restored the expression of CSC (CD49f and Bmi-1) and quiescence (p27) markers in the presence of γ-T3. Each experiment was repeated at least three times, and the results are presented as the mean ± SD. ( p values: **

    Journal: International Journal of Molecular Sciences

    Article Title: Gamma-Tocotrienol Induces Apoptosis in Prostate Cancer Cells by Targeting the Ang-1/Tie-2 Signalling Pathway

    doi: 10.3390/ijms20051164

    Figure Lengend Snippet: γ-T3 downregulates Ang-1 expression in prostate cancer cells. PC-3 cells were treated with 10 μg/mL γ-T3 for 72 h under a serum-free condition. ( A ) qRT-PCR and ( B ) ELISA analyses were performed to examine the effects of γ-T3 on Ang-1 expression in PC-3. ( C ) Addition of exogenous Ang-1 (600 ng/mL) partially restored the expression of CSC (CD49f and Bmi-1) and quiescence (p27) markers in the presence of γ-T3. Each experiment was repeated at least three times, and the results are presented as the mean ± SD. ( p values: **

    Article Snippet: One microgram of RNA was used to synthesize cDNA using the SuperScript® III First-Strand Synthesis Systems (Invitrogen); subsequently, qRT-PCR was carried out with the ViiA™ 7 Real-Time PCR System (Applied Biosystems, Foster City, CA, USA).

    Techniques: Expressing, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay

    TLR2 −/− DCs show enhanced activation after L. braziliensis infection. WT and TLR2 −/− BMDCs were infected with L. braziliensis (Lb) and L. amazonensis (La) promastigotes at an 8:1 parasite-to-cell ratio for 24 h. Expression of DC surface maturation markers CD40 (A) and CD80 (B) was measured by FACS analysis. The levels of IL-12 p40 (C) and IL-10 (D) in the culture supernatants were assayed by ELISA. (E and F) DCs were infected at a 10:1 parasite-to-cell ratio for 4 h before RNA extraction. Total RNA was extracted and used for real-time RT-PCR measurement of SOCS1 (E) and SOCS3 (F) levels. Data are expressed as a fold change of expression relative to uninfected samples and normalized to the 18s ribosomal control. P 3 CSK 4 (Pam 3 CSK 4 ) was used as a quality control for TLR2. Data were pooled from three independent repeats and are shown in the plots. Statistically significant differences between the groups determined by one-way ANOVA and Student's t test are as follows: *, P

    Journal: Infection and Immunity

    Article Title: Distinct Roles for MyD88 and Toll-Like Receptor 2 during Leishmania braziliensis Infection in Mice ▿

    doi: 10.1128/IAI.00154-09

    Figure Lengend Snippet: TLR2 −/− DCs show enhanced activation after L. braziliensis infection. WT and TLR2 −/− BMDCs were infected with L. braziliensis (Lb) and L. amazonensis (La) promastigotes at an 8:1 parasite-to-cell ratio for 24 h. Expression of DC surface maturation markers CD40 (A) and CD80 (B) was measured by FACS analysis. The levels of IL-12 p40 (C) and IL-10 (D) in the culture supernatants were assayed by ELISA. (E and F) DCs were infected at a 10:1 parasite-to-cell ratio for 4 h before RNA extraction. Total RNA was extracted and used for real-time RT-PCR measurement of SOCS1 (E) and SOCS3 (F) levels. Data are expressed as a fold change of expression relative to uninfected samples and normalized to the 18s ribosomal control. P 3 CSK 4 (Pam 3 CSK 4 ) was used as a quality control for TLR2. Data were pooled from three independent repeats and are shown in the plots. Statistically significant differences between the groups determined by one-way ANOVA and Student's t test are as follows: *, P

    Article Snippet: For detecting suppressor or cytokine signaling 1 (SOCS1) and SOCS3 transcripts, cDNA was synthesized from 2 μg of total RNA by using the SuperScript III first-strand system (Invitrogen) primed with random hexamers.

    Techniques: Activation Assay, Infection, Expressing, FACS, Enzyme-linked Immunosorbent Assay, RNA Extraction, Quantitative RT-PCR

    Regulation of mRNA expression of ER stress-related genes ( bZIP17 , bZIP28 , bZIP60 , BiP1 , BiP3 , IRE1a , NAC103 , NAC089 , BAG7 , BAX inhibitor 1 , and ERO1 ) in transgenic Arabidopsis plants expressing mAb CO and mAb COK measured with qRT-PCR analysis. RNA was isolated from the roots of plants from each experimental group (mAb CO and mAb COK) and grown on kanamycin-containing media for 12 days. The ubiquitin 10 ( UBI 10 ) gene was used as a control gene for normalization. Error bars represent the mean ± standard deviation (SD) of three technical replicates with two biological replicates. Statistically significant differences are indicated by asterisks (NS: not significant; *: p

    Journal: PLoS ONE

    Article Title: Endoplasmic reticulum retention motif fused to recombinant anti-cancer monoclonal antibody (mAb) CO17-1A affects mAb expression and plant stress response

    doi: 10.1371/journal.pone.0198978

    Figure Lengend Snippet: Regulation of mRNA expression of ER stress-related genes ( bZIP17 , bZIP28 , bZIP60 , BiP1 , BiP3 , IRE1a , NAC103 , NAC089 , BAG7 , BAX inhibitor 1 , and ERO1 ) in transgenic Arabidopsis plants expressing mAb CO and mAb COK measured with qRT-PCR analysis. RNA was isolated from the roots of plants from each experimental group (mAb CO and mAb COK) and grown on kanamycin-containing media for 12 days. The ubiquitin 10 ( UBI 10 ) gene was used as a control gene for normalization. Error bars represent the mean ± standard deviation (SD) of three technical replicates with two biological replicates. Statistically significant differences are indicated by asterisks (NS: not significant; *: p

    Article Snippet: Total RNA was extracted from Arabidopsis roots using an RNeasy Mini Kit (Qiagen, Valencia, CA), and the extracted RNA samples were the processed with an RNase-free DNase kit (Qiagen, Valencia, CA), according to the supplied protocol. cDNA was synthesized from 2 μg of total RNA using the Superscript III First Strand Synthesis kit (Invitrogen, Carlsbad, CA).

    Techniques: Expressing, Transgenic Assay, Quantitative RT-PCR, Isolation, Standard Deviation

    DYRK1A destabilizes Cyclin D3 by phosphorylating T283. (A and B) Western blots of FACS-purified large and small pre–B cells from Dyrk1a f/f Mx1-Cre − (Control) and Dyrk1a f/f Mx1-Cre + (CKO) mice 4 wk after pI:pC treatment were assessed for the indicated proteins as shown. The same membrane (cut into segments according to molecular weight) from Fig. 7 F was stripped and reprobed, hence the same loading control is shown here. Data are representative of three independent experiments. Densitometry values were normalized to actin. (C) The expression levels of the D-type Cyclin transcripts from RNA-seq data (from Fig. 6 ) are shown for Control and CKO mice. (D and E) Control and CKO thymocytes (D), or WT cultured pre–B cells ± EHT 1610 (E) were treated with cycloheximide (25 µg/ml) for the indicated times before Western blot analysis. Data are representative of three independent experiments. Densitometry values were normalized to actin. (F) An amino acid alignment of C-terminal phosphodegrons in the D-type Cyclins is shown. Conserved residues are highlighted in the blue box; phosphorylated threonines are highlighted in the red box. (G) FLAG-tagged Cyclin D3 (WT or T283A) was immunoprecipitated from transfected 293T cells and used for in vitro kinase assays with recombinant active DYRK1A and ATP-γS. The reaction products were alkylated with PNBM and analyzed for thiophosphate esters by Western blotting. Data are representative of three independent assays. Densitometry values were normalized to FLAG-Cyclin D3.

    Journal: The Journal of Experimental Medicine

    Article Title: DYRK1A controls the transition from proliferation to quiescence during lymphoid development by destabilizing Cyclin D3

    doi: 10.1084/jem.20150002

    Figure Lengend Snippet: DYRK1A destabilizes Cyclin D3 by phosphorylating T283. (A and B) Western blots of FACS-purified large and small pre–B cells from Dyrk1a f/f Mx1-Cre − (Control) and Dyrk1a f/f Mx1-Cre + (CKO) mice 4 wk after pI:pC treatment were assessed for the indicated proteins as shown. The same membrane (cut into segments according to molecular weight) from Fig. 7 F was stripped and reprobed, hence the same loading control is shown here. Data are representative of three independent experiments. Densitometry values were normalized to actin. (C) The expression levels of the D-type Cyclin transcripts from RNA-seq data (from Fig. 6 ) are shown for Control and CKO mice. (D and E) Control and CKO thymocytes (D), or WT cultured pre–B cells ± EHT 1610 (E) were treated with cycloheximide (25 µg/ml) for the indicated times before Western blot analysis. Data are representative of three independent experiments. Densitometry values were normalized to actin. (F) An amino acid alignment of C-terminal phosphodegrons in the D-type Cyclins is shown. Conserved residues are highlighted in the blue box; phosphorylated threonines are highlighted in the red box. (G) FLAG-tagged Cyclin D3 (WT or T283A) was immunoprecipitated from transfected 293T cells and used for in vitro kinase assays with recombinant active DYRK1A and ATP-γS. The reaction products were alkylated with PNBM and analyzed for thiophosphate esters by Western blotting. Data are representative of three independent assays. Densitometry values were normalized to FLAG-Cyclin D3.

    Article Snippet: Total RNA was isolated using RNeasy kits (QIAGEN) and reverse transcribed using SuperScript III First Strand Synthesis kits (Life Technologies).

    Techniques: Western Blot, FACS, Purification, Mouse Assay, Molecular Weight, Expressing, RNA Sequencing Assay, Cell Culture, Immunoprecipitation, Transfection, In Vitro, Recombinant

    Dyrk1a -deficient quiescent DP thymocytes and small pre–B cells fail to repress E2F target gene transcription . (A and B) Up- and down-regulated transcripts were identified by RNA-sequencing of FACS-purified quiescent DP thymocytes (CD4 + /CD8 + /DNA content 2N /Pyronin Y low ), cycling DP thymocytes (CD4 + /CD8 + /DNA content > 2N /Pyronin Y high ), small pre–B cells (IgM − /B220 + /CD43 − /FSC low ), large pre–B cells (IgM − /B220 + /CD43 low /FSC high ), and granulocytes (Gr-1 high /Mac-1/CD11b high ) from Dyrk1a f/f Mx1-Cre − (Control) and Dyrk1a f/f Mx1-Cre + (CKO) mice. Venn diagrams depict shared up-regulated (A) and down-regulated (B) transcripts identified by RNA-sequencing in the three quiescent cell populations. (C–F) mRNA expression of E2F target genes was assessed by qRT-PCR in FACS-purified small pre–B cells (C), quiescent DP thymocytes (D), large pre–B cells (E), and cycling DP thymocytes (F) from Control and CKO mice 2 wk after pI:pC treatment. Transcript levels were normalized to Actb expression. PCRs were performed using 3 independently sorted pairs of samples (each with 1 mouse per genotype) for pre–B cells, and pooled samples from 3 mice per genotype for thymocytes. Error bars depict SD of triplicate wells for representative samples. *, P

    Journal: The Journal of Experimental Medicine

    Article Title: DYRK1A controls the transition from proliferation to quiescence during lymphoid development by destabilizing Cyclin D3

    doi: 10.1084/jem.20150002

    Figure Lengend Snippet: Dyrk1a -deficient quiescent DP thymocytes and small pre–B cells fail to repress E2F target gene transcription . (A and B) Up- and down-regulated transcripts were identified by RNA-sequencing of FACS-purified quiescent DP thymocytes (CD4 + /CD8 + /DNA content 2N /Pyronin Y low ), cycling DP thymocytes (CD4 + /CD8 + /DNA content > 2N /Pyronin Y high ), small pre–B cells (IgM − /B220 + /CD43 − /FSC low ), large pre–B cells (IgM − /B220 + /CD43 low /FSC high ), and granulocytes (Gr-1 high /Mac-1/CD11b high ) from Dyrk1a f/f Mx1-Cre − (Control) and Dyrk1a f/f Mx1-Cre + (CKO) mice. Venn diagrams depict shared up-regulated (A) and down-regulated (B) transcripts identified by RNA-sequencing in the three quiescent cell populations. (C–F) mRNA expression of E2F target genes was assessed by qRT-PCR in FACS-purified small pre–B cells (C), quiescent DP thymocytes (D), large pre–B cells (E), and cycling DP thymocytes (F) from Control and CKO mice 2 wk after pI:pC treatment. Transcript levels were normalized to Actb expression. PCRs were performed using 3 independently sorted pairs of samples (each with 1 mouse per genotype) for pre–B cells, and pooled samples from 3 mice per genotype for thymocytes. Error bars depict SD of triplicate wells for representative samples. *, P

    Article Snippet: Total RNA was isolated using RNeasy kits (QIAGEN) and reverse transcribed using SuperScript III First Strand Synthesis kits (Life Technologies).

    Techniques: RNA Sequencing Assay, FACS, Purification, Mouse Assay, Expressing, Quantitative RT-PCR

    Global changes in infected MDM gene expression. MDMs from four healthy human donors were infected at a 2:1 (parasite:MDM) ratio with each of three isolates of L. braziliensis from clades A, B, or C ( Schriefer et al., 2004 ). The clades A, B, and C isolates were drawn from patients with DL, CL, and ML, respectively. After 4 h, total RNA was extracted and processed for hybridization to Affymetrix human transcript microarrays. Fold changes were calculated by comparing fluorescence data representing the abundance of each transcript in infected versus uninfected MDMs from the same donor. Each dot in the figure represents the average fold change in abundance of each transcript in all four donors. Eighty-nine transcripts were significantly increased, and 471 transcripts were significantly decreased after MDM infection with each of the three L. braziliensis isolates (one-way ANOVA for repressed transcripts among MDM infected with clades A, B, or C, p

    Journal: Frontiers in Microbiology

    Article Title: Early Suppression of Macrophage Gene Expression by Leishmania braziliensis

    doi: 10.3389/fmicb.2018.02464

    Figure Lengend Snippet: Global changes in infected MDM gene expression. MDMs from four healthy human donors were infected at a 2:1 (parasite:MDM) ratio with each of three isolates of L. braziliensis from clades A, B, or C ( Schriefer et al., 2004 ). The clades A, B, and C isolates were drawn from patients with DL, CL, and ML, respectively. After 4 h, total RNA was extracted and processed for hybridization to Affymetrix human transcript microarrays. Fold changes were calculated by comparing fluorescence data representing the abundance of each transcript in infected versus uninfected MDMs from the same donor. Each dot in the figure represents the average fold change in abundance of each transcript in all four donors. Eighty-nine transcripts were significantly increased, and 471 transcripts were significantly decreased after MDM infection with each of the three L. braziliensis isolates (one-way ANOVA for repressed transcripts among MDM infected with clades A, B, or C, p

    Article Snippet: Changes in expression observed on microarrays were validated by reverse transcriptase followed by qPCR to document gene expression in RNA samples from replicate MDM samples incubated without or with the representatives of L. braziliensis clades. cDNA was generated using the Superscript III First Strand Synthesis System kit (Invitrogen/ThermoFisher, Waltham, MA, United States) and random hexamers, followed by RNase H treatment according to the manufacturer’s instructions.

    Techniques: Infection, Expressing, Hybridization, Fluorescence

    Gene expression profiles from the microarrays described and illustrated in Figure 2 were collated according to the number of significantly altered transcripts in MDM infected with isolates from each of the three representatives of L. braziliensis clades (A, B, or C). (A) Venn diagram of the distribution of transcripts with changes in expression that reached statistical significance upon infection of MDMs with each L. braziliensis parasite. Sectors indicate the numbers of transcripts that were uniquely changed due to infection with one parasite clade, or transcripts that were changed by infection with more than one parasite clade (evaluation of gene expression employed ANOVA for detecting transcripts significantly affected by infections, and paired Student’s t -test for comparing the expression elicited by clades of parasites in infected MDM). (B) The magnitude of change in expression of 471 genes in MDM infected with each of the three L. braziliensis isolates is illustrated. Values represent the fold changes in expression of the 471 genes for which transcript abundance was significantly decreased by infection with any of the three parasite isolates tested. L. braziliensis isolates belong to clade A (DL; blue), clade B (CL; green), or clade C (ML; red). Each position on the x-axis corresponds to a single gene, plotted against its fold change in expression on the y-axis (Friedman’s test p

    Journal: Frontiers in Microbiology

    Article Title: Early Suppression of Macrophage Gene Expression by Leishmania braziliensis

    doi: 10.3389/fmicb.2018.02464

    Figure Lengend Snippet: Gene expression profiles from the microarrays described and illustrated in Figure 2 were collated according to the number of significantly altered transcripts in MDM infected with isolates from each of the three representatives of L. braziliensis clades (A, B, or C). (A) Venn diagram of the distribution of transcripts with changes in expression that reached statistical significance upon infection of MDMs with each L. braziliensis parasite. Sectors indicate the numbers of transcripts that were uniquely changed due to infection with one parasite clade, or transcripts that were changed by infection with more than one parasite clade (evaluation of gene expression employed ANOVA for detecting transcripts significantly affected by infections, and paired Student’s t -test for comparing the expression elicited by clades of parasites in infected MDM). (B) The magnitude of change in expression of 471 genes in MDM infected with each of the three L. braziliensis isolates is illustrated. Values represent the fold changes in expression of the 471 genes for which transcript abundance was significantly decreased by infection with any of the three parasite isolates tested. L. braziliensis isolates belong to clade A (DL; blue), clade B (CL; green), or clade C (ML; red). Each position on the x-axis corresponds to a single gene, plotted against its fold change in expression on the y-axis (Friedman’s test p

    Article Snippet: Changes in expression observed on microarrays were validated by reverse transcriptase followed by qPCR to document gene expression in RNA samples from replicate MDM samples incubated without or with the representatives of L. braziliensis clades. cDNA was generated using the Superscript III First Strand Synthesis System kit (Invitrogen/ThermoFisher, Waltham, MA, United States) and random hexamers, followed by RNase H treatment according to the manufacturer’s instructions.

    Techniques: Expressing, Infection

    Changes in LRRK2, TLR8, HSPA1A, and MT1M expression were documented in independent assays of MDMs from eight additional human donors, distinct from those of experiments depicted in Figures 1 – 4 . MDMs were infected with three representative L. braziliensis isolates of each clade (A, B, or C) used in microarray experiments. Data derived from total RNA extracted after 4 h of MDM infection at a MOI of two parasites per macrophage (2:1). The relative abundance of transcripts was assessed by RT-qPCR. p -Values correspond to pair-wise comparisons by one tailed paired Wilcoxon test.

    Journal: Frontiers in Microbiology

    Article Title: Early Suppression of Macrophage Gene Expression by Leishmania braziliensis

    doi: 10.3389/fmicb.2018.02464

    Figure Lengend Snippet: Changes in LRRK2, TLR8, HSPA1A, and MT1M expression were documented in independent assays of MDMs from eight additional human donors, distinct from those of experiments depicted in Figures 1 – 4 . MDMs were infected with three representative L. braziliensis isolates of each clade (A, B, or C) used in microarray experiments. Data derived from total RNA extracted after 4 h of MDM infection at a MOI of two parasites per macrophage (2:1). The relative abundance of transcripts was assessed by RT-qPCR. p -Values correspond to pair-wise comparisons by one tailed paired Wilcoxon test.

    Article Snippet: Changes in expression observed on microarrays were validated by reverse transcriptase followed by qPCR to document gene expression in RNA samples from replicate MDM samples incubated without or with the representatives of L. braziliensis clades. cDNA was generated using the Superscript III First Strand Synthesis System kit (Invitrogen/ThermoFisher, Waltham, MA, United States) and random hexamers, followed by RNase H treatment according to the manufacturer’s instructions.

    Techniques: Expressing, Infection, Microarray, Derivative Assay, Quantitative RT-PCR, One-tailed Test

    Incorrect splicing of ISCU in myoblasts with decreased SRSF3 expression. A lentivirus-mediated expression vector for SRSF3 shRNA (sh3) was introduced into myoblasts from HML patients (P1, P2) and a healthy control (C). A) SRSF3 qRTPCR using cDNA from uninfected myoblasts (-) and myoblasts infected with a shSRSF3 lentivirus-mediated expression vector. The graph present the mean fold change ± SD for the SRSF3 expression from at least three independent experiments. β-actin was used as an internal control. B) Western blot of SRSF3 in non-transduced and transduced myoblasts. ACTIN was used as a loading reference. C) Semi-qRTPCR of human ISCU with incorrect (MUT) and correct (WT) splice variants from uninfected myoblasts, (-) or myoblasts infected with lentivirus-mediated vectors expressing shSRSF3 (sh3). D) Quantification of incorrectly spliced ISCU by qRTPCR in in non-transduced and transduced myoblasts. The graph presents the mean percentage of incorrectly spliced ISCU ± SD from at least three independent experiments (* p

    Journal: PLoS ONE

    Article Title: The High Level of Aberrant Splicing of ISCU in Slow-Twitch Muscle May Involve the Splicing Factor SRSF3

    doi: 10.1371/journal.pone.0165453

    Figure Lengend Snippet: Incorrect splicing of ISCU in myoblasts with decreased SRSF3 expression. A lentivirus-mediated expression vector for SRSF3 shRNA (sh3) was introduced into myoblasts from HML patients (P1, P2) and a healthy control (C). A) SRSF3 qRTPCR using cDNA from uninfected myoblasts (-) and myoblasts infected with a shSRSF3 lentivirus-mediated expression vector. The graph present the mean fold change ± SD for the SRSF3 expression from at least three independent experiments. β-actin was used as an internal control. B) Western blot of SRSF3 in non-transduced and transduced myoblasts. ACTIN was used as a loading reference. C) Semi-qRTPCR of human ISCU with incorrect (MUT) and correct (WT) splice variants from uninfected myoblasts, (-) or myoblasts infected with lentivirus-mediated vectors expressing shSRSF3 (sh3). D) Quantification of incorrectly spliced ISCU by qRTPCR in in non-transduced and transduced myoblasts. The graph presents the mean percentage of incorrectly spliced ISCU ± SD from at least three independent experiments (* p

    Article Snippet: cDNA synthesis cDNA was synthesized using the SuperScript™ III RT First-Strand Synthesis System for RT-PCR (Invitrogen, Waltham, MA, USA) with random hexamers according to the manufacturer’s instructions.

    Techniques: Expressing, Plasmid Preparation, shRNA, Infection, Western Blot

    Induced S-phase arrest of SNB-19 cells enhances ZIKV, but not DENV or WNV replication. (A) Schematic of two treatment conditions used to synchronize SNB-19 cells in S phase. Cells were initially treated with 2 mM thymidine for 16 h and then were released back into the cell cycle for 12 h. Following release, the cells were treated with either 2 mM thymidine or 12 μM aphidicolin for an additional 16 h to induce S-phase arrest prior to infection. (B) Representative flow cytometry analysis of SNB-19 cells treated as indicated in panel A. Samples were stained with propidium iodide (PI) and analyzed by flow cytometry. (C, D, and F) SNB-19 cells were infected for (C) 24 h, (D) 4 to 32 h, or (F) 12 to 24 h with ZIKV PR (MOI of 0.5), ZIKV M (MOI of 0.5), or DENV (MOI of 0.5). (C) Western blot images and quantification of intracellular viral protein expression. Viral protein was detected by anti-ZIKV NS1 or anti-DENV NS3 for each respective sample. Viral protein band intensities were normalized to the DMSO control, representing the mean from three biological replicates ± SD. (D and F) Time course of relative intracellular viral RNA copies in (D) ZIKV M - or (F) DENV-infected SNB-19 cells as measured by qPCR. Error bars are the mean ± SD from three biological replicates. (E and G) Infectivity titers of culture supernatants at (E) 48 h postinfection as measured by focus-forming units (ZIKV PR and ZIKV M [MOI of 0.5]) and at 36 h postinfection as measured by PFU (DENV [MOI of 0.5]) or (G) at 12 and 24 h postinfection as measured by PFU (WNV [MOI of 0.5]) on (E) Vero cells or (F) BHK cells. Error bars are the mean ± SD from three biological replicates. In panels C to G, * indicates P ≤ 0.05, ** indicates P ≤ 0.01, *** indicates P ≤ 0.001, and **** indicates P ≤ 0.0001. (C, E, and G) One-way ANOVA. (D and F) Two-way ANOVA.

    Journal: Journal of Virology

    Article Title: Zika Virus Infection Induces DNA Damage Response in Human Neural Progenitors That Enhances Viral Replication

    doi: 10.1128/JVI.00638-19

    Figure Lengend Snippet: Induced S-phase arrest of SNB-19 cells enhances ZIKV, but not DENV or WNV replication. (A) Schematic of two treatment conditions used to synchronize SNB-19 cells in S phase. Cells were initially treated with 2 mM thymidine for 16 h and then were released back into the cell cycle for 12 h. Following release, the cells were treated with either 2 mM thymidine or 12 μM aphidicolin for an additional 16 h to induce S-phase arrest prior to infection. (B) Representative flow cytometry analysis of SNB-19 cells treated as indicated in panel A. Samples were stained with propidium iodide (PI) and analyzed by flow cytometry. (C, D, and F) SNB-19 cells were infected for (C) 24 h, (D) 4 to 32 h, or (F) 12 to 24 h with ZIKV PR (MOI of 0.5), ZIKV M (MOI of 0.5), or DENV (MOI of 0.5). (C) Western blot images and quantification of intracellular viral protein expression. Viral protein was detected by anti-ZIKV NS1 or anti-DENV NS3 for each respective sample. Viral protein band intensities were normalized to the DMSO control, representing the mean from three biological replicates ± SD. (D and F) Time course of relative intracellular viral RNA copies in (D) ZIKV M - or (F) DENV-infected SNB-19 cells as measured by qPCR. Error bars are the mean ± SD from three biological replicates. (E and G) Infectivity titers of culture supernatants at (E) 48 h postinfection as measured by focus-forming units (ZIKV PR and ZIKV M [MOI of 0.5]) and at 36 h postinfection as measured by PFU (DENV [MOI of 0.5]) or (G) at 12 and 24 h postinfection as measured by PFU (WNV [MOI of 0.5]) on (E) Vero cells or (F) BHK cells. Error bars are the mean ± SD from three biological replicates. In panels C to G, * indicates P ≤ 0.05, ** indicates P ≤ 0.01, *** indicates P ≤ 0.001, and **** indicates P ≤ 0.0001. (C, E, and G) One-way ANOVA. (D and F) Two-way ANOVA.

    Article Snippet: Total cellular RNA was purified from mock-, ZIKVM -, or DENV-infected SNB-19 cells using an RNeasy Plus kit (Qiagen) per the manufacturer’s instructions. cDNA was synthesized from purified RNA using random hexamers and a SuperScript III First-Strand kit (Invitrogen).

    Techniques: Infection, Flow Cytometry, Staining, Western Blot, Expressing, Real-time Polymerase Chain Reaction