qpcr kit  (Kapa Biosystems)


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    Kapa Biosystems qpcr kit
    The IL-17B–IL-17RB signaling pathway is essential for tumorigenic and metastatic abilities of pancreatic cancer cell lines. (A) <t>RT-PCR</t> analysis of IL-17B in pancreatic cancer cell lines. β-actin was used as a loading control. (B) mRNA expression of IL-17B determined by <t>RT-qPCR</t> in CFPAC1 and BxPC3 cells infected with shLacZ or shIL-17RB. (C and D) SACF assay (C) and invasion assay (D) using CFPAC-1 and BxPC3 cells infected with shLacZ or shIL-17B. (E and F) SACF assay (E) and invasion assay (F) using CFPAC-1 and BxPC3 cells supplemented with IgG control, anti–IL-17RB, or anti–IL-17B (1 µg/ml). (G) Tumorigenesis assay of NOD/SCIDγ mice subcutaneously injected with shLacZ-transduced or IL-17B–depleted CFPAC-1 cells. Cell dose: 1 × 10 6 cells per mouse. Four mice were used for each group. (H) Tumor weight of NOD/SCIDγ mice orthotopically implanted with shLacZ-transduced or IL-17B–depleted CFPAC-1 cells. Cell dose: 2.5 × 10 5 cells per mouse. Three mice were used for each group. (I) Summary table of lung and liver metastasis derived from orthotopic xenograft. (J) Lung metastasis of NOD/SCIDγ mice intravenously injected with shLacZ-transduced or IL-17B depleted CFPAC-1 cells. Cell dose: 5 × 10 5 cells per mouse. Four mice were used for each group. (K and L) SACF assay (K) and invasion assay (L) using CFPAC-1 and BxPC3 cells treated with BSA or rIL17B. (M and N) SACF assay (M) and invasion assay (N) using IL-17RB full-length or ΔLBD overexpressing SU.86.86 and HPAC cells treated with BSA or rIL17B. Data shown are means ± SD. *, P
    Qpcr Kit, supplied by Kapa Biosystems, used in various techniques. Bioz Stars score: 94/100, based on 383 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Targeting IL-17B–IL-17RB signaling with an anti–IL-17RB antibody blocks pancreatic cancer metastasis by silencing multiple chemokines"

    Article Title: Targeting IL-17B–IL-17RB signaling with an anti–IL-17RB antibody blocks pancreatic cancer metastasis by silencing multiple chemokines

    Journal: The Journal of Experimental Medicine

    doi: 10.1084/jem.20141702

    The IL-17B–IL-17RB signaling pathway is essential for tumorigenic and metastatic abilities of pancreatic cancer cell lines. (A) RT-PCR analysis of IL-17B in pancreatic cancer cell lines. β-actin was used as a loading control. (B) mRNA expression of IL-17B determined by RT-qPCR in CFPAC1 and BxPC3 cells infected with shLacZ or shIL-17RB. (C and D) SACF assay (C) and invasion assay (D) using CFPAC-1 and BxPC3 cells infected with shLacZ or shIL-17B. (E and F) SACF assay (E) and invasion assay (F) using CFPAC-1 and BxPC3 cells supplemented with IgG control, anti–IL-17RB, or anti–IL-17B (1 µg/ml). (G) Tumorigenesis assay of NOD/SCIDγ mice subcutaneously injected with shLacZ-transduced or IL-17B–depleted CFPAC-1 cells. Cell dose: 1 × 10 6 cells per mouse. Four mice were used for each group. (H) Tumor weight of NOD/SCIDγ mice orthotopically implanted with shLacZ-transduced or IL-17B–depleted CFPAC-1 cells. Cell dose: 2.5 × 10 5 cells per mouse. Three mice were used for each group. (I) Summary table of lung and liver metastasis derived from orthotopic xenograft. (J) Lung metastasis of NOD/SCIDγ mice intravenously injected with shLacZ-transduced or IL-17B depleted CFPAC-1 cells. Cell dose: 5 × 10 5 cells per mouse. Four mice were used for each group. (K and L) SACF assay (K) and invasion assay (L) using CFPAC-1 and BxPC3 cells treated with BSA or rIL17B. (M and N) SACF assay (M) and invasion assay (N) using IL-17RB full-length or ΔLBD overexpressing SU.86.86 and HPAC cells treated with BSA or rIL17B. Data shown are means ± SD. *, P
    Figure Legend Snippet: The IL-17B–IL-17RB signaling pathway is essential for tumorigenic and metastatic abilities of pancreatic cancer cell lines. (A) RT-PCR analysis of IL-17B in pancreatic cancer cell lines. β-actin was used as a loading control. (B) mRNA expression of IL-17B determined by RT-qPCR in CFPAC1 and BxPC3 cells infected with shLacZ or shIL-17RB. (C and D) SACF assay (C) and invasion assay (D) using CFPAC-1 and BxPC3 cells infected with shLacZ or shIL-17B. (E and F) SACF assay (E) and invasion assay (F) using CFPAC-1 and BxPC3 cells supplemented with IgG control, anti–IL-17RB, or anti–IL-17B (1 µg/ml). (G) Tumorigenesis assay of NOD/SCIDγ mice subcutaneously injected with shLacZ-transduced or IL-17B–depleted CFPAC-1 cells. Cell dose: 1 × 10 6 cells per mouse. Four mice were used for each group. (H) Tumor weight of NOD/SCIDγ mice orthotopically implanted with shLacZ-transduced or IL-17B–depleted CFPAC-1 cells. Cell dose: 2.5 × 10 5 cells per mouse. Three mice were used for each group. (I) Summary table of lung and liver metastasis derived from orthotopic xenograft. (J) Lung metastasis of NOD/SCIDγ mice intravenously injected with shLacZ-transduced or IL-17B depleted CFPAC-1 cells. Cell dose: 5 × 10 5 cells per mouse. Four mice were used for each group. (K and L) SACF assay (K) and invasion assay (L) using CFPAC-1 and BxPC3 cells treated with BSA or rIL17B. (M and N) SACF assay (M) and invasion assay (N) using IL-17RB full-length or ΔLBD overexpressing SU.86.86 and HPAC cells treated with BSA or rIL17B. Data shown are means ± SD. *, P

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Expressing, Quantitative RT-PCR, Infection, Invasion Assay, Mouse Assay, Injection, Derivative Assay

    Chemokines CCL20, CXCL1, IL-8, and TFF1 are the downstream targets of the IL-17B–IL-17RB signaling. (A) Summary of cDNA microarray analyses. Total of 71 genes expressed at least 2-fold higher in IL-17RB–overexpressing HPAC cells and 2-fold lower in IL-17RB–depleted CFPAC-1 cells compared with the proper control were identified by Affymetrix microarray analyses. (B) Four chemokines, CCL20, CXCL1, IL-8, and TFF1, were identified among the 71 genes. (C) qRT-PCR analysis to reconfirm the expression profile of the four chemokines in IL-17RB–depleted CFPAC-1, IL-17RB–, and ΔLBD-overexpressing HPAC cells. β-actin was used as an internal control. (D) Immunoblot analysis of IL-17RB and the four chemokines in shLacZ-transduced or IL-17RB–depleted CFPAC-1 cells, and lenti-neo, IL-17RB, or ΔLBD-overexpressing HPAC cells. GAPDH was used as a loading control. RE, relative expression. (E) Representative pictures of the IHC analyses of IL-17RB and TFF1 in serial sections of a PDAC case. Boxes show the enlarged area of IL-17RB high (+++) and negative (−) regions. (F) Correlation between TFF1 and IL-17RB in 111 pancreatic cancer cases from IHC assays. χ 2 test was used. (G) mRNA expression of the four chemokines were measured by RT-qPCR in CFPAC-1 cells treated with 50 ng/ml rIL17B for 0, 1, and 3 h after serum-starvation. β-actin was used as internal control. Data shown are means ± SD. *, P
    Figure Legend Snippet: Chemokines CCL20, CXCL1, IL-8, and TFF1 are the downstream targets of the IL-17B–IL-17RB signaling. (A) Summary of cDNA microarray analyses. Total of 71 genes expressed at least 2-fold higher in IL-17RB–overexpressing HPAC cells and 2-fold lower in IL-17RB–depleted CFPAC-1 cells compared with the proper control were identified by Affymetrix microarray analyses. (B) Four chemokines, CCL20, CXCL1, IL-8, and TFF1, were identified among the 71 genes. (C) qRT-PCR analysis to reconfirm the expression profile of the four chemokines in IL-17RB–depleted CFPAC-1, IL-17RB–, and ΔLBD-overexpressing HPAC cells. β-actin was used as an internal control. (D) Immunoblot analysis of IL-17RB and the four chemokines in shLacZ-transduced or IL-17RB–depleted CFPAC-1 cells, and lenti-neo, IL-17RB, or ΔLBD-overexpressing HPAC cells. GAPDH was used as a loading control. RE, relative expression. (E) Representative pictures of the IHC analyses of IL-17RB and TFF1 in serial sections of a PDAC case. Boxes show the enlarged area of IL-17RB high (+++) and negative (−) regions. (F) Correlation between TFF1 and IL-17RB in 111 pancreatic cancer cases from IHC assays. χ 2 test was used. (G) mRNA expression of the four chemokines were measured by RT-qPCR in CFPAC-1 cells treated with 50 ng/ml rIL17B for 0, 1, and 3 h after serum-starvation. β-actin was used as internal control. Data shown are means ± SD. *, P

    Techniques Used: Microarray, Quantitative RT-PCR, Expressing, Immunohistochemistry

    2) Product Images from "Lack of Activation Marker Induction and Chemokine Receptor Switch in Human Neonatal Myeloid Dendritic Cells in Response to Human Respiratory Syncytial Virus"

    Article Title: Lack of Activation Marker Induction and Chemokine Receptor Switch in Human Neonatal Myeloid Dendritic Cells in Response to Human Respiratory Syncytial Virus

    Journal: Journal of Virology

    doi: 10.1128/JVI.01216-19

    Expression of 62 activation-related host genes in CB or AB mDC inoculated with rgRSV or IAV. CB or AB mDC ( n = 3 different CB or AB donors) were mock inoculated, inoculated with rgRSV or IAV at an MOI of 3 PFU/cell, or stimulated with 1 μg/ml of LPS. At 24 hpi, total cellular RNA was prepared and reverse transcribed using random primers. The cDNA was analyzed by qPCR using a custom-made 62-gene TaqMan array. qPCR results were analyzed using the comparative threshold cycle (ΔΔ C T ) method and normalized to the amount of 18S rRNA. The results are presented as the fold increase (red) or decrease (green) (in log 2 ) in gene expression over that for mock-inoculated cells from the same donor. Treg, regulatory T cells.
    Figure Legend Snippet: Expression of 62 activation-related host genes in CB or AB mDC inoculated with rgRSV or IAV. CB or AB mDC ( n = 3 different CB or AB donors) were mock inoculated, inoculated with rgRSV or IAV at an MOI of 3 PFU/cell, or stimulated with 1 μg/ml of LPS. At 24 hpi, total cellular RNA was prepared and reverse transcribed using random primers. The cDNA was analyzed by qPCR using a custom-made 62-gene TaqMan array. qPCR results were analyzed using the comparative threshold cycle (ΔΔ C T ) method and normalized to the amount of 18S rRNA. The results are presented as the fold increase (red) or decrease (green) (in log 2 ) in gene expression over that for mock-inoculated cells from the same donor. Treg, regulatory T cells.

    Techniques Used: Expressing, Activation Assay, Real-time Polymerase Chain Reaction

    3) Product Images from "Phyllanthus emblica Fruit Extract Activates Spindle Assembly Checkpoint, Prevents Mitotic Aberrations and Genomic Instability in Human Colon Epithelial NCM460 Cells"

    Article Title: Phyllanthus emblica Fruit Extract Activates Spindle Assembly Checkpoint, Prevents Mitotic Aberrations and Genomic Instability in Human Colon Epithelial NCM460 Cells

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms17091437

    P. emblica  (PE) treatment activated the spindle assembly checkpoint (SAC) and upregulated the expression of budding uninhibited by benzimidazoles related 1 ( BubR1 ) in NCM460 cells. ( A ) NCM460 cells were treated with indicated doses of PE or 300 ng/mL nocodozale (ND, positive control) for 4 h followed by recovery for 1 h before cells were harvested for determining anaphase-to-metaphase ratio (AMR), an estimated measurement of SAC activity. AMR was obtained by dividing the total number of anaphase cells by the total number of metaphase cells counted for each PE concentration; ( B ) NCM460 cells were treated with indicated doses of PE for 72 h,  BubR1  (gray bars) and mitotic arrest deficient 2 ( Mad2 ; black bars) mRNA levels were measured by real-time quantitative polymerase chain reaction (PCR) after mRNA extraction and reserve transcription. Samples for each experimental group were run in triplicates and normalized to glyceraldehyde-3-phosphate dehydrogenase ( GAPDH ) mRNA levels. Results are expressed as fold increase in treated cells vs. untreated control. Values represent the means ± SEM of the fold induction taken from three independent experiments. *  p
    Figure Legend Snippet: P. emblica (PE) treatment activated the spindle assembly checkpoint (SAC) and upregulated the expression of budding uninhibited by benzimidazoles related 1 ( BubR1 ) in NCM460 cells. ( A ) NCM460 cells were treated with indicated doses of PE or 300 ng/mL nocodozale (ND, positive control) for 4 h followed by recovery for 1 h before cells were harvested for determining anaphase-to-metaphase ratio (AMR), an estimated measurement of SAC activity. AMR was obtained by dividing the total number of anaphase cells by the total number of metaphase cells counted for each PE concentration; ( B ) NCM460 cells were treated with indicated doses of PE for 72 h, BubR1 (gray bars) and mitotic arrest deficient 2 ( Mad2 ; black bars) mRNA levels were measured by real-time quantitative polymerase chain reaction (PCR) after mRNA extraction and reserve transcription. Samples for each experimental group were run in triplicates and normalized to glyceraldehyde-3-phosphate dehydrogenase ( GAPDH ) mRNA levels. Results are expressed as fold increase in treated cells vs. untreated control. Values represent the means ± SEM of the fold induction taken from three independent experiments. * p

    Techniques Used: Expressing, Positive Control, Activity Assay, Concentration Assay, Real-time Polymerase Chain Reaction, Polymerase Chain Reaction

    4) Product Images from "Combinatorial treatment increases IKAP levels in human cells generated from Familial Dysautonomia patients"

    Article Title: Combinatorial treatment increases IKAP levels in human cells generated from Familial Dysautonomia patients

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0211602

    Phosphatidylserine activates the BDNF-mediated signaling pathway. (A-H) FD cells were treated with 50 μg/ml PS or a vehicle only control, and RNA was extracted 5 days after the treatment. qPCR was used to quantify (A) WT IKBKAP , (B) CDKN1A , (C) HOMER1 , (D) RASSF8 , (E) EGR1 , (F) SRXN1 , (G) RGS17 , and (H) BAIAP2 mRNA transcripts. All values were normalized to LZIC , which did not change as a result of PS treatment. Asterisks denote statistically significant differences (*P ≤ 0.05 and **P ≤ 0.01) relative to control (vehicle only); Student’s t -test.
    Figure Legend Snippet: Phosphatidylserine activates the BDNF-mediated signaling pathway. (A-H) FD cells were treated with 50 μg/ml PS or a vehicle only control, and RNA was extracted 5 days after the treatment. qPCR was used to quantify (A) WT IKBKAP , (B) CDKN1A , (C) HOMER1 , (D) RASSF8 , (E) EGR1 , (F) SRXN1 , (G) RGS17 , and (H) BAIAP2 mRNA transcripts. All values were normalized to LZIC , which did not change as a result of PS treatment. Asterisks denote statistically significant differences (*P ≤ 0.05 and **P ≤ 0.01) relative to control (vehicle only); Student’s t -test.

    Techniques Used: Real-time Polymerase Chain Reaction

    5) Product Images from "Metronidazole Causes Skeletal Muscle Atrophy and Modulates Muscle Chronometabolism"

    Article Title: Metronidazole Causes Skeletal Muscle Atrophy and Modulates Muscle Chronometabolism

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms19082418

    Metronidazole disrupts skeletal muscle RNA epigenetics. Real-time quantitative PCR analysis of RNA m 6 A methyltransferases Mettl3 and Mettl14 expression in gastrocnemius muscle of metronidazole-treated and nontreated ( a ) SPF and ( b ) GF mice. N = 5 mice per group. Data presented as means ± SEM. Asterisks indicate statistically significant differences (*, p
    Figure Legend Snippet: Metronidazole disrupts skeletal muscle RNA epigenetics. Real-time quantitative PCR analysis of RNA m 6 A methyltransferases Mettl3 and Mettl14 expression in gastrocnemius muscle of metronidazole-treated and nontreated ( a ) SPF and ( b ) GF mice. N = 5 mice per group. Data presented as means ± SEM. Asterisks indicate statistically significant differences (*, p

    Techniques Used: Real-time Polymerase Chain Reaction, Expressing, Mouse Assay

    Metronidazole causes changes in the expression of skeletal muscle atrophy genes. Real-time quantitative PCR analysis of Hdac4 , myogenin , MuRF1 , atrogin1 , and FoxO3 expression in gastrocnemius of metronidazole-treated and nontreated ( a ) SPF and ( b ) GF mice. N = 5 mice per group. Data presented as means ± SEM. Asterisks indicate statistically significant differences (*, p
    Figure Legend Snippet: Metronidazole causes changes in the expression of skeletal muscle atrophy genes. Real-time quantitative PCR analysis of Hdac4 , myogenin , MuRF1 , atrogin1 , and FoxO3 expression in gastrocnemius of metronidazole-treated and nontreated ( a ) SPF and ( b ) GF mice. N = 5 mice per group. Data presented as means ± SEM. Asterisks indicate statistically significant differences (*, p

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction, Mouse Assay

    Metronidazole affects the expression of skeletal muscle metabolism genes. Real-time quantitative PCR analysis of FoxO1 and Pdk4 expression in gastrocnemius of metronidazole-treated and nontreated ( a ) SPF and ( b ) GF mice. N = 5 mice per group. Data presented as means ± SEM. Asterisks indicate statistically significant differences (*, p
    Figure Legend Snippet: Metronidazole affects the expression of skeletal muscle metabolism genes. Real-time quantitative PCR analysis of FoxO1 and Pdk4 expression in gastrocnemius of metronidazole-treated and nontreated ( a ) SPF and ( b ) GF mice. N = 5 mice per group. Data presented as means ± SEM. Asterisks indicate statistically significant differences (*, p

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction, Mouse Assay

    Metronidazole alters skeletal muscle core clock and clock effector gene expression. Real-time quantitative PCR analysis of Bmal1 , Per2 , Cry2 , Ror - β , and E4BP4 expression in gastrocnemius of metronidazole-treated and nontreated ( a ) SPF and ( b ) GF mice. N = 5 mice per group. Data presented as means ± SEM. Asterisks indicate statistically significant differences (*, p
    Figure Legend Snippet: Metronidazole alters skeletal muscle core clock and clock effector gene expression. Real-time quantitative PCR analysis of Bmal1 , Per2 , Cry2 , Ror - β , and E4BP4 expression in gastrocnemius of metronidazole-treated and nontreated ( a ) SPF and ( b ) GF mice. N = 5 mice per group. Data presented as means ± SEM. Asterisks indicate statistically significant differences (*, p

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction, Mouse Assay

    Metronidazole modulates skeletal muscle adiponectin and PPARγ expression. Real-time quantitative PCR analysis of PPARγ and adiponectin expression in gastrocnemius muscle of metronidazole-treated and nontreated ( a ) SPF ( a ) and ( b ) GF mice. N = 5 mice per group. Data presented as means ± SEM. Asterisks indicate statistically significant differences (*, p
    Figure Legend Snippet: Metronidazole modulates skeletal muscle adiponectin and PPARγ expression. Real-time quantitative PCR analysis of PPARγ and adiponectin expression in gastrocnemius muscle of metronidazole-treated and nontreated ( a ) SPF ( a ) and ( b ) GF mice. N = 5 mice per group. Data presented as means ± SEM. Asterisks indicate statistically significant differences (*, p

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction, Mouse Assay

    6) Product Images from "A New STAT3-binding Partner, ARL3, Enhances the Phosphorylation and Nuclear Accumulation of STAT3 *"

    Article Title: A New STAT3-binding Partner, ARL3, Enhances the Phosphorylation and Nuclear Accumulation of STAT3 *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M116.724849

    ARL3 regulates the STAT3-mediated cell growth in BaF-G133 cells. A, BaF-G133 cells were transfected with control and muARL3 siRNAs (20 pmol). Total RNA was extracted at 48 h post-transfection, and target mRNA levels were quantified by qPCR. Data represent the levels of target mRNA normalized to that of a G3PDH internal control and are expressed relative to the value of control siRNA-treated samples. The results are representative of three independent experiments, and the error bars represent the S.D. ***, p
    Figure Legend Snippet: ARL3 regulates the STAT3-mediated cell growth in BaF-G133 cells. A, BaF-G133 cells were transfected with control and muARL3 siRNAs (20 pmol). Total RNA was extracted at 48 h post-transfection, and target mRNA levels were quantified by qPCR. Data represent the levels of target mRNA normalized to that of a G3PDH internal control and are expressed relative to the value of control siRNA-treated samples. The results are representative of three independent experiments, and the error bars represent the S.D. ***, p

    Techniques Used: Transfection, Real-time Polymerase Chain Reaction

    ARL3 enhances the transcriptional activity of STAT3 after IL-6-stimulation. A, HeLa cells in a 24-well plate were transfected with control, huARL3#1, or huARL3#2 siRNAs (20 pmol). Total RNA was extracted at 48 h post-transfection, and target mRNA levels were quantified by qPCR. Data represent the levels of target mRNA normalized to that of a G3PDH internal control and are expressed relative to the value of control siRNA-treated samples. The results are representative of three independent experiments, and the error bars represent the S.D. **, p
    Figure Legend Snippet: ARL3 enhances the transcriptional activity of STAT3 after IL-6-stimulation. A, HeLa cells in a 24-well plate were transfected with control, huARL3#1, or huARL3#2 siRNAs (20 pmol). Total RNA was extracted at 48 h post-transfection, and target mRNA levels were quantified by qPCR. Data represent the levels of target mRNA normalized to that of a G3PDH internal control and are expressed relative to the value of control siRNA-treated samples. The results are representative of three independent experiments, and the error bars represent the S.D. **, p

    Techniques Used: Activity Assay, Transfection, Real-time Polymerase Chain Reaction

    7) Product Images from "Role of N Terminus-Truncated NS1 Proteins of Influenza A Virus in Inhibiting IRF3 Activation"

    Article Title: Role of N Terminus-Truncated NS1 Proteins of Influenza A Virus in Inhibiting IRF3 Activation

    Journal: Journal of Virology

    doi: 10.1128/JVI.02843-15

    Mutations at the second and third in-frame AUG codons abolish the expression of the truncated NS1 protein in infected cells. A549 cells were infected with WT or mutated Ud viruses that express NS1 with codon 79 or 81 single mutations or 79 and 81 double mutations (A) or with WT or mutated PR8 viruses that are mutated at codons 79 and 81 in the NS1 protein (B) at an MOI of 2. At 3, 6, and 9 h postinfection, cell extracts were collected and analyzed by immunoblotting with anti-NS1 and anti-β actin antibodies.
    Figure Legend Snippet: Mutations at the second and third in-frame AUG codons abolish the expression of the truncated NS1 protein in infected cells. A549 cells were infected with WT or mutated Ud viruses that express NS1 with codon 79 or 81 single mutations or 79 and 81 double mutations (A) or with WT or mutated PR8 viruses that are mutated at codons 79 and 81 in the NS1 protein (B) at an MOI of 2. At 3, 6, and 9 h postinfection, cell extracts were collected and analyzed by immunoblotting with anti-NS1 and anti-β actin antibodies.

    Techniques Used: Expressing, Infection

    N-terminally truncated NS1 protein of PR8 virus inhibits IFN-β promoter activation. (A) 293T cells were cotransfected with a reporter plasmid containing IFN-β promoter fused to the firefly luciferase gene, a renilla luciferase control plasmid, a plasmid expressing WT or truncated NS1 protein (containing amino acids 79 to 230 derived from PR8 virus), and either the plasmid expressing Myc-tagged RIG-I CARD domains or an empty vector. At 24 h posttransfection, cell extracts were collected and analyzed by dual-luciferase activity assay. (B) 293T cells were cotransfected with the aforementioned luciferase reporters and the plasmid expressing PR8 WT or truncated NS1 for 18 h, followed by Sendai virus (SeV) infection for another 24 h. IFN-β promoter activity was determined by dual-luciferase activity assay. A549 cells that constitutionally express RFP or PR8 truncated NS1 were infected with Sendai virus at a final concentration of 13 HAU/ml (C) or with PR/M79.81I (D) or Ud/M79.81I (E) mutant virus at an MOI of 0.2. At 9 h postinfection, the activation of IRF3 was examined by anti-phosphorylated IRF3 antibody. The relative levels of IFN-β mRNA or pre-mRNA in the infected cells were analyzed by real-time RT-PCR. Protein expression was monitored by immunoblotting with anti-NS1, β-actin, c-Myc, or total IRF3 antibodies. *, P
    Figure Legend Snippet: N-terminally truncated NS1 protein of PR8 virus inhibits IFN-β promoter activation. (A) 293T cells were cotransfected with a reporter plasmid containing IFN-β promoter fused to the firefly luciferase gene, a renilla luciferase control plasmid, a plasmid expressing WT or truncated NS1 protein (containing amino acids 79 to 230 derived from PR8 virus), and either the plasmid expressing Myc-tagged RIG-I CARD domains or an empty vector. At 24 h posttransfection, cell extracts were collected and analyzed by dual-luciferase activity assay. (B) 293T cells were cotransfected with the aforementioned luciferase reporters and the plasmid expressing PR8 WT or truncated NS1 for 18 h, followed by Sendai virus (SeV) infection for another 24 h. IFN-β promoter activity was determined by dual-luciferase activity assay. A549 cells that constitutionally express RFP or PR8 truncated NS1 were infected with Sendai virus at a final concentration of 13 HAU/ml (C) or with PR/M79.81I (D) or Ud/M79.81I (E) mutant virus at an MOI of 0.2. At 9 h postinfection, the activation of IRF3 was examined by anti-phosphorylated IRF3 antibody. The relative levels of IFN-β mRNA or pre-mRNA in the infected cells were analyzed by real-time RT-PCR. Protein expression was monitored by immunoblotting with anti-NS1, β-actin, c-Myc, or total IRF3 antibodies. *, P

    Techniques Used: Activation Assay, Plasmid Preparation, Luciferase, Expressing, Derivative Assay, Activity Assay, Infection, Concentration Assay, Mutagenesis, Quantitative RT-PCR

    Mutations at the second and third in-frame AUG codons in NS1 ORF of PR8 virus increase IRF3 activation and IFN-β production in infected cells. A549 cells were infected with WT or mutated influenza A virus that expresses NS1 with mutations at the second and third AUG (codons 79 and 81, respectively) in the PR8 backbone (A and B) or Ud backbone (E and F) at an MOI of 2. (A and E) At 9 h postinfection, cell extracts were collected and analyzed by immunoblotting with anti-NS1, anti-phospho-IRF3 (serine 396), anti-total IRF3, and anti-β actin antibodies. (B and F) Relative amount of IFN-β mRNA of infected cells was quantitated by real-time RT-PCR. HEL-299 cells were infected with the WT or the PR8 virus mutated at codons 79 and 81 at an MOI of 2. (C) At 9 h postinfection, cell extracts were collected and analyzed for the proteins of NS1, phosphorylated IRF3, and total IRF3. (D) Relative amount of IFN-β pre-mRNA and mRNA of infected cells was quantitated by real-time RT-PCR. tNS1, truncated NS1 protein. *,  P
    Figure Legend Snippet: Mutations at the second and third in-frame AUG codons in NS1 ORF of PR8 virus increase IRF3 activation and IFN-β production in infected cells. A549 cells were infected with WT or mutated influenza A virus that expresses NS1 with mutations at the second and third AUG (codons 79 and 81, respectively) in the PR8 backbone (A and B) or Ud backbone (E and F) at an MOI of 2. (A and E) At 9 h postinfection, cell extracts were collected and analyzed by immunoblotting with anti-NS1, anti-phospho-IRF3 (serine 396), anti-total IRF3, and anti-β actin antibodies. (B and F) Relative amount of IFN-β mRNA of infected cells was quantitated by real-time RT-PCR. HEL-299 cells were infected with the WT or the PR8 virus mutated at codons 79 and 81 at an MOI of 2. (C) At 9 h postinfection, cell extracts were collected and analyzed for the proteins of NS1, phosphorylated IRF3, and total IRF3. (D) Relative amount of IFN-β pre-mRNA and mRNA of infected cells was quantitated by real-time RT-PCR. tNS1, truncated NS1 protein. *, P

    Techniques Used: Activation Assay, Infection, Quantitative RT-PCR

    8) Product Images from "Hypoxia and Macrophages Act in Concert Towards a Beneficial Outcome in Colon Cancer"

    Article Title: Hypoxia and Macrophages Act in Concert Towards a Beneficial Outcome in Colon Cancer

    Journal: Cancers

    doi: 10.3390/cancers12040818

    Hypoxia impacts colon cancer cell behavior, increasing invasion and glucose consumption. ( A – C ) Macrophages were indirectly co-cultured with RKO, at 20% or 1% O 2 for 72 h, and ( A ) proliferation of cancer cells was assessed for that period. Data are presented as percentage of proliferative cells. ( B ) AnnexinV/PI assay was used to quantify the percentage of viable (AnnexinV/PI negative), early apoptotic (AnnexinV positive/PI negative), and late apoptotic/necrotic (Annexin V/PI positive) cells. ( C ) Lactate production, glucose consumption, and pH measurements were made on co-cultures CM. Lactate/glucose ratio was calculated with the glucose and lactate concentration values of the same experiments. Cancer cells SLC2A1 and LDHA mRNA levels were measured by qRT–PCR. Relative expression changes are presented as fold variation SLC2A1 / ACTB and LDHA/ACTB relatively to 20% O 2 condition. ( D ) Cells maintained at 20% or 1% O 2 , and stimulated or not with macrophages previously maintained at 20% or 1% O 2 were cultured for 24 h on Matrigel coated filters, at 20% or 1% O 2 . Data are presented as fold variation in the number of invasive cells relative to RKO cells cultured at 20% O 2 without any stimulation with macrophages. Graphs represent the mean values with standard deviations, and are representative of n = 5 (proliferation assay); n = 6 (Annexin V/PI assay); n = 10 (glucose quantification); n = 11 (lactate quantification); n = 6 ( SLC2A1 ); n = 5 ( LDHA ); n = 9 (pH); n = 10 (invasion assay) independent experiments. The statistical tests Friedman, RM one-way ANOVA, Wilcox or paired t -test were used *** p
    Figure Legend Snippet: Hypoxia impacts colon cancer cell behavior, increasing invasion and glucose consumption. ( A – C ) Macrophages were indirectly co-cultured with RKO, at 20% or 1% O 2 for 72 h, and ( A ) proliferation of cancer cells was assessed for that period. Data are presented as percentage of proliferative cells. ( B ) AnnexinV/PI assay was used to quantify the percentage of viable (AnnexinV/PI negative), early apoptotic (AnnexinV positive/PI negative), and late apoptotic/necrotic (Annexin V/PI positive) cells. ( C ) Lactate production, glucose consumption, and pH measurements were made on co-cultures CM. Lactate/glucose ratio was calculated with the glucose and lactate concentration values of the same experiments. Cancer cells SLC2A1 and LDHA mRNA levels were measured by qRT–PCR. Relative expression changes are presented as fold variation SLC2A1 / ACTB and LDHA/ACTB relatively to 20% O 2 condition. ( D ) Cells maintained at 20% or 1% O 2 , and stimulated or not with macrophages previously maintained at 20% or 1% O 2 were cultured for 24 h on Matrigel coated filters, at 20% or 1% O 2 . Data are presented as fold variation in the number of invasive cells relative to RKO cells cultured at 20% O 2 without any stimulation with macrophages. Graphs represent the mean values with standard deviations, and are representative of n = 5 (proliferation assay); n = 6 (Annexin V/PI assay); n = 10 (glucose quantification); n = 11 (lactate quantification); n = 6 ( SLC2A1 ); n = 5 ( LDHA ); n = 9 (pH); n = 10 (invasion assay) independent experiments. The statistical tests Friedman, RM one-way ANOVA, Wilcox or paired t -test were used *** p

    Techniques Used: Cell Culture, Concentration Assay, Quantitative RT-PCR, Expressing, Proliferation Assay, Invasion Assay

    9) Product Images from "Tumor-suppressing effects of microRNA-612 in bladder cancer cells by targeting malic enzyme 1 expression"

    Article Title: Tumor-suppressing effects of microRNA-612 in bladder cancer cells by targeting malic enzyme 1 expression

    Journal: International Journal of Oncology

    doi: 10.3892/ijo.2018.4342

    ME1 knockdown mimicked the inhibitory effect of miR-612 overexpression in bladder cancer cells. (A) mRNA and (B) protein levels of ME1 in T24 cells following transfection with si-ME1 or si-NC were measured using reverse transcription-quantitative polymerase chain reaction and western blot analysis, respectively. GAPDH and β-actin were used as internal controls, respectively. (C) Cell proliferation, (D) colony formation, (E) Transwell tumor cell migration assay and (F) Transwell tumor cell invasion assays in bladder cancer T24 cells transfected with si-ME1 or si-NC. (G) Protein levels of EMT-associated markers in T24 cells transfected with si-ME1 or si-NC were evaluated by western blot analysis. * P
    Figure Legend Snippet: ME1 knockdown mimicked the inhibitory effect of miR-612 overexpression in bladder cancer cells. (A) mRNA and (B) protein levels of ME1 in T24 cells following transfection with si-ME1 or si-NC were measured using reverse transcription-quantitative polymerase chain reaction and western blot analysis, respectively. GAPDH and β-actin were used as internal controls, respectively. (C) Cell proliferation, (D) colony formation, (E) Transwell tumor cell migration assay and (F) Transwell tumor cell invasion assays in bladder cancer T24 cells transfected with si-ME1 or si-NC. (G) Protein levels of EMT-associated markers in T24 cells transfected with si-ME1 or si-NC were evaluated by western blot analysis. * P

    Techniques Used: Over Expression, Transfection, Real-time Polymerase Chain Reaction, Western Blot, Cell Migration Assay

    ME1 overexpression partially rescues the inhibitory effects of miR-612 expression on bladder cancer cells. (A) mRNA and (B) protein levels of ME1 in T24 cells co-transfected with miR-612 mimic or miR-NC, and with or without ME1 cDNA were measured using reverse transcription-quantitative polymerase chain reaction and western blot analysis, respectively. (C) Cell proliferation, (D) colony formation, (E) Transwell tumor cell migration and (F) Transwell tumor cell invasion assays in T24 cells co-transfected with miR-612 mimic or miR-NC, and with or without ME1 cDNA. * P
    Figure Legend Snippet: ME1 overexpression partially rescues the inhibitory effects of miR-612 expression on bladder cancer cells. (A) mRNA and (B) protein levels of ME1 in T24 cells co-transfected with miR-612 mimic or miR-NC, and with or without ME1 cDNA were measured using reverse transcription-quantitative polymerase chain reaction and western blot analysis, respectively. (C) Cell proliferation, (D) colony formation, (E) Transwell tumor cell migration and (F) Transwell tumor cell invasion assays in T24 cells co-transfected with miR-612 mimic or miR-NC, and with or without ME1 cDNA. * P

    Techniques Used: Over Expression, Expressing, Transfection, Real-time Polymerase Chain Reaction, Western Blot, Migration

    ME1 is a direct target of miR-612. (A) Bioinformatic analysis indicated that ME1 is the target of miR-612. The presumable miR-612-binding sites in the 3′-UTR of ME1 cDNA and the mutant binding sites are shown. (B) The relative luciferase activity was determined in T24 cells following co-transfection with a luciferase reporter plasmid (WT or MUT 3′-UTR ME1 cDNA), and miR-612 mimic or miR-NC. (C) mRNA and (D) protein levels of ME1 in T24 cells following transfection with miR-612 mimic or miR-NC were analyzed using RT-qPCR and western blot analysis, respectively. GAPDH and β-actin served as the internal controls, respectively. (E) The levels of ME1 mRNA were measured using RT-qPCR in 46 paired bladder cancer and ANT specimens. GAPDH was used as an internal control. (F) The correlation between ME1 and miR-612 expression in bladder cancer tissue samples (n=46) was analyzed using Spearman's correlation test. ** P
    Figure Legend Snippet: ME1 is a direct target of miR-612. (A) Bioinformatic analysis indicated that ME1 is the target of miR-612. The presumable miR-612-binding sites in the 3′-UTR of ME1 cDNA and the mutant binding sites are shown. (B) The relative luciferase activity was determined in T24 cells following co-transfection with a luciferase reporter plasmid (WT or MUT 3′-UTR ME1 cDNA), and miR-612 mimic or miR-NC. (C) mRNA and (D) protein levels of ME1 in T24 cells following transfection with miR-612 mimic or miR-NC were analyzed using RT-qPCR and western blot analysis, respectively. GAPDH and β-actin served as the internal controls, respectively. (E) The levels of ME1 mRNA were measured using RT-qPCR in 46 paired bladder cancer and ANT specimens. GAPDH was used as an internal control. (F) The correlation between ME1 and miR-612 expression in bladder cancer tissue samples (n=46) was analyzed using Spearman's correlation test. ** P

    Techniques Used: Binding Assay, Mutagenesis, Luciferase, Activity Assay, Cotransfection, Plasmid Preparation, Transfection, Quantitative RT-PCR, Western Blot, Expressing

    10) Product Images from "Phyllanthus emblica Fruit Extract Activates Spindle Assembly Checkpoint, Prevents Mitotic Aberrations and Genomic Instability in Human Colon Epithelial NCM460 Cells"

    Article Title: Phyllanthus emblica Fruit Extract Activates Spindle Assembly Checkpoint, Prevents Mitotic Aberrations and Genomic Instability in Human Colon Epithelial NCM460 Cells

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms17091437

    P. emblica (PE) treatment activated the spindle assembly checkpoint (SAC) and upregulated the expression of budding uninhibited by benzimidazoles related 1 ( BubR1 ) in NCM460 cells. ( A ) NCM460 cells were treated with indicated doses of PE or 300 ng/mL nocodozale (ND, positive control) for 4 h followed by recovery for 1 h before cells were harvested for determining anaphase-to-metaphase ratio (AMR), an estimated measurement of SAC activity. AMR was obtained by dividing the total number of anaphase cells by the total number of metaphase cells counted for each PE concentration; ( B ) NCM460 cells were treated with indicated doses of PE for 72 h, BubR1 (gray bars) and mitotic arrest deficient 2 ( Mad2 ; black bars) mRNA levels were measured by real-time quantitative polymerase chain reaction (PCR) after mRNA extraction and reserve transcription. Samples for each experimental group were run in triplicates and normalized to glyceraldehyde-3-phosphate dehydrogenase ( GAPDH ) mRNA levels. Results are expressed as fold increase in treated cells vs. untreated control. Values represent the means ± SEM of the fold induction taken from three independent experiments. * p
    Figure Legend Snippet: P. emblica (PE) treatment activated the spindle assembly checkpoint (SAC) and upregulated the expression of budding uninhibited by benzimidazoles related 1 ( BubR1 ) in NCM460 cells. ( A ) NCM460 cells were treated with indicated doses of PE or 300 ng/mL nocodozale (ND, positive control) for 4 h followed by recovery for 1 h before cells were harvested for determining anaphase-to-metaphase ratio (AMR), an estimated measurement of SAC activity. AMR was obtained by dividing the total number of anaphase cells by the total number of metaphase cells counted for each PE concentration; ( B ) NCM460 cells were treated with indicated doses of PE for 72 h, BubR1 (gray bars) and mitotic arrest deficient 2 ( Mad2 ; black bars) mRNA levels were measured by real-time quantitative polymerase chain reaction (PCR) after mRNA extraction and reserve transcription. Samples for each experimental group were run in triplicates and normalized to glyceraldehyde-3-phosphate dehydrogenase ( GAPDH ) mRNA levels. Results are expressed as fold increase in treated cells vs. untreated control. Values represent the means ± SEM of the fold induction taken from three independent experiments. * p

    Techniques Used: Expressing, Positive Control, Activity Assay, Concentration Assay, Real-time Polymerase Chain Reaction, Polymerase Chain Reaction

    11) Product Images from "Tumor-suppressing effects of microRNA-612 in bladder cancer cells by targeting malic enzyme 1 expression"

    Article Title: Tumor-suppressing effects of microRNA-612 in bladder cancer cells by targeting malic enzyme 1 expression

    Journal: International Journal of Oncology

    doi: 10.3892/ijo.2018.4342

    ME1 knockdown mimicked the inhibitory effect of miR-612 overexpression in bladder cancer cells. (A) mRNA and (B) protein levels of ME1 in T24 cells following transfection with si-ME1 or si-NC were measured using reverse transcription-quantitative polymerase chain reaction and western blot analysis, respectively. GAPDH and β-actin were used as internal controls, respectively. (C) Cell proliferation, (D) colony formation, (E) Transwell tumor cell migration assay and (F) Transwell tumor cell invasion assays in bladder cancer T24 cells transfected with si-ME1 or si-NC. (G) Protein levels of EMT-associated markers in T24 cells transfected with si-ME1 or si-NC were evaluated by western blot analysis. * P
    Figure Legend Snippet: ME1 knockdown mimicked the inhibitory effect of miR-612 overexpression in bladder cancer cells. (A) mRNA and (B) protein levels of ME1 in T24 cells following transfection with si-ME1 or si-NC were measured using reverse transcription-quantitative polymerase chain reaction and western blot analysis, respectively. GAPDH and β-actin were used as internal controls, respectively. (C) Cell proliferation, (D) colony formation, (E) Transwell tumor cell migration assay and (F) Transwell tumor cell invasion assays in bladder cancer T24 cells transfected with si-ME1 or si-NC. (G) Protein levels of EMT-associated markers in T24 cells transfected with si-ME1 or si-NC were evaluated by western blot analysis. * P

    Techniques Used: Over Expression, Transfection, Real-time Polymerase Chain Reaction, Western Blot, Cell Migration Assay

    ME1 overexpression partially rescues the inhibitory effects of miR-612 expression on bladder cancer cells. (A) mRNA and (B) protein levels of ME1 in T24 cells co-transfected with miR-612 mimic or miR-NC, and with or without ME1 cDNA were measured using reverse transcription-quantitative polymerase chain reaction and western blot analysis, respectively. (C) Cell proliferation, (D) colony formation, (E) Transwell tumor cell migration and (F) Transwell tumor cell invasion assays in T24 cells co-transfected with miR-612 mimic or miR-NC, and with or without ME1 cDNA. * P
    Figure Legend Snippet: ME1 overexpression partially rescues the inhibitory effects of miR-612 expression on bladder cancer cells. (A) mRNA and (B) protein levels of ME1 in T24 cells co-transfected with miR-612 mimic or miR-NC, and with or without ME1 cDNA were measured using reverse transcription-quantitative polymerase chain reaction and western blot analysis, respectively. (C) Cell proliferation, (D) colony formation, (E) Transwell tumor cell migration and (F) Transwell tumor cell invasion assays in T24 cells co-transfected with miR-612 mimic or miR-NC, and with or without ME1 cDNA. * P

    Techniques Used: Over Expression, Expressing, Transfection, Real-time Polymerase Chain Reaction, Western Blot, Migration

    ME1 is a direct target of miR-612. (A) Bioinformatic analysis indicated that ME1 is the target of miR-612. The presumable miR-612-binding sites in the 3′-UTR of ME1 cDNA and the mutant binding sites are shown. (B) The relative luciferase activity was determined in T24 cells following co-transfection with a luciferase reporter plasmid (WT or MUT 3′-UTR ME1 cDNA), and miR-612 mimic or miR-NC. (C) mRNA and (D) protein levels of ME1 in T24 cells following transfection with miR-612 mimic or miR-NC were analyzed using RT-qPCR and western blot analysis, respectively. GAPDH and β-actin served as the internal controls, respectively. (E) The levels of ME1 mRNA were measured using RT-qPCR in 46 paired bladder cancer and ANT specimens. GAPDH was used as an internal control. (F) The correlation between ME1 and miR-612 expression in bladder cancer tissue samples (n=46) was analyzed using Spearman's correlation test. ** P
    Figure Legend Snippet: ME1 is a direct target of miR-612. (A) Bioinformatic analysis indicated that ME1 is the target of miR-612. The presumable miR-612-binding sites in the 3′-UTR of ME1 cDNA and the mutant binding sites are shown. (B) The relative luciferase activity was determined in T24 cells following co-transfection with a luciferase reporter plasmid (WT or MUT 3′-UTR ME1 cDNA), and miR-612 mimic or miR-NC. (C) mRNA and (D) protein levels of ME1 in T24 cells following transfection with miR-612 mimic or miR-NC were analyzed using RT-qPCR and western blot analysis, respectively. GAPDH and β-actin served as the internal controls, respectively. (E) The levels of ME1 mRNA were measured using RT-qPCR in 46 paired bladder cancer and ANT specimens. GAPDH was used as an internal control. (F) The correlation between ME1 and miR-612 expression in bladder cancer tissue samples (n=46) was analyzed using Spearman's correlation test. ** P

    Techniques Used: Binding Assay, Mutagenesis, Luciferase, Activity Assay, Cotransfection, Plasmid Preparation, Transfection, Quantitative RT-PCR, Western Blot, Expressing

    12) Product Images from "Identification of the miRNA-mRNA regulatory network of small cell osteosarcoma based on RNA-seq"

    Article Title: Identification of the miRNA-mRNA regulatory network of small cell osteosarcoma based on RNA-seq

    Journal: Oncotarget

    doi: 10.18632/oncotarget.17208

    The expression levels of candidate mRNAs and miRNAs in peripheral blood samples and tumor tissues from patients with SCO were verified by qRT-PCR ( A ): hsa-miR-221-5p; ( B ): hsa-miR-26b-5p; ( C ): hsa-miR-21-5p; ( D ): hsa-miR-5706; ( E ): hsa-miR-656-3p; ( F ): RIF1; ( G ): FAM89A. Blood N: peripheral blood samples of healthy individuals; Blood C: peripheral blood samples of patients with SCO; Tissue N: adjacent non-tumor tissues of SCO patients; Tissue C: SCO tumor tissues. At least three independent experiments were performed for the statistical evaluation.
    Figure Legend Snippet: The expression levels of candidate mRNAs and miRNAs in peripheral blood samples and tumor tissues from patients with SCO were verified by qRT-PCR ( A ): hsa-miR-221-5p; ( B ): hsa-miR-26b-5p; ( C ): hsa-miR-21-5p; ( D ): hsa-miR-5706; ( E ): hsa-miR-656-3p; ( F ): RIF1; ( G ): FAM89A. Blood N: peripheral blood samples of healthy individuals; Blood C: peripheral blood samples of patients with SCO; Tissue N: adjacent non-tumor tissues of SCO patients; Tissue C: SCO tumor tissues. At least three independent experiments were performed for the statistical evaluation.

    Techniques Used: Expressing, Quantitative RT-PCR

    13) Product Images from "Identification of differentially expressed genes in the development of osteosarcoma using RNA-seq"

    Article Title: Identification of differentially expressed genes in the development of osteosarcoma using RNA-seq

    Journal: Oncotarget

    doi: 10.18632/oncotarget.13554

    The expression levels of DEGs between primary OS and healthy controls were verified by qRT-PCR ( A ): AURKB; ( B ): CD177; ( C ): ZDHHC19; ( D ): PPP2R2B; ( E ): CKMT2; ( F ): CEACAM8; ( G ): SMN1. NC represents tissues of normal control and OS represents osteosarcoma. At least three independent experiments were performed for statistical evaluation.
    Figure Legend Snippet: The expression levels of DEGs between primary OS and healthy controls were verified by qRT-PCR ( A ): AURKB; ( B ): CD177; ( C ): ZDHHC19; ( D ): PPP2R2B; ( E ): CKMT2; ( F ): CEACAM8; ( G ): SMN1. NC represents tissues of normal control and OS represents osteosarcoma. At least three independent experiments were performed for statistical evaluation.

    Techniques Used: Expressing, Quantitative RT-PCR

    The expression levels of DEGs between primary OS and metastatic OS were verified by qRT-PCR ( A ): ALAS2; ( B ): ISG15; ( C ): PPP2R2B; ( D ): BTRC; ( E ): CD177; ( F ) ZDHHC19. Primary OS represents primary osteosarcoma and metastatic OS represents metastatic osteosarcoma. At least three independent experiments were performed for statistical evaluation.
    Figure Legend Snippet: The expression levels of DEGs between primary OS and metastatic OS were verified by qRT-PCR ( A ): ALAS2; ( B ): ISG15; ( C ): PPP2R2B; ( D ): BTRC; ( E ): CD177; ( F ) ZDHHC19. Primary OS represents primary osteosarcoma and metastatic OS represents metastatic osteosarcoma. At least three independent experiments were performed for statistical evaluation.

    Techniques Used: Expressing, Quantitative RT-PCR

    14) Product Images from "Role of N Terminus-Truncated NS1 Proteins of Influenza A Virus in Inhibiting IRF3 Activation"

    Article Title: Role of N Terminus-Truncated NS1 Proteins of Influenza A Virus in Inhibiting IRF3 Activation

    Journal: Journal of Virology

    doi: 10.1128/JVI.02843-15

    N-terminally truncated NS1 protein of PR8 virus inhibits IFN-β promoter activation. (A) 293T cells were cotransfected with a reporter plasmid containing IFN-β promoter fused to the firefly luciferase gene, a renilla luciferase control plasmid, a plasmid expressing WT or truncated NS1 protein (containing amino acids 79 to 230 derived from PR8 virus), and either the plasmid expressing Myc-tagged RIG-I CARD domains or an empty vector. At 24 h posttransfection, cell extracts were collected and analyzed by dual-luciferase activity assay. (B) 293T cells were cotransfected with the aforementioned luciferase reporters and the plasmid expressing PR8 WT or truncated NS1 for 18 h, followed by Sendai virus (SeV) infection for another 24 h. IFN-β promoter activity was determined by dual-luciferase activity assay. A549 cells that constitutionally express RFP or PR8 truncated NS1 were infected with Sendai virus at a final concentration of 13 HAU/ml (C) or with PR/M79.81I (D) or Ud/M79.81I (E) mutant virus at an MOI of 0.2. At 9 h postinfection, the activation of IRF3 was examined by anti-phosphorylated IRF3 antibody. The relative levels of IFN-β mRNA or pre-mRNA in the infected cells were analyzed by real-time RT-PCR. Protein expression was monitored by immunoblotting with anti-NS1, β-actin, c-Myc, or total IRF3 antibodies. *, P
    Figure Legend Snippet: N-terminally truncated NS1 protein of PR8 virus inhibits IFN-β promoter activation. (A) 293T cells were cotransfected with a reporter plasmid containing IFN-β promoter fused to the firefly luciferase gene, a renilla luciferase control plasmid, a plasmid expressing WT or truncated NS1 protein (containing amino acids 79 to 230 derived from PR8 virus), and either the plasmid expressing Myc-tagged RIG-I CARD domains or an empty vector. At 24 h posttransfection, cell extracts were collected and analyzed by dual-luciferase activity assay. (B) 293T cells were cotransfected with the aforementioned luciferase reporters and the plasmid expressing PR8 WT or truncated NS1 for 18 h, followed by Sendai virus (SeV) infection for another 24 h. IFN-β promoter activity was determined by dual-luciferase activity assay. A549 cells that constitutionally express RFP or PR8 truncated NS1 were infected with Sendai virus at a final concentration of 13 HAU/ml (C) or with PR/M79.81I (D) or Ud/M79.81I (E) mutant virus at an MOI of 0.2. At 9 h postinfection, the activation of IRF3 was examined by anti-phosphorylated IRF3 antibody. The relative levels of IFN-β mRNA or pre-mRNA in the infected cells were analyzed by real-time RT-PCR. Protein expression was monitored by immunoblotting with anti-NS1, β-actin, c-Myc, or total IRF3 antibodies. *, P

    Techniques Used: Activation Assay, Plasmid Preparation, Luciferase, Expressing, Derivative Assay, Activity Assay, Infection, Concentration Assay, Mutagenesis, Quantitative RT-PCR

    Mutations at the second and third in-frame AUG codons in NS1 ORF of PR8 virus increase IRF3 activation and IFN-β production in infected cells. A549 cells were infected with WT or mutated influenza A virus that expresses NS1 with mutations at the second and third AUG (codons 79 and 81, respectively) in the PR8 backbone (A and B) or Ud backbone (E and F) at an MOI of 2. (A and E) At 9 h postinfection, cell extracts were collected and analyzed by immunoblotting with anti-NS1, anti-phospho-IRF3 (serine 396), anti-total IRF3, and anti-β actin antibodies. (B and F) Relative amount of IFN-β mRNA of infected cells was quantitated by real-time RT-PCR. HEL-299 cells were infected with the WT or the PR8 virus mutated at codons 79 and 81 at an MOI of 2. (C) At 9 h postinfection, cell extracts were collected and analyzed for the proteins of NS1, phosphorylated IRF3, and total IRF3. (D) Relative amount of IFN-β pre-mRNA and mRNA of infected cells was quantitated by real-time RT-PCR. tNS1, truncated NS1 protein. *, P
    Figure Legend Snippet: Mutations at the second and third in-frame AUG codons in NS1 ORF of PR8 virus increase IRF3 activation and IFN-β production in infected cells. A549 cells were infected with WT or mutated influenza A virus that expresses NS1 with mutations at the second and third AUG (codons 79 and 81, respectively) in the PR8 backbone (A and B) or Ud backbone (E and F) at an MOI of 2. (A and E) At 9 h postinfection, cell extracts were collected and analyzed by immunoblotting with anti-NS1, anti-phospho-IRF3 (serine 396), anti-total IRF3, and anti-β actin antibodies. (B and F) Relative amount of IFN-β mRNA of infected cells was quantitated by real-time RT-PCR. HEL-299 cells were infected with the WT or the PR8 virus mutated at codons 79 and 81 at an MOI of 2. (C) At 9 h postinfection, cell extracts were collected and analyzed for the proteins of NS1, phosphorylated IRF3, and total IRF3. (D) Relative amount of IFN-β pre-mRNA and mRNA of infected cells was quantitated by real-time RT-PCR. tNS1, truncated NS1 protein. *, P

    Techniques Used: Activation Assay, Infection, Quantitative RT-PCR

    15) Product Images from "Tumor-suppressing effects of microRNA-612 in bladder cancer cells by targeting malic enzyme 1 expression"

    Article Title: Tumor-suppressing effects of microRNA-612 in bladder cancer cells by targeting malic enzyme 1 expression

    Journal: International Journal of Oncology

    doi: 10.3892/ijo.2018.4342

    ME1 knockdown mimicked the inhibitory effect of miR-612 overexpression in bladder cancer cells. (A) mRNA and (B) protein levels of ME1 in T24 cells following transfection with si-ME1 or si-NC were measured using reverse transcription-quantitative polymerase chain reaction and western blot analysis, respectively. GAPDH and β-actin were used as internal controls, respectively. (C) Cell proliferation, (D) colony formation, (E) Transwell tumor cell migration assay and (F) Transwell tumor cell invasion assays in bladder cancer T24 cells transfected with si-ME1 or si-NC. (G) Protein levels of EMT-associated markers in T24 cells transfected with si-ME1 or si-NC were evaluated by western blot analysis. * P
    Figure Legend Snippet: ME1 knockdown mimicked the inhibitory effect of miR-612 overexpression in bladder cancer cells. (A) mRNA and (B) protein levels of ME1 in T24 cells following transfection with si-ME1 or si-NC were measured using reverse transcription-quantitative polymerase chain reaction and western blot analysis, respectively. GAPDH and β-actin were used as internal controls, respectively. (C) Cell proliferation, (D) colony formation, (E) Transwell tumor cell migration assay and (F) Transwell tumor cell invasion assays in bladder cancer T24 cells transfected with si-ME1 or si-NC. (G) Protein levels of EMT-associated markers in T24 cells transfected with si-ME1 or si-NC were evaluated by western blot analysis. * P

    Techniques Used: Over Expression, Transfection, Real-time Polymerase Chain Reaction, Western Blot, Cell Migration Assay

    ME1 overexpression partially rescues the inhibitory effects of miR-612 expression on bladder cancer cells. (A) mRNA and (B) protein levels of ME1 in T24 cells co-transfected with miR-612 mimic or miR-NC, and with or without ME1 cDNA were measured using reverse transcription-quantitative polymerase chain reaction and western blot analysis, respectively. (C) Cell proliferation, (D) colony formation, (E) Transwell tumor cell migration and (F) Transwell tumor cell invasion assays in T24 cells co-transfected with miR-612 mimic or miR-NC, and with or without ME1 cDNA. * P
    Figure Legend Snippet: ME1 overexpression partially rescues the inhibitory effects of miR-612 expression on bladder cancer cells. (A) mRNA and (B) protein levels of ME1 in T24 cells co-transfected with miR-612 mimic or miR-NC, and with or without ME1 cDNA were measured using reverse transcription-quantitative polymerase chain reaction and western blot analysis, respectively. (C) Cell proliferation, (D) colony formation, (E) Transwell tumor cell migration and (F) Transwell tumor cell invasion assays in T24 cells co-transfected with miR-612 mimic or miR-NC, and with or without ME1 cDNA. * P

    Techniques Used: Over Expression, Expressing, Transfection, Real-time Polymerase Chain Reaction, Western Blot, Migration

    ME1 is a direct target of miR-612. (A) Bioinformatic analysis indicated that ME1 is the target of miR-612. The presumable miR-612-binding sites in the 3′-UTR of ME1 cDNA and the mutant binding sites are shown. (B) The relative luciferase activity was determined in T24 cells following co-transfection with a luciferase reporter plasmid (WT or MUT 3′-UTR ME1 cDNA), and miR-612 mimic or miR-NC. (C) mRNA and (D) protein levels of ME1 in T24 cells following transfection with miR-612 mimic or miR-NC were analyzed using RT-qPCR and western blot analysis, respectively. GAPDH and β-actin served as the internal controls, respectively. (E) The levels of ME1 mRNA were measured using RT-qPCR in 46 paired bladder cancer and ANT specimens. GAPDH was used as an internal control. (F) The correlation between ME1 and miR-612 expression in bladder cancer tissue samples (n=46) was analyzed using Spearman's correlation test. ** P
    Figure Legend Snippet: ME1 is a direct target of miR-612. (A) Bioinformatic analysis indicated that ME1 is the target of miR-612. The presumable miR-612-binding sites in the 3′-UTR of ME1 cDNA and the mutant binding sites are shown. (B) The relative luciferase activity was determined in T24 cells following co-transfection with a luciferase reporter plasmid (WT or MUT 3′-UTR ME1 cDNA), and miR-612 mimic or miR-NC. (C) mRNA and (D) protein levels of ME1 in T24 cells following transfection with miR-612 mimic or miR-NC were analyzed using RT-qPCR and western blot analysis, respectively. GAPDH and β-actin served as the internal controls, respectively. (E) The levels of ME1 mRNA were measured using RT-qPCR in 46 paired bladder cancer and ANT specimens. GAPDH was used as an internal control. (F) The correlation between ME1 and miR-612 expression in bladder cancer tissue samples (n=46) was analyzed using Spearman's correlation test. ** P

    Techniques Used: Binding Assay, Mutagenesis, Luciferase, Activity Assay, Cotransfection, Plasmid Preparation, Transfection, Quantitative RT-PCR, Western Blot, Expressing

    16) Product Images from "Inhibitory Effects of a Novel PPAR-γ Agonist MEKT1 on Pomc Expression/ACTH Secretion in AtT20 Cells"

    Article Title: Inhibitory Effects of a Novel PPAR-γ Agonist MEKT1 on Pomc Expression/ACTH Secretion in AtT20 Cells

    Journal: PPAR Research

    doi: 10.1155/2018/5346272

    Effects of MEKT1 on the interaction between Nur77/Nurr1 and NurRE, Tpit and TpitRE, and Nur77 and NBRE on Pomc promoter (a) in AtT20 cells. Effects of MEKT1 on the interaction between Nur77/Nurr1 and NurRE (b), Tpit and TpitRE (c), and Nur77 and NBRE (d) on Pomc promoter examined by ChIP assay using NurRE, TpitRE, and NBRE primer. ChIP assay was carried out using digested chromatin extracted from the cells cultured in the presence of either 10 μ M MEKT1 or 0.1% DMSO (control) for 24 hours. Chromatin fragments were immunoprecipitated either by normal rabbit IgG (negative control), anti-Nur77/Nurr1 antibody, or anti-Tpit (anti TBX 19) antibody. Purified DNA was analyzed by qPCR using primers specific for NurRE, TpitRE, and NBRE containing sequence on Pomc promoter. The primer product sizes of NurRE, TpitRE, and NBRE were 211 bp, 146 bp, and 102 bp, respectively. Immunoprecipitated DNA was amplified by qPCR and then normalized to the values obtained after amplification of immunoprecipitated 1% input DNA. Data represent mean ± SEM ( n = 3). NS means “not significant.” ∗ P
    Figure Legend Snippet: Effects of MEKT1 on the interaction between Nur77/Nurr1 and NurRE, Tpit and TpitRE, and Nur77 and NBRE on Pomc promoter (a) in AtT20 cells. Effects of MEKT1 on the interaction between Nur77/Nurr1 and NurRE (b), Tpit and TpitRE (c), and Nur77 and NBRE (d) on Pomc promoter examined by ChIP assay using NurRE, TpitRE, and NBRE primer. ChIP assay was carried out using digested chromatin extracted from the cells cultured in the presence of either 10 μ M MEKT1 or 0.1% DMSO (control) for 24 hours. Chromatin fragments were immunoprecipitated either by normal rabbit IgG (negative control), anti-Nur77/Nurr1 antibody, or anti-Tpit (anti TBX 19) antibody. Purified DNA was analyzed by qPCR using primers specific for NurRE, TpitRE, and NBRE containing sequence on Pomc promoter. The primer product sizes of NurRE, TpitRE, and NBRE were 211 bp, 146 bp, and 102 bp, respectively. Immunoprecipitated DNA was amplified by qPCR and then normalized to the values obtained after amplification of immunoprecipitated 1% input DNA. Data represent mean ± SEM ( n = 3). NS means “not significant.” ∗ P

    Techniques Used: Chromatin Immunoprecipitation, Cell Culture, Immunoprecipitation, Negative Control, Purification, Real-time Polymerase Chain Reaction, Sequencing, Amplification

    17) Product Images from "Role of N Terminus-Truncated NS1 Proteins of Influenza A Virus in Inhibiting IRF3 Activation"

    Article Title: Role of N Terminus-Truncated NS1 Proteins of Influenza A Virus in Inhibiting IRF3 Activation

    Journal: Journal of Virology

    doi: 10.1128/JVI.02843-15

    N-terminally truncated NS1 protein of PR8 virus inhibits IFN-β promoter activation. (A) 293T cells were cotransfected with a reporter plasmid containing IFN-β promoter fused to the firefly luciferase gene, a renilla luciferase control plasmid, a plasmid expressing WT or truncated NS1 protein (containing amino acids 79 to 230 derived from PR8 virus), and either the plasmid expressing Myc-tagged RIG-I CARD domains or an empty vector. At 24 h posttransfection, cell extracts were collected and analyzed by dual-luciferase activity assay. (B) 293T cells were cotransfected with the aforementioned luciferase reporters and the plasmid expressing PR8 WT or truncated NS1 for 18 h, followed by Sendai virus (SeV) infection for another 24 h. IFN-β promoter activity was determined by dual-luciferase activity assay. A549 cells that constitutionally express RFP or PR8 truncated NS1 were infected with Sendai virus at a final concentration of 13 HAU/ml (C) or with PR/M79.81I (D) or Ud/M79.81I (E) mutant virus at an MOI of 0.2. At 9 h postinfection, the activation of IRF3 was examined by anti-phosphorylated IRF3 antibody. The relative levels of IFN-β mRNA or pre-mRNA in the infected cells were analyzed by real-time RT-PCR. Protein expression was monitored by immunoblotting with anti-NS1, β-actin, c-Myc, or total IRF3 antibodies. *, P
    Figure Legend Snippet: N-terminally truncated NS1 protein of PR8 virus inhibits IFN-β promoter activation. (A) 293T cells were cotransfected with a reporter plasmid containing IFN-β promoter fused to the firefly luciferase gene, a renilla luciferase control plasmid, a plasmid expressing WT or truncated NS1 protein (containing amino acids 79 to 230 derived from PR8 virus), and either the plasmid expressing Myc-tagged RIG-I CARD domains or an empty vector. At 24 h posttransfection, cell extracts were collected and analyzed by dual-luciferase activity assay. (B) 293T cells were cotransfected with the aforementioned luciferase reporters and the plasmid expressing PR8 WT or truncated NS1 for 18 h, followed by Sendai virus (SeV) infection for another 24 h. IFN-β promoter activity was determined by dual-luciferase activity assay. A549 cells that constitutionally express RFP or PR8 truncated NS1 were infected with Sendai virus at a final concentration of 13 HAU/ml (C) or with PR/M79.81I (D) or Ud/M79.81I (E) mutant virus at an MOI of 0.2. At 9 h postinfection, the activation of IRF3 was examined by anti-phosphorylated IRF3 antibody. The relative levels of IFN-β mRNA or pre-mRNA in the infected cells were analyzed by real-time RT-PCR. Protein expression was monitored by immunoblotting with anti-NS1, β-actin, c-Myc, or total IRF3 antibodies. *, P

    Techniques Used: Activation Assay, Plasmid Preparation, Luciferase, Expressing, Derivative Assay, Activity Assay, Infection, Concentration Assay, Mutagenesis, Quantitative RT-PCR

    Mutations at the second and third in-frame AUG codons in NS1 ORF of PR8 virus increase IRF3 activation and IFN-β production in infected cells. A549 cells were infected with WT or mutated influenza A virus that expresses NS1 with mutations at the second and third AUG (codons 79 and 81, respectively) in the PR8 backbone (A and B) or Ud backbone (E and F) at an MOI of 2. (A and E) At 9 h postinfection, cell extracts were collected and analyzed by immunoblotting with anti-NS1, anti-phospho-IRF3 (serine 396), anti-total IRF3, and anti-β actin antibodies. (B and F) Relative amount of IFN-β mRNA of infected cells was quantitated by real-time RT-PCR. HEL-299 cells were infected with the WT or the PR8 virus mutated at codons 79 and 81 at an MOI of 2. (C) At 9 h postinfection, cell extracts were collected and analyzed for the proteins of NS1, phosphorylated IRF3, and total IRF3. (D) Relative amount of IFN-β pre-mRNA and mRNA of infected cells was quantitated by real-time RT-PCR. tNS1, truncated NS1 protein. *, P
    Figure Legend Snippet: Mutations at the second and third in-frame AUG codons in NS1 ORF of PR8 virus increase IRF3 activation and IFN-β production in infected cells. A549 cells were infected with WT or mutated influenza A virus that expresses NS1 with mutations at the second and third AUG (codons 79 and 81, respectively) in the PR8 backbone (A and B) or Ud backbone (E and F) at an MOI of 2. (A and E) At 9 h postinfection, cell extracts were collected and analyzed by immunoblotting with anti-NS1, anti-phospho-IRF3 (serine 396), anti-total IRF3, and anti-β actin antibodies. (B and F) Relative amount of IFN-β mRNA of infected cells was quantitated by real-time RT-PCR. HEL-299 cells were infected with the WT or the PR8 virus mutated at codons 79 and 81 at an MOI of 2. (C) At 9 h postinfection, cell extracts were collected and analyzed for the proteins of NS1, phosphorylated IRF3, and total IRF3. (D) Relative amount of IFN-β pre-mRNA and mRNA of infected cells was quantitated by real-time RT-PCR. tNS1, truncated NS1 protein. *, P

    Techniques Used: Activation Assay, Infection, Quantitative RT-PCR

    18) Product Images from "MicroRNA-429/Cxcl1 Axis Protective Against Oxygen Glucose Deprivation/Reoxygenation-Induced Injury in Brain Microvascular Endothelial Cells"

    Article Title: MicroRNA-429/Cxcl1 Axis Protective Against Oxygen Glucose Deprivation/Reoxygenation-Induced Injury in Brain Microvascular Endothelial Cells

    Journal: Dose-Response

    doi: 10.1177/1559325820913785

    Effects of Cxcl1 and miR-429 on cell viability in OGD/R-treated BMECs. A, The cell viability in sham group, OGD/R-treated group, vector group, and overexpression of Cxcl1 group, ** P
    Figure Legend Snippet: Effects of Cxcl1 and miR-429 on cell viability in OGD/R-treated BMECs. A, The cell viability in sham group, OGD/R-treated group, vector group, and overexpression of Cxcl1 group, ** P

    Techniques Used: Plasmid Preparation, Over Expression

    MicroRNA-429 directly targets Cxcl1 in OGD/R-treated BMECs. A, Comparison of the sequences of miR-429 and Cxcl1. B, The fluorescence intensity of Cxcl1-WT and miR-429 mimic cotransected cells was markedly declined, ** P
    Figure Legend Snippet: MicroRNA-429 directly targets Cxcl1 in OGD/R-treated BMECs. A, Comparison of the sequences of miR-429 and Cxcl1. B, The fluorescence intensity of Cxcl1-WT and miR-429 mimic cotransected cells was markedly declined, ** P

    Techniques Used: Fluorescence

    The effect of miR-429/Cxcl1 axis on biological behavior of OGD/R-treated BMECs. A and B, Overexpression of miR-429 can significantly reduce the apoptotic rate of cells compared with the control group, whereas overexpression of Cxcl1 can significantly reverse this effect, ** P
    Figure Legend Snippet: The effect of miR-429/Cxcl1 axis on biological behavior of OGD/R-treated BMECs. A and B, Overexpression of miR-429 can significantly reduce the apoptotic rate of cells compared with the control group, whereas overexpression of Cxcl1 can significantly reverse this effect, ** P

    Techniques Used: Over Expression

    The expression level of Cxcl1 and miR-429 in OGD/R-treated BMECs. A, The expression level of Cxcl1 in I/R-2h group, I/R-8h group, and I/R-24 h group was significantly increased compared with the sham group, P
    Figure Legend Snippet: The expression level of Cxcl1 and miR-429 in OGD/R-treated BMECs. A, The expression level of Cxcl1 in I/R-2h group, I/R-8h group, and I/R-24 h group was significantly increased compared with the sham group, P

    Techniques Used: Expressing

    The transfect ion efficiency of Cxcl1 and miR-429 in OGD/R-treated BMECs. A, The expression of Cxcl1 in cells transected with pcDNA3.1-Cxcl1 or vector compared with control. ** P
    Figure Legend Snippet: The transfect ion efficiency of Cxcl1 and miR-429 in OGD/R-treated BMECs. A, The expression of Cxcl1 in cells transected with pcDNA3.1-Cxcl1 or vector compared with control. ** P

    Techniques Used: Expressing, Plasmid Preparation

    19) Product Images from "miR-125b inhibited epithelial–mesenchymal transition of triple-negative breast cancer by targeting MAP2K7"

    Article Title: miR-125b inhibited epithelial–mesenchymal transition of triple-negative breast cancer by targeting MAP2K7

    Journal: OncoTargets and therapy

    doi: 10.2147/OTT.S102713

    miR-125b targets 3′UTR of MAP2K7 and hinders EMT. Notes: ( A ) The binding site between miR-125b and MAP2K7 3′UTR was identified by TargetScan. The region of the MAP2K7 3′UTR that interacted with miR-125b is highlighted in red. ( B ) 293T cells were transfected with Wt reporter plasmid and miR-125b or control. After transfection for 48 hours, luciferase activity was measured by a dual luciferase reporter assay. The result is presented as Rluc/Fluc% (Renilla/Firefly luciferase), the Rluc/Fluc% of control group is set as 100%. Each bar represents the mean ± SD of three independent experiments; ** P
    Figure Legend Snippet: miR-125b targets 3′UTR of MAP2K7 and hinders EMT. Notes: ( A ) The binding site between miR-125b and MAP2K7 3′UTR was identified by TargetScan. The region of the MAP2K7 3′UTR that interacted with miR-125b is highlighted in red. ( B ) 293T cells were transfected with Wt reporter plasmid and miR-125b or control. After transfection for 48 hours, luciferase activity was measured by a dual luciferase reporter assay. The result is presented as Rluc/Fluc% (Renilla/Firefly luciferase), the Rluc/Fluc% of control group is set as 100%. Each bar represents the mean ± SD of three independent experiments; ** P

    Techniques Used: Binding Assay, Transfection, Plasmid Preparation, Luciferase, Activity Assay, Reporter Assay

    The correlation between MAP2K7 and miR-125b in TNBC specimens. Notes: ( A ) Shows the expression of MAP2K7 in clinical specimens by immunohistochemistry. Representative IHC photos (200×) of MAP2K7 expression in normal and TNBC specimens are presented. MAP2K7 staining was mainly localized within the cytoplasm of cells in the form of yellow brown granules. All sections were counterstained with hematoxylin and eosin. ( B ) Shows negative correlation between the expression of miR-125b and MAP2K7 in 23 TNBC samples ( r 2 =0.2465; P
    Figure Legend Snippet: The correlation between MAP2K7 and miR-125b in TNBC specimens. Notes: ( A ) Shows the expression of MAP2K7 in clinical specimens by immunohistochemistry. Representative IHC photos (200×) of MAP2K7 expression in normal and TNBC specimens are presented. MAP2K7 staining was mainly localized within the cytoplasm of cells in the form of yellow brown granules. All sections were counterstained with hematoxylin and eosin. ( B ) Shows negative correlation between the expression of miR-125b and MAP2K7 in 23 TNBC samples ( r 2 =0.2465; P

    Techniques Used: Expressing, Immunohistochemistry, Staining

    MAP2K7 overexpression partly rescues the EMT suppression of miR-125b. Notes: ( A – D ) Hs578T cells were transfected with blank or MAP2K7-overexpressing plasmid and miR-125b, or negative control (NC) and processed with ( A ) transwell migration assay, ( B ) transwell invasion assay, ( C ) qPCR, and ( D ) Western blot. * P
    Figure Legend Snippet: MAP2K7 overexpression partly rescues the EMT suppression of miR-125b. Notes: ( A – D ) Hs578T cells were transfected with blank or MAP2K7-overexpressing plasmid and miR-125b, or negative control (NC) and processed with ( A ) transwell migration assay, ( B ) transwell invasion assay, ( C ) qPCR, and ( D ) Western blot. * P

    Techniques Used: Over Expression, Transfection, Plasmid Preparation, Negative Control, Transwell Migration Assay, Transwell Invasion Assay, Real-time Polymerase Chain Reaction, Western Blot

    MAP2K7 depletion mimics the effect of miR-125b. Notes: ( A – C ) Hs578T cells were transfected with siMAP2K7 or negative control (NC) and then processed with ( A ) wound healing assay, ( B ) transwell migration assay, and ( C ) transwell invasion assay; *** P
    Figure Legend Snippet: MAP2K7 depletion mimics the effect of miR-125b. Notes: ( A – C ) Hs578T cells were transfected with siMAP2K7 or negative control (NC) and then processed with ( A ) wound healing assay, ( B ) transwell migration assay, and ( C ) transwell invasion assay; *** P

    Techniques Used: Transfection, Negative Control, Wound Healing Assay, Transwell Migration Assay, Transwell Invasion Assay

    20) Product Images from "Role of N Terminus-Truncated NS1 Proteins of Influenza A Virus in Inhibiting IRF3 Activation"

    Article Title: Role of N Terminus-Truncated NS1 Proteins of Influenza A Virus in Inhibiting IRF3 Activation

    Journal: Journal of Virology

    doi: 10.1128/JVI.02843-15

    N-terminally truncated NS1 protein of PR8 virus inhibits IFN-β promoter activation. (A) 293T cells were cotransfected with a reporter plasmid containing IFN-β promoter fused to the firefly luciferase gene, a renilla luciferase control plasmid, a plasmid expressing WT or truncated NS1 protein (containing amino acids 79 to 230 derived from PR8 virus), and either the plasmid expressing Myc-tagged RIG-I CARD domains or an empty vector. At 24 h posttransfection, cell extracts were collected and analyzed by dual-luciferase activity assay. (B) 293T cells were cotransfected with the aforementioned luciferase reporters and the plasmid expressing PR8 WT or truncated NS1 for 18 h, followed by Sendai virus (SeV) infection for another 24 h. IFN-β promoter activity was determined by dual-luciferase activity assay. A549 cells that constitutionally express RFP or PR8 truncated NS1 were infected with Sendai virus at a final concentration of 13 HAU/ml (C) or with PR/M79.81I (D) or Ud/M79.81I (E) mutant virus at an MOI of 0.2. At 9 h postinfection, the activation of IRF3 was examined by anti-phosphorylated IRF3 antibody. The relative levels of IFN-β mRNA or pre-mRNA in the infected cells were analyzed by real-time RT-PCR. Protein expression was monitored by immunoblotting with anti-NS1, β-actin, c-Myc, or total IRF3 antibodies. *, P
    Figure Legend Snippet: N-terminally truncated NS1 protein of PR8 virus inhibits IFN-β promoter activation. (A) 293T cells were cotransfected with a reporter plasmid containing IFN-β promoter fused to the firefly luciferase gene, a renilla luciferase control plasmid, a plasmid expressing WT or truncated NS1 protein (containing amino acids 79 to 230 derived from PR8 virus), and either the plasmid expressing Myc-tagged RIG-I CARD domains or an empty vector. At 24 h posttransfection, cell extracts were collected and analyzed by dual-luciferase activity assay. (B) 293T cells were cotransfected with the aforementioned luciferase reporters and the plasmid expressing PR8 WT or truncated NS1 for 18 h, followed by Sendai virus (SeV) infection for another 24 h. IFN-β promoter activity was determined by dual-luciferase activity assay. A549 cells that constitutionally express RFP or PR8 truncated NS1 were infected with Sendai virus at a final concentration of 13 HAU/ml (C) or with PR/M79.81I (D) or Ud/M79.81I (E) mutant virus at an MOI of 0.2. At 9 h postinfection, the activation of IRF3 was examined by anti-phosphorylated IRF3 antibody. The relative levels of IFN-β mRNA or pre-mRNA in the infected cells were analyzed by real-time RT-PCR. Protein expression was monitored by immunoblotting with anti-NS1, β-actin, c-Myc, or total IRF3 antibodies. *, P

    Techniques Used: Activation Assay, Plasmid Preparation, Luciferase, Expressing, Derivative Assay, Activity Assay, Infection, Concentration Assay, Mutagenesis, Quantitative RT-PCR

    Mutations at the second and third in-frame AUG codons in NS1 ORF of PR8 virus increase IRF3 activation and IFN-β production in infected cells. A549 cells were infected with WT or mutated influenza A virus that expresses NS1 with mutations at the second and third AUG (codons 79 and 81, respectively) in the PR8 backbone (A and B) or Ud backbone (E and F) at an MOI of 2. (A and E) At 9 h postinfection, cell extracts were collected and analyzed by immunoblotting with anti-NS1, anti-phospho-IRF3 (serine 396), anti-total IRF3, and anti-β actin antibodies. (B and F) Relative amount of IFN-β mRNA of infected cells was quantitated by real-time RT-PCR. HEL-299 cells were infected with the WT or the PR8 virus mutated at codons 79 and 81 at an MOI of 2. (C) At 9 h postinfection, cell extracts were collected and analyzed for the proteins of NS1, phosphorylated IRF3, and total IRF3. (D) Relative amount of IFN-β pre-mRNA and mRNA of infected cells was quantitated by real-time RT-PCR. tNS1, truncated NS1 protein. *, P
    Figure Legend Snippet: Mutations at the second and third in-frame AUG codons in NS1 ORF of PR8 virus increase IRF3 activation and IFN-β production in infected cells. A549 cells were infected with WT or mutated influenza A virus that expresses NS1 with mutations at the second and third AUG (codons 79 and 81, respectively) in the PR8 backbone (A and B) or Ud backbone (E and F) at an MOI of 2. (A and E) At 9 h postinfection, cell extracts were collected and analyzed by immunoblotting with anti-NS1, anti-phospho-IRF3 (serine 396), anti-total IRF3, and anti-β actin antibodies. (B and F) Relative amount of IFN-β mRNA of infected cells was quantitated by real-time RT-PCR. HEL-299 cells were infected with the WT or the PR8 virus mutated at codons 79 and 81 at an MOI of 2. (C) At 9 h postinfection, cell extracts were collected and analyzed for the proteins of NS1, phosphorylated IRF3, and total IRF3. (D) Relative amount of IFN-β pre-mRNA and mRNA of infected cells was quantitated by real-time RT-PCR. tNS1, truncated NS1 protein. *, P

    Techniques Used: Activation Assay, Infection, Quantitative RT-PCR

    21) Product Images from "Role of N Terminus-Truncated NS1 Proteins of Influenza A Virus in Inhibiting IRF3 Activation"

    Article Title: Role of N Terminus-Truncated NS1 Proteins of Influenza A Virus in Inhibiting IRF3 Activation

    Journal: Journal of Virology

    doi: 10.1128/JVI.02843-15

    N-terminally truncated NS1 protein of PR8 virus inhibits IFN-β promoter activation. (A) 293T cells were cotransfected with a reporter plasmid containing IFN-β promoter fused to the firefly luciferase gene, a renilla luciferase control plasmid, a plasmid expressing WT or truncated NS1 protein (containing amino acids 79 to 230 derived from PR8 virus), and either the plasmid expressing Myc-tagged RIG-I CARD domains or an empty vector. At 24 h posttransfection, cell extracts were collected and analyzed by dual-luciferase activity assay. (B) 293T cells were cotransfected with the aforementioned luciferase reporters and the plasmid expressing PR8 WT or truncated NS1 for 18 h, followed by Sendai virus (SeV) infection for another 24 h. IFN-β promoter activity was determined by dual-luciferase activity assay. A549 cells that constitutionally express RFP or PR8 truncated NS1 were infected with Sendai virus at a final concentration of 13 HAU/ml (C) or with PR/M79.81I (D) or Ud/M79.81I (E) mutant virus at an MOI of 0.2. At 9 h postinfection, the activation of IRF3 was examined by anti-phosphorylated IRF3 antibody. The relative levels of IFN-β mRNA or pre-mRNA in the infected cells were analyzed by real-time RT-PCR. Protein expression was monitored by immunoblotting with anti-NS1, β-actin, c-Myc, or total IRF3 antibodies. *, P
    Figure Legend Snippet: N-terminally truncated NS1 protein of PR8 virus inhibits IFN-β promoter activation. (A) 293T cells were cotransfected with a reporter plasmid containing IFN-β promoter fused to the firefly luciferase gene, a renilla luciferase control plasmid, a plasmid expressing WT or truncated NS1 protein (containing amino acids 79 to 230 derived from PR8 virus), and either the plasmid expressing Myc-tagged RIG-I CARD domains or an empty vector. At 24 h posttransfection, cell extracts were collected and analyzed by dual-luciferase activity assay. (B) 293T cells were cotransfected with the aforementioned luciferase reporters and the plasmid expressing PR8 WT or truncated NS1 for 18 h, followed by Sendai virus (SeV) infection for another 24 h. IFN-β promoter activity was determined by dual-luciferase activity assay. A549 cells that constitutionally express RFP or PR8 truncated NS1 were infected with Sendai virus at a final concentration of 13 HAU/ml (C) or with PR/M79.81I (D) or Ud/M79.81I (E) mutant virus at an MOI of 0.2. At 9 h postinfection, the activation of IRF3 was examined by anti-phosphorylated IRF3 antibody. The relative levels of IFN-β mRNA or pre-mRNA in the infected cells were analyzed by real-time RT-PCR. Protein expression was monitored by immunoblotting with anti-NS1, β-actin, c-Myc, or total IRF3 antibodies. *, P

    Techniques Used: Activation Assay, Plasmid Preparation, Luciferase, Expressing, Derivative Assay, Activity Assay, Infection, Concentration Assay, Mutagenesis, Quantitative RT-PCR

    Mutations at the second and third in-frame AUG codons in NS1 ORF of PR8 virus increase IRF3 activation and IFN-β production in infected cells. A549 cells were infected with WT or mutated influenza A virus that expresses NS1 with mutations at the second and third AUG (codons 79 and 81, respectively) in the PR8 backbone (A and B) or Ud backbone (E and F) at an MOI of 2. (A and E) At 9 h postinfection, cell extracts were collected and analyzed by immunoblotting with anti-NS1, anti-phospho-IRF3 (serine 396), anti-total IRF3, and anti-β actin antibodies. (B and F) Relative amount of IFN-β mRNA of infected cells was quantitated by real-time RT-PCR. HEL-299 cells were infected with the WT or the PR8 virus mutated at codons 79 and 81 at an MOI of 2. (C) At 9 h postinfection, cell extracts were collected and analyzed for the proteins of NS1, phosphorylated IRF3, and total IRF3. (D) Relative amount of IFN-β pre-mRNA and mRNA of infected cells was quantitated by real-time RT-PCR. tNS1, truncated NS1 protein. *, P
    Figure Legend Snippet: Mutations at the second and third in-frame AUG codons in NS1 ORF of PR8 virus increase IRF3 activation and IFN-β production in infected cells. A549 cells were infected with WT or mutated influenza A virus that expresses NS1 with mutations at the second and third AUG (codons 79 and 81, respectively) in the PR8 backbone (A and B) or Ud backbone (E and F) at an MOI of 2. (A and E) At 9 h postinfection, cell extracts were collected and analyzed by immunoblotting with anti-NS1, anti-phospho-IRF3 (serine 396), anti-total IRF3, and anti-β actin antibodies. (B and F) Relative amount of IFN-β mRNA of infected cells was quantitated by real-time RT-PCR. HEL-299 cells were infected with the WT or the PR8 virus mutated at codons 79 and 81 at an MOI of 2. (C) At 9 h postinfection, cell extracts were collected and analyzed for the proteins of NS1, phosphorylated IRF3, and total IRF3. (D) Relative amount of IFN-β pre-mRNA and mRNA of infected cells was quantitated by real-time RT-PCR. tNS1, truncated NS1 protein. *, P

    Techniques Used: Activation Assay, Infection, Quantitative RT-PCR

    22) Product Images from "Origin of fundus hyperautofluorescent spots and their role in retinal degeneration in a mouse model of Goldmann-Favre syndrome"

    Article Title: Origin of fundus hyperautofluorescent spots and their role in retinal degeneration in a mouse model of Goldmann-Favre syndrome

    Journal: Disease Models & Mechanisms

    doi: 10.1242/dmm.012112

    Relative gene expression after systemic depletion of circulating BM-derived microglia. Quantification of gene expression changes by qRT-PCR at P60 (4 weeks after being treated with AP20187 or vehicle; n =3 in each group), expressed as mean fold change in comparison with wild type (WT) ± 1 s.d., showing a trend of downregulation of the gene encoding the anti-inflammatory cytokine TGFβ1, and upregulation of genes encoding the inflammatory cytokines IL-1β, IL-6, TNFα and monocyte chemotactic protein (MCP-1). Asterisks indicate significant difference: * P
    Figure Legend Snippet: Relative gene expression after systemic depletion of circulating BM-derived microglia. Quantification of gene expression changes by qRT-PCR at P60 (4 weeks after being treated with AP20187 or vehicle; n =3 in each group), expressed as mean fold change in comparison with wild type (WT) ± 1 s.d., showing a trend of downregulation of the gene encoding the anti-inflammatory cytokine TGFβ1, and upregulation of genes encoding the inflammatory cytokines IL-1β, IL-6, TNFα and monocyte chemotactic protein (MCP-1). Asterisks indicate significant difference: * P

    Techniques Used: Expressing, Derivative Assay, Quantitative RT-PCR

    23) Product Images from "MIV-150/Zinc Acetate Gel Inhibits Cell-Associated Simian-Human Immunodeficiency Virus Reverse Transcriptase Infection in a Macaque Vaginal Explant Model"

    Article Title: MIV-150/Zinc Acetate Gel Inhibits Cell-Associated Simian-Human Immunodeficiency Virus Reverse Transcriptase Infection in a Macaque Vaginal Explant Model

    Journal: Antimicrobial Agents and Chemotherapy

    doi: 10.1128/AAC.00073-15

    De novo viral production in SHIV-RT-infected PBMCs is inhibited by mitomycin-C. Infected PBMCs were thawed, cultured overnight in the presence of IL-2, and then treated with mitomycin-C (versus untreated control PBMCs) and cultured for 14 days in the presence of IL-2 ( n = 3 replicates per condition). Supernatants were collected at days 0 (18 h after culture setup), 3, 7, 11, and 14, and replicates were pooled for analysis by qRT-PCR. Shown are the SIV gag copies per milliliter from two independent experiments.
    Figure Legend Snippet: De novo viral production in SHIV-RT-infected PBMCs is inhibited by mitomycin-C. Infected PBMCs were thawed, cultured overnight in the presence of IL-2, and then treated with mitomycin-C (versus untreated control PBMCs) and cultured for 14 days in the presence of IL-2 ( n = 3 replicates per condition). Supernatants were collected at days 0 (18 h after culture setup), 3, 7, 11, and 14, and replicates were pooled for analysis by qRT-PCR. Shown are the SIV gag copies per milliliter from two independent experiments.

    Techniques Used: Infection, Cell Culture, Quantitative RT-PCR

    Characterization of cell-associated SHIV-RT stock. (A) Total DNA and RNA were isolated from 10 6 infected PBMCs. SIV gag qPCR and qRT-PCR were run using 50 μg of DNA and RNA as the templates, respectively. Shown are calculated per cell SIV gag copies (single experiment). (B) Infected PBMCs were thawed and immediately used in the p27 ELISA. Shown is p27 content in lysed PBMCs (single experiment).
    Figure Legend Snippet: Characterization of cell-associated SHIV-RT stock. (A) Total DNA and RNA were isolated from 10 6 infected PBMCs. SIV gag qPCR and qRT-PCR were run using 50 μg of DNA and RNA as the templates, respectively. Shown are calculated per cell SIV gag copies (single experiment). (B) Infected PBMCs were thawed and immediately used in the p27 ELISA. Shown is p27 content in lysed PBMCs (single experiment).

    Techniques Used: Isolation, Infection, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay

    24) Product Images from "Tumor-suppressing effects of microRNA-612 in bladder cancer cells by targeting malic enzyme 1 expression"

    Article Title: Tumor-suppressing effects of microRNA-612 in bladder cancer cells by targeting malic enzyme 1 expression

    Journal: International Journal of Oncology

    doi: 10.3892/ijo.2018.4342

    ME1 knockdown mimicked the inhibitory effect of miR-612 overexpression in bladder cancer cells. (A) mRNA and (B) protein levels of ME1 in T24 cells following transfection with si-ME1 or si-NC were measured using reverse transcription-quantitative polymerase chain reaction and western blot analysis, respectively. GAPDH and β-actin were used as internal controls, respectively. (C) Cell proliferation, (D) colony formation, (E) Transwell tumor cell migration assay and (F) Transwell tumor cell invasion assays in bladder cancer T24 cells transfected with si-ME1 or si-NC. (G) Protein levels of EMT-associated markers in T24 cells transfected with si-ME1 or si-NC were evaluated by western blot analysis. * P
    Figure Legend Snippet: ME1 knockdown mimicked the inhibitory effect of miR-612 overexpression in bladder cancer cells. (A) mRNA and (B) protein levels of ME1 in T24 cells following transfection with si-ME1 or si-NC were measured using reverse transcription-quantitative polymerase chain reaction and western blot analysis, respectively. GAPDH and β-actin were used as internal controls, respectively. (C) Cell proliferation, (D) colony formation, (E) Transwell tumor cell migration assay and (F) Transwell tumor cell invasion assays in bladder cancer T24 cells transfected with si-ME1 or si-NC. (G) Protein levels of EMT-associated markers in T24 cells transfected with si-ME1 or si-NC were evaluated by western blot analysis. * P

    Techniques Used: Over Expression, Transfection, Real-time Polymerase Chain Reaction, Western Blot, Cell Migration Assay

    ME1 overexpression partially rescues the inhibitory effects of miR-612 expression on bladder cancer cells. (A) mRNA and (B) protein levels of ME1 in T24 cells co-transfected with miR-612 mimic or miR-NC, and with or without ME1 cDNA were measured using reverse transcription-quantitative polymerase chain reaction and western blot analysis, respectively. (C) Cell proliferation, (D) colony formation, (E) Transwell tumor cell migration and (F) Transwell tumor cell invasion assays in T24 cells co-transfected with miR-612 mimic or miR-NC, and with or without ME1 cDNA. * P
    Figure Legend Snippet: ME1 overexpression partially rescues the inhibitory effects of miR-612 expression on bladder cancer cells. (A) mRNA and (B) protein levels of ME1 in T24 cells co-transfected with miR-612 mimic or miR-NC, and with or without ME1 cDNA were measured using reverse transcription-quantitative polymerase chain reaction and western blot analysis, respectively. (C) Cell proliferation, (D) colony formation, (E) Transwell tumor cell migration and (F) Transwell tumor cell invasion assays in T24 cells co-transfected with miR-612 mimic or miR-NC, and with or without ME1 cDNA. * P

    Techniques Used: Over Expression, Expressing, Transfection, Real-time Polymerase Chain Reaction, Western Blot, Migration

    ME1 is a direct target of miR-612. (A) Bioinformatic analysis indicated that ME1 is the target of miR-612. The presumable miR-612-binding sites in the 3′-UTR of ME1 cDNA and the mutant binding sites are shown. (B) The relative luciferase activity was determined in T24 cells following co-transfection with a luciferase reporter plasmid (WT or MUT 3′-UTR ME1 cDNA), and miR-612 mimic or miR-NC. (C) mRNA and (D) protein levels of ME1 in T24 cells following transfection with miR-612 mimic or miR-NC were analyzed using RT-qPCR and western blot analysis, respectively. GAPDH and β-actin served as the internal controls, respectively. (E) The levels of ME1 mRNA were measured using RT-qPCR in 46 paired bladder cancer and ANT specimens. GAPDH was used as an internal control. (F) The correlation between ME1 and miR-612 expression in bladder cancer tissue samples (n=46) was analyzed using Spearman's correlation test. ** P
    Figure Legend Snippet: ME1 is a direct target of miR-612. (A) Bioinformatic analysis indicated that ME1 is the target of miR-612. The presumable miR-612-binding sites in the 3′-UTR of ME1 cDNA and the mutant binding sites are shown. (B) The relative luciferase activity was determined in T24 cells following co-transfection with a luciferase reporter plasmid (WT or MUT 3′-UTR ME1 cDNA), and miR-612 mimic or miR-NC. (C) mRNA and (D) protein levels of ME1 in T24 cells following transfection with miR-612 mimic or miR-NC were analyzed using RT-qPCR and western blot analysis, respectively. GAPDH and β-actin served as the internal controls, respectively. (E) The levels of ME1 mRNA were measured using RT-qPCR in 46 paired bladder cancer and ANT specimens. GAPDH was used as an internal control. (F) The correlation between ME1 and miR-612 expression in bladder cancer tissue samples (n=46) was analyzed using Spearman's correlation test. ** P

    Techniques Used: Binding Assay, Mutagenesis, Luciferase, Activity Assay, Cotransfection, Plasmid Preparation, Transfection, Quantitative RT-PCR, Western Blot, Expressing

    25) Product Images from "A New STAT3-binding Partner, ARL3, Enhances the Phosphorylation and Nuclear Accumulation of STAT3 *"

    Article Title: A New STAT3-binding Partner, ARL3, Enhances the Phosphorylation and Nuclear Accumulation of STAT3 *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M116.724849

    ARL3 regulates the STAT3-mediated cell growth in BaF-G133 cells. A, BaF-G133 cells were transfected with control and muARL3 siRNAs (20 pmol). Total RNA was extracted at 48 h post-transfection, and target mRNA levels were quantified by qPCR. Data represent the levels of target mRNA normalized to that of a G3PDH internal control and are expressed relative to the value of control siRNA-treated samples. The results are representative of three independent experiments, and the error bars represent the S.D. ***, p
    Figure Legend Snippet: ARL3 regulates the STAT3-mediated cell growth in BaF-G133 cells. A, BaF-G133 cells were transfected with control and muARL3 siRNAs (20 pmol). Total RNA was extracted at 48 h post-transfection, and target mRNA levels were quantified by qPCR. Data represent the levels of target mRNA normalized to that of a G3PDH internal control and are expressed relative to the value of control siRNA-treated samples. The results are representative of three independent experiments, and the error bars represent the S.D. ***, p

    Techniques Used: Transfection, Real-time Polymerase Chain Reaction

    ARL3 enhances the transcriptional activity of STAT3 after IL-6-stimulation. A, HeLa cells in a 24-well plate were transfected with control, huARL3#1, or huARL3#2 siRNAs (20 pmol). Total RNA was extracted at 48 h post-transfection, and target mRNA levels were quantified by qPCR. Data represent the levels of target mRNA normalized to that of a G3PDH internal control and are expressed relative to the value of control siRNA-treated samples. The results are representative of three independent experiments, and the error bars represent the S.D. **, p
    Figure Legend Snippet: ARL3 enhances the transcriptional activity of STAT3 after IL-6-stimulation. A, HeLa cells in a 24-well plate were transfected with control, huARL3#1, or huARL3#2 siRNAs (20 pmol). Total RNA was extracted at 48 h post-transfection, and target mRNA levels were quantified by qPCR. Data represent the levels of target mRNA normalized to that of a G3PDH internal control and are expressed relative to the value of control siRNA-treated samples. The results are representative of three independent experiments, and the error bars represent the S.D. **, p

    Techniques Used: Activity Assay, Transfection, Real-time Polymerase Chain Reaction

    26) Product Images from "CHUK/IKK-α loss in lung epithelial cells enhances NSCLC growth associated with HIF up-regulation"

    Article Title: CHUK/IKK-α loss in lung epithelial cells enhances NSCLC growth associated with HIF up-regulation

    Journal: Life Science Alliance

    doi: 10.26508/lsa.201900460

    Expression of direct HIF target genes. (A, B) qRT-PCR results showing up-regulation of direct HIF-1α target genes HK2 (A) and Scl2a1/Glut1 (B) in each of five independent IKKα KO urethane-induced large adenomas (L1–L5) compared with three independent IKKα WT control urethane-induced small lung adenomas. ** P
    Figure Legend Snippet: Expression of direct HIF target genes. (A, B) qRT-PCR results showing up-regulation of direct HIF-1α target genes HK2 (A) and Scl2a1/Glut1 (B) in each of five independent IKKα KO urethane-induced large adenomas (L1–L5) compared with three independent IKKα WT control urethane-induced small lung adenomas. ** P

    Techniques Used: Expressing, Quantitative RT-PCR

    Tamoxifen induction of CreER T2 in TMX-urethane–treated IKKα KO mouse lung suppresses CHUK/IKKα mRNA levels in large IKKα KO lung adenomas. (A) Representative X-gal–stained IKKα f/f :ROSA-fLacz:Sftpc-CreER T2 mouse lung after TMX and urethane IP injections verifying TMX-mediated CreER T2 activation with a LacZ-positive adenoma (AD) as indicated. (B) Reduced expression levels of CHUK/IKKα mRNA in five independent urethane-induced large adenomas (L1–L5) of tamoxifen-treated IKKα f/f :Sftpc-CreER T2 mice compared with the urethane-induced small adenomas in three independent IKKα WT control mice. The qRT-PCR was performed with primers that uniquely amplify mouse CHUK exons 6 and 7, which would be deleted by Cre-mediated lox P site recombination. 18S mouse rRNA was used as a reference control standard to correct for variations in PCR efficiency. *** P
    Figure Legend Snippet: Tamoxifen induction of CreER T2 in TMX-urethane–treated IKKα KO mouse lung suppresses CHUK/IKKα mRNA levels in large IKKα KO lung adenomas. (A) Representative X-gal–stained IKKα f/f :ROSA-fLacz:Sftpc-CreER T2 mouse lung after TMX and urethane IP injections verifying TMX-mediated CreER T2 activation with a LacZ-positive adenoma (AD) as indicated. (B) Reduced expression levels of CHUK/IKKα mRNA in five independent urethane-induced large adenomas (L1–L5) of tamoxifen-treated IKKα f/f :Sftpc-CreER T2 mice compared with the urethane-induced small adenomas in three independent IKKα WT control mice. The qRT-PCR was performed with primers that uniquely amplify mouse CHUK exons 6 and 7, which would be deleted by Cre-mediated lox P site recombination. 18S mouse rRNA was used as a reference control standard to correct for variations in PCR efficiency. *** P

    Techniques Used: Staining, Activation Assay, Expressing, Mouse Assay, Quantitative RT-PCR, Polymerase Chain Reaction

    27) Product Images from "Role of N Terminus-Truncated NS1 Proteins of Influenza A Virus in Inhibiting IRF3 Activation"

    Article Title: Role of N Terminus-Truncated NS1 Proteins of Influenza A Virus in Inhibiting IRF3 Activation

    Journal: Journal of Virology

    doi: 10.1128/JVI.02843-15

    Mutations at the second and third in-frame AUG codons abolish the expression of the truncated NS1 protein in infected cells. A549 cells were infected with WT or mutated Ud viruses that express NS1 with codon 79 or 81 single mutations or 79 and 81 double mutations (A) or with WT or mutated PR8 viruses that are mutated at codons 79 and 81 in the NS1 protein (B) at an MOI of 2. At 3, 6, and 9 h postinfection, cell extracts were collected and analyzed by immunoblotting with anti-NS1 and anti-β actin antibodies.
    Figure Legend Snippet: Mutations at the second and third in-frame AUG codons abolish the expression of the truncated NS1 protein in infected cells. A549 cells were infected with WT or mutated Ud viruses that express NS1 with codon 79 or 81 single mutations or 79 and 81 double mutations (A) or with WT or mutated PR8 viruses that are mutated at codons 79 and 81 in the NS1 protein (B) at an MOI of 2. At 3, 6, and 9 h postinfection, cell extracts were collected and analyzed by immunoblotting with anti-NS1 and anti-β actin antibodies.

    Techniques Used: Expressing, Infection

    N-terminally truncated NS1 protein of PR8 virus inhibits IFN-β promoter activation. (A) 293T cells were cotransfected with a reporter plasmid containing IFN-β promoter fused to the firefly luciferase gene, a renilla luciferase control plasmid, a plasmid expressing WT or truncated NS1 protein (containing amino acids 79 to 230 derived from PR8 virus), and either the plasmid expressing Myc-tagged RIG-I CARD domains or an empty vector. At 24 h posttransfection, cell extracts were collected and analyzed by dual-luciferase activity assay. (B) 293T cells were cotransfected with the aforementioned luciferase reporters and the plasmid expressing PR8 WT or truncated NS1 for 18 h, followed by Sendai virus (SeV) infection for another 24 h. IFN-β promoter activity was determined by dual-luciferase activity assay. A549 cells that constitutionally express RFP or PR8 truncated NS1 were infected with Sendai virus at a final concentration of 13 HAU/ml (C) or with PR/M79.81I (D) or Ud/M79.81I (E) mutant virus at an MOI of 0.2. At 9 h postinfection, the activation of IRF3 was examined by anti-phosphorylated IRF3 antibody. The relative levels of IFN-β mRNA or pre-mRNA in the infected cells were analyzed by real-time RT-PCR. Protein expression was monitored by immunoblotting with anti-NS1, β-actin, c-Myc, or total IRF3 antibodies. *, P
    Figure Legend Snippet: N-terminally truncated NS1 protein of PR8 virus inhibits IFN-β promoter activation. (A) 293T cells were cotransfected with a reporter plasmid containing IFN-β promoter fused to the firefly luciferase gene, a renilla luciferase control plasmid, a plasmid expressing WT or truncated NS1 protein (containing amino acids 79 to 230 derived from PR8 virus), and either the plasmid expressing Myc-tagged RIG-I CARD domains or an empty vector. At 24 h posttransfection, cell extracts were collected and analyzed by dual-luciferase activity assay. (B) 293T cells were cotransfected with the aforementioned luciferase reporters and the plasmid expressing PR8 WT or truncated NS1 for 18 h, followed by Sendai virus (SeV) infection for another 24 h. IFN-β promoter activity was determined by dual-luciferase activity assay. A549 cells that constitutionally express RFP or PR8 truncated NS1 were infected with Sendai virus at a final concentration of 13 HAU/ml (C) or with PR/M79.81I (D) or Ud/M79.81I (E) mutant virus at an MOI of 0.2. At 9 h postinfection, the activation of IRF3 was examined by anti-phosphorylated IRF3 antibody. The relative levels of IFN-β mRNA or pre-mRNA in the infected cells were analyzed by real-time RT-PCR. Protein expression was monitored by immunoblotting with anti-NS1, β-actin, c-Myc, or total IRF3 antibodies. *, P

    Techniques Used: Activation Assay, Plasmid Preparation, Luciferase, Expressing, Derivative Assay, Activity Assay, Infection, Concentration Assay, Mutagenesis, Quantitative RT-PCR

    Mutations at the second and third in-frame AUG codons in NS1 ORF of PR8 virus increase IRF3 activation and IFN-β production in infected cells. A549 cells were infected with WT or mutated influenza A virus that expresses NS1 with mutations at the second and third AUG (codons 79 and 81, respectively) in the PR8 backbone (A and B) or Ud backbone (E and F) at an MOI of 2. (A and E) At 9 h postinfection, cell extracts were collected and analyzed by immunoblotting with anti-NS1, anti-phospho-IRF3 (serine 396), anti-total IRF3, and anti-β actin antibodies. (B and F) Relative amount of IFN-β mRNA of infected cells was quantitated by real-time RT-PCR. HEL-299 cells were infected with the WT or the PR8 virus mutated at codons 79 and 81 at an MOI of 2. (C) At 9 h postinfection, cell extracts were collected and analyzed for the proteins of NS1, phosphorylated IRF3, and total IRF3. (D) Relative amount of IFN-β pre-mRNA and mRNA of infected cells was quantitated by real-time RT-PCR. tNS1, truncated NS1 protein. *,  P
    Figure Legend Snippet: Mutations at the second and third in-frame AUG codons in NS1 ORF of PR8 virus increase IRF3 activation and IFN-β production in infected cells. A549 cells were infected with WT or mutated influenza A virus that expresses NS1 with mutations at the second and third AUG (codons 79 and 81, respectively) in the PR8 backbone (A and B) or Ud backbone (E and F) at an MOI of 2. (A and E) At 9 h postinfection, cell extracts were collected and analyzed by immunoblotting with anti-NS1, anti-phospho-IRF3 (serine 396), anti-total IRF3, and anti-β actin antibodies. (B and F) Relative amount of IFN-β mRNA of infected cells was quantitated by real-time RT-PCR. HEL-299 cells were infected with the WT or the PR8 virus mutated at codons 79 and 81 at an MOI of 2. (C) At 9 h postinfection, cell extracts were collected and analyzed for the proteins of NS1, phosphorylated IRF3, and total IRF3. (D) Relative amount of IFN-β pre-mRNA and mRNA of infected cells was quantitated by real-time RT-PCR. tNS1, truncated NS1 protein. *, P

    Techniques Used: Activation Assay, Infection, Quantitative RT-PCR

    28) Product Images from "Delineation of the HPV11E6 and HPV18E6 Pathways in Initiating Cellular Transformation"

    Article Title: Delineation of the HPV11E6 and HPV18E6 Pathways in Initiating Cellular Transformation

    Journal: Frontiers in Oncology

    doi: 10.3389/fonc.2017.00258

    HPVE6 induces cellular transformation. (A) Quantitation of p21 and p53 mRNA in the adenovirus-HPVE6-infected cells as determined by qRT-PCR. A reduction in the mRNA levels of both genes in HPV18E6-infected HaCaT cells and the positive control (PC) HeLa cells was seen as opposed to the unchanged levels in the HPV11E6 infected cells. (B) Analysis of HPV18E6 (18 kDa), P21 (21 kDa), and P53 (53 kDa) protein levels by western blot analysis showed a marked reduction of both P53 and P21 in the HPV18E6 infected HaCaT cells but only a marginal decrease in the HPV11E6 cells. Uninfected HaCaT cells served as negative control (Nc) and HeLa cells are known to constitutively express the HPV18E6 protein served as PC. (C) The localization of HPV18E6 and P53 in HaCaT cells infected with HPV11E6 and HP18E6 was determined by confocal microscopy. In HPV11E6-expressing cells, p53 was localized primarily in the nucleus with some diffused staining in the cytoplasm, whereas in HPV18E6-expressing cells, p53 was observed at lower levels than either in the control or the HPV11E6 infected cells, with increased cytoplasmic staining. (D,E) Anchorage-independent cell growth as determined by colony formation in soft agar. Cells were plated at a density of 2.5 × 10 3 cells/well and colony formation determined after 21 days as described in the Section “ Materials and Methods .” Analysis of the colonies was done using the ImageJ software (33), with colonies larger than 150 μm in diameter being scored as positive. Graph shows mean number of colonies ( ± SE) obtained in three independent experiments, with each experiment performed in six replicate wells.
    Figure Legend Snippet: HPVE6 induces cellular transformation. (A) Quantitation of p21 and p53 mRNA in the adenovirus-HPVE6-infected cells as determined by qRT-PCR. A reduction in the mRNA levels of both genes in HPV18E6-infected HaCaT cells and the positive control (PC) HeLa cells was seen as opposed to the unchanged levels in the HPV11E6 infected cells. (B) Analysis of HPV18E6 (18 kDa), P21 (21 kDa), and P53 (53 kDa) protein levels by western blot analysis showed a marked reduction of both P53 and P21 in the HPV18E6 infected HaCaT cells but only a marginal decrease in the HPV11E6 cells. Uninfected HaCaT cells served as negative control (Nc) and HeLa cells are known to constitutively express the HPV18E6 protein served as PC. (C) The localization of HPV18E6 and P53 in HaCaT cells infected with HPV11E6 and HP18E6 was determined by confocal microscopy. In HPV11E6-expressing cells, p53 was localized primarily in the nucleus with some diffused staining in the cytoplasm, whereas in HPV18E6-expressing cells, p53 was observed at lower levels than either in the control or the HPV11E6 infected cells, with increased cytoplasmic staining. (D,E) Anchorage-independent cell growth as determined by colony formation in soft agar. Cells were plated at a density of 2.5 × 10 3 cells/well and colony formation determined after 21 days as described in the Section “ Materials and Methods .” Analysis of the colonies was done using the ImageJ software (33), with colonies larger than 150 μm in diameter being scored as positive. Graph shows mean number of colonies ( ± SE) obtained in three independent experiments, with each experiment performed in six replicate wells.

    Techniques Used: Transformation Assay, Quantitation Assay, Infection, Quantitative RT-PCR, Positive Control, Western Blot, Negative Control, Confocal Microscopy, Expressing, Staining, Software

    The effect of HPVE6 on cellular gene expression patterns. (A) A Venn diagram showing the number of significantly differentially expressed genes (DEGs) (both up- and downregulated). A total of 3,210 genes were significantly differentially expressed, of which 1,416 genes were uniquely deregulated in HPV18E6 cells, while 923 genes were uniquely deregulated in HPV11E6 cells and 871 genes were deregulated by both HPVs compared to the control HaCaT cells. (B) HPV11E6 and (C) HPV18E6, qRT-PCR validation of the microarray data showing the selected top 10 DEGs based on either the extent of up- or downregulation or on their known involvement in cellular transformation. The fold change shown was normalized to GAPDH mRNA levels and qRT-PCR figures represent the average of three replicates for each infection with the error bars indicating SD.
    Figure Legend Snippet: The effect of HPVE6 on cellular gene expression patterns. (A) A Venn diagram showing the number of significantly differentially expressed genes (DEGs) (both up- and downregulated). A total of 3,210 genes were significantly differentially expressed, of which 1,416 genes were uniquely deregulated in HPV18E6 cells, while 923 genes were uniquely deregulated in HPV11E6 cells and 871 genes were deregulated by both HPVs compared to the control HaCaT cells. (B) HPV11E6 and (C) HPV18E6, qRT-PCR validation of the microarray data showing the selected top 10 DEGs based on either the extent of up- or downregulation or on their known involvement in cellular transformation. The fold change shown was normalized to GAPDH mRNA levels and qRT-PCR figures represent the average of three replicates for each infection with the error bars indicating SD.

    Techniques Used: Expressing, Quantitative RT-PCR, Microarray, Transformation Assay, Infection

    29) Product Images from "miR-125b inhibited epithelial–mesenchymal transition of triple-negative breast cancer by targeting MAP2K7"

    Article Title: miR-125b inhibited epithelial–mesenchymal transition of triple-negative breast cancer by targeting MAP2K7

    Journal: OncoTargets and therapy

    doi: 10.2147/OTT.S102713

    miR-125b targets 3′UTR of MAP2K7 and hinders EMT. Notes: ( A ) The binding site between miR-125b and MAP2K7 3′UTR was identified by TargetScan. The region of the MAP2K7 3′UTR that interacted with miR-125b is highlighted in red. ( B ) 293T cells were transfected with Wt reporter plasmid and miR-125b or control. After transfection for 48 hours, luciferase activity was measured by a dual luciferase reporter assay. The result is presented as Rluc/Fluc% (Renilla/Firefly luciferase), the Rluc/Fluc% of control group is set as 100%. Each bar represents the mean ± SD of three independent experiments; ** P
    Figure Legend Snippet: miR-125b targets 3′UTR of MAP2K7 and hinders EMT. Notes: ( A ) The binding site between miR-125b and MAP2K7 3′UTR was identified by TargetScan. The region of the MAP2K7 3′UTR that interacted with miR-125b is highlighted in red. ( B ) 293T cells were transfected with Wt reporter plasmid and miR-125b or control. After transfection for 48 hours, luciferase activity was measured by a dual luciferase reporter assay. The result is presented as Rluc/Fluc% (Renilla/Firefly luciferase), the Rluc/Fluc% of control group is set as 100%. Each bar represents the mean ± SD of three independent experiments; ** P

    Techniques Used: Binding Assay, Transfection, Plasmid Preparation, Luciferase, Activity Assay, Reporter Assay

    The correlation between MAP2K7 and miR-125b in TNBC specimens. Notes: ( A ) Shows the expression of MAP2K7 in clinical specimens by immunohistochemistry. Representative IHC photos (200×) of MAP2K7 expression in normal and TNBC specimens are presented. MAP2K7 staining was mainly localized within the cytoplasm of cells in the form of yellow brown granules. All sections were counterstained with hematoxylin and eosin. ( B ) Shows negative correlation between the expression of miR-125b and MAP2K7 in 23 TNBC samples ( r 2 =0.2465; P
    Figure Legend Snippet: The correlation between MAP2K7 and miR-125b in TNBC specimens. Notes: ( A ) Shows the expression of MAP2K7 in clinical specimens by immunohistochemistry. Representative IHC photos (200×) of MAP2K7 expression in normal and TNBC specimens are presented. MAP2K7 staining was mainly localized within the cytoplasm of cells in the form of yellow brown granules. All sections were counterstained with hematoxylin and eosin. ( B ) Shows negative correlation between the expression of miR-125b and MAP2K7 in 23 TNBC samples ( r 2 =0.2465; P

    Techniques Used: Expressing, Immunohistochemistry, Staining

    miR-125b is downregulated in TNBC tissues. Notes: ( A ) The figure shows fold changes of the relative expression of miR-125b (T/N). miR-125b expression in TNBC samples (T, n=23) and adjacent breast cancer tissues (N, n=23) was analyzed by qPCR. U6 served as an internal control. The value of each sample was calculated as 2 −ΔΔCt : 2 −(Ct miR-125b-Ct U6) . The relative expression of miR-125b showed underexpression (T/N ≤0.5 fold) in 19 of the TNBC tissues. ( B ) The figure shows the distribution of miR-125b expression in clinical specimens. miR-125b relative expression showed underexpression (T/N ≤0.5 fold) in 83% of the TNBC tissues, overexpression (T/N ≥2 fold) in 4% of the TNBC tissues, and was unchanged (0.5
    Figure Legend Snippet: miR-125b is downregulated in TNBC tissues. Notes: ( A ) The figure shows fold changes of the relative expression of miR-125b (T/N). miR-125b expression in TNBC samples (T, n=23) and adjacent breast cancer tissues (N, n=23) was analyzed by qPCR. U6 served as an internal control. The value of each sample was calculated as 2 −ΔΔCt : 2 −(Ct miR-125b-Ct U6) . The relative expression of miR-125b showed underexpression (T/N ≤0.5 fold) in 19 of the TNBC tissues. ( B ) The figure shows the distribution of miR-125b expression in clinical specimens. miR-125b relative expression showed underexpression (T/N ≤0.5 fold) in 83% of the TNBC tissues, overexpression (T/N ≥2 fold) in 4% of the TNBC tissues, and was unchanged (0.5

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction, Over Expression

    miR-125b suppresses migration and invasion of TNBC cells. Notes: ( A ) The figure shows miR-125b relative expression in Hs578T cells expressing miR-125b. U6 was used as an internal control. The value of each sample was calculated as 2 −ΔΔCt and Student’s t -test was used. Data were present as mean ± SD, n=3, and *** P
    Figure Legend Snippet: miR-125b suppresses migration and invasion of TNBC cells. Notes: ( A ) The figure shows miR-125b relative expression in Hs578T cells expressing miR-125b. U6 was used as an internal control. The value of each sample was calculated as 2 −ΔΔCt and Student’s t -test was used. Data were present as mean ± SD, n=3, and *** P

    Techniques Used: Migration, Expressing

    MAP2K7 overexpression partly rescues the EMT suppression of miR-125b. Notes: ( A – D ) Hs578T cells were transfected with blank or MAP2K7-overexpressing plasmid and miR-125b, or negative control (NC) and processed with ( A ) transwell migration assay, ( B ) transwell invasion assay, ( C ) qPCR, and ( D ) Western blot. * P
    Figure Legend Snippet: MAP2K7 overexpression partly rescues the EMT suppression of miR-125b. Notes: ( A – D ) Hs578T cells were transfected with blank or MAP2K7-overexpressing plasmid and miR-125b, or negative control (NC) and processed with ( A ) transwell migration assay, ( B ) transwell invasion assay, ( C ) qPCR, and ( D ) Western blot. * P

    Techniques Used: Over Expression, Transfection, Plasmid Preparation, Negative Control, Transwell Migration Assay, Transwell Invasion Assay, Real-time Polymerase Chain Reaction, Western Blot

    MAP2K7 depletion mimics the effect of miR-125b. Notes: ( A – C ) Hs578T cells were transfected with siMAP2K7 or negative control (NC) and then processed with ( A ) wound healing assay, ( B ) transwell migration assay, and ( C ) transwell invasion assay; *** P
    Figure Legend Snippet: MAP2K7 depletion mimics the effect of miR-125b. Notes: ( A – C ) Hs578T cells were transfected with siMAP2K7 or negative control (NC) and then processed with ( A ) wound healing assay, ( B ) transwell migration assay, and ( C ) transwell invasion assay; *** P

    Techniques Used: Transfection, Negative Control, Wound Healing Assay, Transwell Migration Assay, Transwell Invasion Assay

    30) Product Images from "IL-34 promotes foam cell formation by enhancing CD36 expression through p38 MAPK pathway"

    Article Title: IL-34 promotes foam cell formation by enhancing CD36 expression through p38 MAPK pathway

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-35485-2

    IL-34 increased Dil-oxLDL uptake in BMDMs through up-regulation of expression of CD36. BMDMs were treated either with medium alone (control), with Dil-oxLDL (50 μg/ml), or with Dil-oxLDL (50 μg/ml) + IL-34 (50 ng/ml) for 4 hr at 37 °C followed by confocal microscopy ( a , b ) or flow cytometry ( c , d ) to determine the uptake of Dil-oxLDL. ( e ) Expression of scavenger receptors CD36, SR-A and Lox-1 protein in BMDMs by western blot. ( f ) Analysis of mRNA levels of CD36 was carried out by real-time quantitative PCR. For a neutralizing assay, BMDMs were pre-incubated with 10 μg/ml anti-CD36 antibody or normal goat IgG for 1 hr followed by treated with either Dil-oxLDL or Dil-oxLDL + IL-34. Dil-oxLDL uptake was determined by confocal microscopy ( g , h ) or flow cytometry ( i , j ). Data represent mean ± SD of n = 3 biologically independent experiments. * P
    Figure Legend Snippet: IL-34 increased Dil-oxLDL uptake in BMDMs through up-regulation of expression of CD36. BMDMs were treated either with medium alone (control), with Dil-oxLDL (50 μg/ml), or with Dil-oxLDL (50 μg/ml) + IL-34 (50 ng/ml) for 4 hr at 37 °C followed by confocal microscopy ( a , b ) or flow cytometry ( c , d ) to determine the uptake of Dil-oxLDL. ( e ) Expression of scavenger receptors CD36, SR-A and Lox-1 protein in BMDMs by western blot. ( f ) Analysis of mRNA levels of CD36 was carried out by real-time quantitative PCR. For a neutralizing assay, BMDMs were pre-incubated with 10 μg/ml anti-CD36 antibody or normal goat IgG for 1 hr followed by treated with either Dil-oxLDL or Dil-oxLDL + IL-34. Dil-oxLDL uptake was determined by confocal microscopy ( g , h ) or flow cytometry ( i , j ). Data represent mean ± SD of n = 3 biologically independent experiments. * P

    Techniques Used: Expressing, Confocal Microscopy, Flow Cytometry, Western Blot, Real-time Polymerase Chain Reaction, Neutralizing Assay, Incubation

    31) Product Images from "Origin of fundus hyperautofluorescent spots and their role in retinal degeneration in a mouse model of Goldmann-Favre syndrome"

    Article Title: Origin of fundus hyperautofluorescent spots and their role in retinal degeneration in a mouse model of Goldmann-Favre syndrome

    Journal: Disease Models & Mechanisms

    doi: 10.1242/dmm.012112

    Relative gene expression after systemic depletion of circulating BM-derived microglia. Quantification of gene expression changes by qRT-PCR at P60 (4 weeks after being treated with AP20187 or vehicle; n =3 in each group), expressed as mean fold change in comparison with wild type (WT) ± 1 s.d., showing a trend of downregulation of the gene encoding the anti-inflammatory cytokine TGFβ1, and upregulation of genes encoding the inflammatory cytokines IL-1β, IL-6, TNFα and monocyte chemotactic protein (MCP-1). Asterisks indicate significant difference: * P
    Figure Legend Snippet: Relative gene expression after systemic depletion of circulating BM-derived microglia. Quantification of gene expression changes by qRT-PCR at P60 (4 weeks after being treated with AP20187 or vehicle; n =3 in each group), expressed as mean fold change in comparison with wild type (WT) ± 1 s.d., showing a trend of downregulation of the gene encoding the anti-inflammatory cytokine TGFβ1, and upregulation of genes encoding the inflammatory cytokines IL-1β, IL-6, TNFα and monocyte chemotactic protein (MCP-1). Asterisks indicate significant difference: * P

    Techniques Used: Expressing, Derivative Assay, Quantitative RT-PCR

    32) Product Images from "Caspase-1 Promotes Epstein-Barr Virus Replication by Targeting the Large Tegument Protein Deneddylase to the Nucleus of Productively Infected Cells"

    Article Title: Caspase-1 Promotes Epstein-Barr Virus Replication by Targeting the Large Tegument Protein Deneddylase to the Nucleus of Productively Infected Cells

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1003664

    Inhibition of caspase-1 prevents the inactivation of nuclear CRLs and hampers EBV DNA replication. A. Caspase-1 is constitutively active in Akata-Bx1 and is further activated during virus replication but is not required for expression of the lytic cycle genes. Akata-Bx1 cells were induced in the presence or absence of caspase-1 inhibitors and western blots were probed as indicated. The asterisks in the caspase-1 blot indicate residual IgG heavy chains detected by the secondary antibody. One representative experiment out of three is shown. B. Inhibition of caspase-1 prevents the degradation of cullins and stabilization of CRL substrates. Western blots of cell lysates produced as described in Figure 7A were probed with the indicated antibodies. One representative experiment out of three is shown. C. Inhibition of caspase-1 hampers EBV DNA replication. The amount of viral DNA was measure by BZLF1 and EBNA1 specific Q-PCR in control induced Akata-Bx1 and cells induced in the presence of the indicated caspase-1 inhibitors. Mean ± SE of three experiments. D. The productive virus cycle was induced in the EBV positive B95.8 cell line by culture in medium supplemented with 2% FCS and 20 ng/ml TPA in the absence or presence of increasing concentrations of the caspase-1 inhibitor VX-765. The synthesis of viral DNA was monitored after 48 h by BZLF1 and EBNA1 specific Q-PCR. Mean ± SE of three experiments. E. Spent supernatants were collected after 2 weeks and the presence of infectious virus was measured by the induction of EBNA2 specific immunofluorescence in infected EBV negative BJAB cells. The mean % EBNA2 positive cells recorded in two independent experiments is shown.
    Figure Legend Snippet: Inhibition of caspase-1 prevents the inactivation of nuclear CRLs and hampers EBV DNA replication. A. Caspase-1 is constitutively active in Akata-Bx1 and is further activated during virus replication but is not required for expression of the lytic cycle genes. Akata-Bx1 cells were induced in the presence or absence of caspase-1 inhibitors and western blots were probed as indicated. The asterisks in the caspase-1 blot indicate residual IgG heavy chains detected by the secondary antibody. One representative experiment out of three is shown. B. Inhibition of caspase-1 prevents the degradation of cullins and stabilization of CRL substrates. Western blots of cell lysates produced as described in Figure 7A were probed with the indicated antibodies. One representative experiment out of three is shown. C. Inhibition of caspase-1 hampers EBV DNA replication. The amount of viral DNA was measure by BZLF1 and EBNA1 specific Q-PCR in control induced Akata-Bx1 and cells induced in the presence of the indicated caspase-1 inhibitors. Mean ± SE of three experiments. D. The productive virus cycle was induced in the EBV positive B95.8 cell line by culture in medium supplemented with 2% FCS and 20 ng/ml TPA in the absence or presence of increasing concentrations of the caspase-1 inhibitor VX-765. The synthesis of viral DNA was monitored after 48 h by BZLF1 and EBNA1 specific Q-PCR. Mean ± SE of three experiments. E. Spent supernatants were collected after 2 weeks and the presence of infectious virus was measured by the induction of EBNA2 specific immunofluorescence in infected EBV negative BJAB cells. The mean % EBNA2 positive cells recorded in two independent experiments is shown.

    Techniques Used: Inhibition, Expressing, Western Blot, Produced, Polymerase Chain Reaction, Immunofluorescence, Infection

    CAND1 rescues the degradation of cullins and inhibits virus replication. A . The degradation of cullins is rescued in cells transfected with the full-length and N-terminus of CAND1. The productive cycle was induced in Akata-Bx1 transfected with plasmids expressing the full-length, N-terminal or C-terminal domain of CAND1 or, as control, the empty vector. Twenty-four after transfection the cells were analyzed in western blots with the indicated antibodies. One representative experiment out of three is shown. B . CAND1 and the CAND1 N-terminus inhibit virus replication. The amount of EBV DNA was quantified by Q-PCR specific for unique sequence in the BZLF1 and EBNA1 genes. The mean ± SE of three independent experiments is shown.
    Figure Legend Snippet: CAND1 rescues the degradation of cullins and inhibits virus replication. A . The degradation of cullins is rescued in cells transfected with the full-length and N-terminus of CAND1. The productive cycle was induced in Akata-Bx1 transfected with plasmids expressing the full-length, N-terminal or C-terminal domain of CAND1 or, as control, the empty vector. Twenty-four after transfection the cells were analyzed in western blots with the indicated antibodies. One representative experiment out of three is shown. B . CAND1 and the CAND1 N-terminus inhibit virus replication. The amount of EBV DNA was quantified by Q-PCR specific for unique sequence in the BZLF1 and EBNA1 genes. The mean ± SE of three independent experiments is shown.

    Techniques Used: Transfection, Expressing, Plasmid Preparation, Western Blot, Polymerase Chain Reaction, Sequencing

    33) Product Images from "Role of N Terminus-Truncated NS1 Proteins of Influenza A Virus in Inhibiting IRF3 Activation"

    Article Title: Role of N Terminus-Truncated NS1 Proteins of Influenza A Virus in Inhibiting IRF3 Activation

    Journal: Journal of Virology

    doi: 10.1128/JVI.02843-15

    N-terminally truncated NS1 protein of PR8 virus inhibits IFN-β promoter activation. (A) 293T cells were cotransfected with a reporter plasmid containing IFN-β promoter fused to the firefly luciferase gene, a renilla luciferase control plasmid, a plasmid expressing WT or truncated NS1 protein (containing amino acids 79 to 230 derived from PR8 virus), and either the plasmid expressing Myc-tagged RIG-I CARD domains or an empty vector. At 24 h posttransfection, cell extracts were collected and analyzed by dual-luciferase activity assay. (B) 293T cells were cotransfected with the aforementioned luciferase reporters and the plasmid expressing PR8 WT or truncated NS1 for 18 h, followed by Sendai virus (SeV) infection for another 24 h. IFN-β promoter activity was determined by dual-luciferase activity assay. A549 cells that constitutionally express RFP or PR8 truncated NS1 were infected with Sendai virus at a final concentration of 13 HAU/ml (C) or with PR/M79.81I (D) or Ud/M79.81I (E) mutant virus at an MOI of 0.2. At 9 h postinfection, the activation of IRF3 was examined by anti-phosphorylated IRF3 antibody. The relative levels of IFN-β mRNA or pre-mRNA in the infected cells were analyzed by real-time RT-PCR. Protein expression was monitored by immunoblotting with anti-NS1, β-actin, c-Myc, or total IRF3 antibodies. *, P
    Figure Legend Snippet: N-terminally truncated NS1 protein of PR8 virus inhibits IFN-β promoter activation. (A) 293T cells were cotransfected with a reporter plasmid containing IFN-β promoter fused to the firefly luciferase gene, a renilla luciferase control plasmid, a plasmid expressing WT or truncated NS1 protein (containing amino acids 79 to 230 derived from PR8 virus), and either the plasmid expressing Myc-tagged RIG-I CARD domains or an empty vector. At 24 h posttransfection, cell extracts were collected and analyzed by dual-luciferase activity assay. (B) 293T cells were cotransfected with the aforementioned luciferase reporters and the plasmid expressing PR8 WT or truncated NS1 for 18 h, followed by Sendai virus (SeV) infection for another 24 h. IFN-β promoter activity was determined by dual-luciferase activity assay. A549 cells that constitutionally express RFP or PR8 truncated NS1 were infected with Sendai virus at a final concentration of 13 HAU/ml (C) or with PR/M79.81I (D) or Ud/M79.81I (E) mutant virus at an MOI of 0.2. At 9 h postinfection, the activation of IRF3 was examined by anti-phosphorylated IRF3 antibody. The relative levels of IFN-β mRNA or pre-mRNA in the infected cells were analyzed by real-time RT-PCR. Protein expression was monitored by immunoblotting with anti-NS1, β-actin, c-Myc, or total IRF3 antibodies. *, P

    Techniques Used: Activation Assay, Plasmid Preparation, Luciferase, Expressing, Derivative Assay, Activity Assay, Infection, Concentration Assay, Mutagenesis, Quantitative RT-PCR

    Mutations at the second and third in-frame AUG codons in NS1 ORF of PR8 virus increase IRF3 activation and IFN-β production in infected cells. A549 cells were infected with WT or mutated influenza A virus that expresses NS1 with mutations at the second and third AUG (codons 79 and 81, respectively) in the PR8 backbone (A and B) or Ud backbone (E and F) at an MOI of 2. (A and E) At 9 h postinfection, cell extracts were collected and analyzed by immunoblotting with anti-NS1, anti-phospho-IRF3 (serine 396), anti-total IRF3, and anti-β actin antibodies. (B and F) Relative amount of IFN-β mRNA of infected cells was quantitated by real-time RT-PCR. HEL-299 cells were infected with the WT or the PR8 virus mutated at codons 79 and 81 at an MOI of 2. (C) At 9 h postinfection, cell extracts were collected and analyzed for the proteins of NS1, phosphorylated IRF3, and total IRF3. (D) Relative amount of IFN-β pre-mRNA and mRNA of infected cells was quantitated by real-time RT-PCR. tNS1, truncated NS1 protein. *, P
    Figure Legend Snippet: Mutations at the second and third in-frame AUG codons in NS1 ORF of PR8 virus increase IRF3 activation and IFN-β production in infected cells. A549 cells were infected with WT or mutated influenza A virus that expresses NS1 with mutations at the second and third AUG (codons 79 and 81, respectively) in the PR8 backbone (A and B) or Ud backbone (E and F) at an MOI of 2. (A and E) At 9 h postinfection, cell extracts were collected and analyzed by immunoblotting with anti-NS1, anti-phospho-IRF3 (serine 396), anti-total IRF3, and anti-β actin antibodies. (B and F) Relative amount of IFN-β mRNA of infected cells was quantitated by real-time RT-PCR. HEL-299 cells were infected with the WT or the PR8 virus mutated at codons 79 and 81 at an MOI of 2. (C) At 9 h postinfection, cell extracts were collected and analyzed for the proteins of NS1, phosphorylated IRF3, and total IRF3. (D) Relative amount of IFN-β pre-mRNA and mRNA of infected cells was quantitated by real-time RT-PCR. tNS1, truncated NS1 protein. *, P

    Techniques Used: Activation Assay, Infection, Quantitative RT-PCR

    34) Product Images from "Role of N Terminus-Truncated NS1 Proteins of Influenza A Virus in Inhibiting IRF3 Activation"

    Article Title: Role of N Terminus-Truncated NS1 Proteins of Influenza A Virus in Inhibiting IRF3 Activation

    Journal: Journal of Virology

    doi: 10.1128/JVI.02843-15

    N-terminally truncated NS1 protein of PR8 virus inhibits IFN-β promoter activation. (A) 293T cells were cotransfected with a reporter plasmid containing IFN-β promoter fused to the firefly luciferase gene, a renilla luciferase control plasmid, a plasmid expressing WT or truncated NS1 protein (containing amino acids 79 to 230 derived from PR8 virus), and either the plasmid expressing Myc-tagged RIG-I CARD domains or an empty vector. At 24 h posttransfection, cell extracts were collected and analyzed by dual-luciferase activity assay. (B) 293T cells were cotransfected with the aforementioned luciferase reporters and the plasmid expressing PR8 WT or truncated NS1 for 18 h, followed by Sendai virus (SeV) infection for another 24 h. IFN-β promoter activity was determined by dual-luciferase activity assay. A549 cells that constitutionally express RFP or PR8 truncated NS1 were infected with Sendai virus at a final concentration of 13 HAU/ml (C) or with PR/M79.81I (D) or Ud/M79.81I (E) mutant virus at an MOI of 0.2. At 9 h postinfection, the activation of IRF3 was examined by anti-phosphorylated IRF3 antibody. The relative levels of IFN-β mRNA or pre-mRNA in the infected cells were analyzed by real-time RT-PCR. Protein expression was monitored by immunoblotting with anti-NS1, β-actin, c-Myc, or total IRF3 antibodies. *, P
    Figure Legend Snippet: N-terminally truncated NS1 protein of PR8 virus inhibits IFN-β promoter activation. (A) 293T cells were cotransfected with a reporter plasmid containing IFN-β promoter fused to the firefly luciferase gene, a renilla luciferase control plasmid, a plasmid expressing WT or truncated NS1 protein (containing amino acids 79 to 230 derived from PR8 virus), and either the plasmid expressing Myc-tagged RIG-I CARD domains or an empty vector. At 24 h posttransfection, cell extracts were collected and analyzed by dual-luciferase activity assay. (B) 293T cells were cotransfected with the aforementioned luciferase reporters and the plasmid expressing PR8 WT or truncated NS1 for 18 h, followed by Sendai virus (SeV) infection for another 24 h. IFN-β promoter activity was determined by dual-luciferase activity assay. A549 cells that constitutionally express RFP or PR8 truncated NS1 were infected with Sendai virus at a final concentration of 13 HAU/ml (C) or with PR/M79.81I (D) or Ud/M79.81I (E) mutant virus at an MOI of 0.2. At 9 h postinfection, the activation of IRF3 was examined by anti-phosphorylated IRF3 antibody. The relative levels of IFN-β mRNA or pre-mRNA in the infected cells were analyzed by real-time RT-PCR. Protein expression was monitored by immunoblotting with anti-NS1, β-actin, c-Myc, or total IRF3 antibodies. *, P

    Techniques Used: Activation Assay, Plasmid Preparation, Luciferase, Expressing, Derivative Assay, Activity Assay, Infection, Concentration Assay, Mutagenesis, Quantitative RT-PCR

    Mutations at the second and third in-frame AUG codons in NS1 ORF of PR8 virus increase IRF3 activation and IFN-β production in infected cells. A549 cells were infected with WT or mutated influenza A virus that expresses NS1 with mutations at the second and third AUG (codons 79 and 81, respectively) in the PR8 backbone (A and B) or Ud backbone (E and F) at an MOI of 2. (A and E) At 9 h postinfection, cell extracts were collected and analyzed by immunoblotting with anti-NS1, anti-phospho-IRF3 (serine 396), anti-total IRF3, and anti-β actin antibodies. (B and F) Relative amount of IFN-β mRNA of infected cells was quantitated by real-time RT-PCR. HEL-299 cells were infected with the WT or the PR8 virus mutated at codons 79 and 81 at an MOI of 2. (C) At 9 h postinfection, cell extracts were collected and analyzed for the proteins of NS1, phosphorylated IRF3, and total IRF3. (D) Relative amount of IFN-β pre-mRNA and mRNA of infected cells was quantitated by real-time RT-PCR. tNS1, truncated NS1 protein. *, P
    Figure Legend Snippet: Mutations at the second and third in-frame AUG codons in NS1 ORF of PR8 virus increase IRF3 activation and IFN-β production in infected cells. A549 cells were infected with WT or mutated influenza A virus that expresses NS1 with mutations at the second and third AUG (codons 79 and 81, respectively) in the PR8 backbone (A and B) or Ud backbone (E and F) at an MOI of 2. (A and E) At 9 h postinfection, cell extracts were collected and analyzed by immunoblotting with anti-NS1, anti-phospho-IRF3 (serine 396), anti-total IRF3, and anti-β actin antibodies. (B and F) Relative amount of IFN-β mRNA of infected cells was quantitated by real-time RT-PCR. HEL-299 cells were infected with the WT or the PR8 virus mutated at codons 79 and 81 at an MOI of 2. (C) At 9 h postinfection, cell extracts were collected and analyzed for the proteins of NS1, phosphorylated IRF3, and total IRF3. (D) Relative amount of IFN-β pre-mRNA and mRNA of infected cells was quantitated by real-time RT-PCR. tNS1, truncated NS1 protein. *, P

    Techniques Used: Activation Assay, Infection, Quantitative RT-PCR

    35) Product Images from "Combinatorial treatment increases IKAP levels in human cells generated from Familial Dysautonomia patients"

    Article Title: Combinatorial treatment increases IKAP levels in human cells generated from Familial Dysautonomia patients

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0211602

    Phosphatidylserine activates the BDNF-mediated signaling pathway. (A-H) FD cells were treated with 50 μg/ml PS or a vehicle only control, and RNA was extracted 5 days after the treatment. qPCR was used to quantify (A) WT IKBKAP , (B) CDKN1A , (C) HOMER1 , (D) RASSF8 , (E) EGR1 , (F) SRXN1 , (G) RGS17 , and (H) BAIAP2 mRNA transcripts. All values were normalized to LZIC , which did not change as a result of PS treatment. Asterisks denote statistically significant differences (*P ≤ 0.05 and **P ≤ 0.01) relative to control (vehicle only); Student’s t -test.
    Figure Legend Snippet: Phosphatidylserine activates the BDNF-mediated signaling pathway. (A-H) FD cells were treated with 50 μg/ml PS or a vehicle only control, and RNA was extracted 5 days after the treatment. qPCR was used to quantify (A) WT IKBKAP , (B) CDKN1A , (C) HOMER1 , (D) RASSF8 , (E) EGR1 , (F) SRXN1 , (G) RGS17 , and (H) BAIAP2 mRNA transcripts. All values were normalized to LZIC , which did not change as a result of PS treatment. Asterisks denote statistically significant differences (*P ≤ 0.05 and **P ≤ 0.01) relative to control (vehicle only); Student’s t -test.

    Techniques Used: Real-time Polymerase Chain Reaction

    36) Product Images from "Activity of vitamin D receptor agonists against dengue virus"

    Article Title: Activity of vitamin D receptor agonists against dengue virus

    Journal: Scientific Reports

    doi: 10.1038/s41598-020-67783-z

    VDR agonist activity on DENV E protein and VDR expression assessed by immunofluorescence assay. Mock or DENV 2 infected HEK293T/17 cells were treated with 0.01% DMSO or 10 μM of VDR agonists (ZD-1, ZD-2, ZD-3, ZD-5 and ZD-6). After 24 h of treatment, cells were processed under the standard procedure of immunofluorescence assay. DENV E protein (green) and VDR (red) were detected using specific antibodies. Nuclei were stained with DAPI (blue). All the signal was observed under a LSM 800 w Airyscan (ZEISS, Oberkochen, Germany) confocal microscope with 60X magnification.
    Figure Legend Snippet: VDR agonist activity on DENV E protein and VDR expression assessed by immunofluorescence assay. Mock or DENV 2 infected HEK293T/17 cells were treated with 0.01% DMSO or 10 μM of VDR agonists (ZD-1, ZD-2, ZD-3, ZD-5 and ZD-6). After 24 h of treatment, cells were processed under the standard procedure of immunofluorescence assay. DENV E protein (green) and VDR (red) were detected using specific antibodies. Nuclei were stained with DAPI (blue). All the signal was observed under a LSM 800 w Airyscan (ZEISS, Oberkochen, Germany) confocal microscope with 60X magnification.

    Techniques Used: Activity Assay, Expressing, Immunofluorescence, Infection, Staining, Microscopy

    The antiviral and agonist activity of commercial VDR agonist EB1089. ( A , B ) Mock and DENV 2 infected HEK293T/17 cells were treated with 20 µM EB1089 for 24 after which ( A ) the level of infection was determined by flow cytometry and ( B ) the virus titer in the supernatant was determined by standard plaque assay. ( C ) HEK293T/17 cells were treated with 20 µM EB1089 or DMSO vehicle for 24 h, after which cells were collected and RNA extracted and gene expression of VDR, CYP27B1, CYP24A1 and actin determined by qRT-PCR. The relative gene expression of VDR, CYP27B1 and CYP24A1 was normalized against actin and untreated cells (0.02% DMSO), respectively. p value; *
    Figure Legend Snippet: The antiviral and agonist activity of commercial VDR agonist EB1089. ( A , B ) Mock and DENV 2 infected HEK293T/17 cells were treated with 20 µM EB1089 for 24 after which ( A ) the level of infection was determined by flow cytometry and ( B ) the virus titer in the supernatant was determined by standard plaque assay. ( C ) HEK293T/17 cells were treated with 20 µM EB1089 or DMSO vehicle for 24 h, after which cells were collected and RNA extracted and gene expression of VDR, CYP27B1, CYP24A1 and actin determined by qRT-PCR. The relative gene expression of VDR, CYP27B1 and CYP24A1 was normalized against actin and untreated cells (0.02% DMSO), respectively. p value; *

    Techniques Used: Activity Assay, Infection, Flow Cytometry, Plaque Assay, Expressing, Quantitative RT-PCR

    VDR agonist activity on viral protein and viral genome copy number. ( A – E ) HEK293T/17 cells were mock or DENV 2 infected followed by treatment with 0.01% DMSO or 10 µM of VDR agonists (ZD-1, ZD-2, ZD-3, ZD-5 and ZD-6) at the indicated time points. Expression of DENV structural protein ( E ) and non-structural proteins (NS1, NS3 and NS5) was determined by western blot assay, using GAPDH as an internal control. White spaces separate different antibody probing. ( F ) In parallel, the supernatant were collected the level of the viral genome copy number determined by qRT-PCR. Viral genome copy number was calculated by comparing with tenfold serial dilution of a DENV NS1 standard control. p value; *
    Figure Legend Snippet: VDR agonist activity on viral protein and viral genome copy number. ( A – E ) HEK293T/17 cells were mock or DENV 2 infected followed by treatment with 0.01% DMSO or 10 µM of VDR agonists (ZD-1, ZD-2, ZD-3, ZD-5 and ZD-6) at the indicated time points. Expression of DENV structural protein ( E ) and non-structural proteins (NS1, NS3 and NS5) was determined by western blot assay, using GAPDH as an internal control. White spaces separate different antibody probing. ( F ) In parallel, the supernatant were collected the level of the viral genome copy number determined by qRT-PCR. Viral genome copy number was calculated by comparing with tenfold serial dilution of a DENV NS1 standard control. p value; *

    Techniques Used: Activity Assay, Infection, Expressing, Western Blot, Quantitative RT-PCR, Serial Dilution

    Antiviral activity of VDR agonists against DENV infection on HepG2 cells. HepG2 cells were mock-infected or infected with DENV 2 at MOI 5 for 2 h, followed by the treatment with 10 μM of ZD-1, ZD-2, ZD-3, ZD-5 and ZD-6 for 24 h.p.i. The treated cells were collected and level of infection determined by flow cytometry. p value; *
    Figure Legend Snippet: Antiviral activity of VDR agonists against DENV infection on HepG2 cells. HepG2 cells were mock-infected or infected with DENV 2 at MOI 5 for 2 h, followed by the treatment with 10 μM of ZD-1, ZD-2, ZD-3, ZD-5 and ZD-6 for 24 h.p.i. The treated cells were collected and level of infection determined by flow cytometry. p value; *

    Techniques Used: Activity Assay, Infection, Flow Cytometry

    Antiviral activity of VDR agonists. Mock and DENV 2 infected HEK293T/17 cells were treated with 0.01% DMSO or 10 µM of ZD-1, ZD-2, ZD-3, ZD-5, ZD-6 and ZD-20. ( A ) At 24 h.p.i. the treated cells were collected and the levels of infection determined using flow cytometry. ( B ) In parallel, the supernatants of the treated cells were evaluated for virus titer by standard plaque assay. p value; *
    Figure Legend Snippet: Antiviral activity of VDR agonists. Mock and DENV 2 infected HEK293T/17 cells were treated with 0.01% DMSO or 10 µM of ZD-1, ZD-2, ZD-3, ZD-5, ZD-6 and ZD-20. ( A ) At 24 h.p.i. the treated cells were collected and the levels of infection determined using flow cytometry. ( B ) In parallel, the supernatants of the treated cells were evaluated for virus titer by standard plaque assay. p value; *

    Techniques Used: Activity Assay, Infection, Flow Cytometry, Plaque Assay

    37) Product Images from "Low-dose radiation decreases tumor progression via the inhibition of the JAK1/STAT3 signaling axis in breast cancer cell lines"

    Article Title: Low-dose radiation decreases tumor progression via the inhibition of the JAK1/STAT3 signaling axis in breast cancer cell lines

    Journal: Scientific Reports

    doi: 10.1038/srep43361

    Low-dose radiation reduces the migration and invasion in MDA-MB231 cells via EMT. ( A ) Migration and invasion Transwell assays of MDA-MB231 breast cancer cells after LDR at doses of 0.01 Gy × 10 (fractionated) and 0.1 Gy (single dose). ( B ) Representative graphs of the migration and invasion of LDR-exposed MDA-MB231 cells. ( C ) Western blot for vimentin and SNAI2 (SLUG) in MDA-MB231 breast cancer cells after irradiation at 0.01Gy × 10 and 0.1Gy. ( D ) qRT-PCR analysis of vimentin and SNAI2 gene expression after irradiation in MDA-MB231 cells. ( E ) Immunocytochemistry for a vimentin mesenchymal marker in MDA-MB231 breast cancer cells after irradiation. ( F ) Phase-contrast images of control and LDR-treated MDA-MB231 cells. Representative graph shows the percentage of mesenchymal-like cells in respective each group. β-actin was used as a loading control. Error bars denote the mean ± S.D. of triplicate samples. * p
    Figure Legend Snippet: Low-dose radiation reduces the migration and invasion in MDA-MB231 cells via EMT. ( A ) Migration and invasion Transwell assays of MDA-MB231 breast cancer cells after LDR at doses of 0.01 Gy × 10 (fractionated) and 0.1 Gy (single dose). ( B ) Representative graphs of the migration and invasion of LDR-exposed MDA-MB231 cells. ( C ) Western blot for vimentin and SNAI2 (SLUG) in MDA-MB231 breast cancer cells after irradiation at 0.01Gy × 10 and 0.1Gy. ( D ) qRT-PCR analysis of vimentin and SNAI2 gene expression after irradiation in MDA-MB231 cells. ( E ) Immunocytochemistry for a vimentin mesenchymal marker in MDA-MB231 breast cancer cells after irradiation. ( F ) Phase-contrast images of control and LDR-treated MDA-MB231 cells. Representative graph shows the percentage of mesenchymal-like cells in respective each group. β-actin was used as a loading control. Error bars denote the mean ± S.D. of triplicate samples. * p

    Techniques Used: Migration, Multiple Displacement Amplification, Western Blot, Irradiation, Quantitative RT-PCR, Expressing, Immunocytochemistry, Marker

    Low-dose radiation decreases the cancer stem-cell maintenance in breast cancer cell lines. ( A ) Flow cytometer analysis of CD44+/CD24− cells in control and LDR-exposed MDA-MB231 cells at 0.01 Gy × 10 (fractionated) and 0.1 Gy (single dose). ( B ) Determination of the CD44 fluorescence intensity in LDR treated and untreated control cells using an Elisa reader. ( C ) Western blot of the CD44 and OCT4 protein levels LDR treated and untreated control MDA-MB231 cells. ( D ) qRT-PCR analyses results of CD44 and OCT4 gene expression levels in LDR treated and untreated control MDA-MB231 cells. ( E ) Immunocytochemistry of CD24 and OCT4 expression levels in LDR treated and untreated control cells. ( F ) Determination of the sphere-forming ability of LDR-exposed and control MCF7 breast cancer cells cultured in a sphere-conditioned medium. ( G , H ) Single-cell assay of LDR-exposed and control MCF7 sphere cells as observed from days 1 to 11 days after treatment. Quantification of the average size of each single cell is shown in the representative graph. ( I ) Immunocytochemistry of CD24 and CD24 in LDR treated and untreated control MCF7 sphere-cultured cells. ( J ) Limiting dilution assay performed on MCF7 cells after LDR exposure and compared with LDR unexposed control cells. Solid lines represents the average value of samples. β-actin was used as a loading control. Error bars denote the mean ± S.D. of triplicate samples. * p
    Figure Legend Snippet: Low-dose radiation decreases the cancer stem-cell maintenance in breast cancer cell lines. ( A ) Flow cytometer analysis of CD44+/CD24− cells in control and LDR-exposed MDA-MB231 cells at 0.01 Gy × 10 (fractionated) and 0.1 Gy (single dose). ( B ) Determination of the CD44 fluorescence intensity in LDR treated and untreated control cells using an Elisa reader. ( C ) Western blot of the CD44 and OCT4 protein levels LDR treated and untreated control MDA-MB231 cells. ( D ) qRT-PCR analyses results of CD44 and OCT4 gene expression levels in LDR treated and untreated control MDA-MB231 cells. ( E ) Immunocytochemistry of CD24 and OCT4 expression levels in LDR treated and untreated control cells. ( F ) Determination of the sphere-forming ability of LDR-exposed and control MCF7 breast cancer cells cultured in a sphere-conditioned medium. ( G , H ) Single-cell assay of LDR-exposed and control MCF7 sphere cells as observed from days 1 to 11 days after treatment. Quantification of the average size of each single cell is shown in the representative graph. ( I ) Immunocytochemistry of CD24 and CD24 in LDR treated and untreated control MCF7 sphere-cultured cells. ( J ) Limiting dilution assay performed on MCF7 cells after LDR exposure and compared with LDR unexposed control cells. Solid lines represents the average value of samples. β-actin was used as a loading control. Error bars denote the mean ± S.D. of triplicate samples. * p

    Techniques Used: Flow Cytometry, Multiple Displacement Amplification, Fluorescence, Enzyme-linked Immunosorbent Assay, Western Blot, Quantitative RT-PCR, Expressing, Immunocytochemistry, Cell Culture, Limiting Dilution Assay

    38) Product Images from "Tumor-suppressing effects of microRNA-612 in bladder cancer cells by targeting malic enzyme 1 expression"

    Article Title: Tumor-suppressing effects of microRNA-612 in bladder cancer cells by targeting malic enzyme 1 expression

    Journal: International Journal of Oncology

    doi: 10.3892/ijo.2018.4342

    ME1 knockdown mimicked the inhibitory effect of miR-612 overexpression in bladder cancer cells. (A) mRNA and (B) protein levels of ME1 in T24 cells following transfection with si-ME1 or si-NC were measured using reverse transcription-quantitative polymerase chain reaction and western blot analysis, respectively. GAPDH and β-actin were used as internal controls, respectively. (C) Cell proliferation, (D) colony formation, (E) Transwell tumor cell migration assay and (F) Transwell tumor cell invasion assays in bladder cancer T24 cells transfected with si-ME1 or si-NC. (G) Protein levels of EMT-associated markers in T24 cells transfected with si-ME1 or si-NC were evaluated by western blot analysis. * P
    Figure Legend Snippet: ME1 knockdown mimicked the inhibitory effect of miR-612 overexpression in bladder cancer cells. (A) mRNA and (B) protein levels of ME1 in T24 cells following transfection with si-ME1 or si-NC were measured using reverse transcription-quantitative polymerase chain reaction and western blot analysis, respectively. GAPDH and β-actin were used as internal controls, respectively. (C) Cell proliferation, (D) colony formation, (E) Transwell tumor cell migration assay and (F) Transwell tumor cell invasion assays in bladder cancer T24 cells transfected with si-ME1 or si-NC. (G) Protein levels of EMT-associated markers in T24 cells transfected with si-ME1 or si-NC were evaluated by western blot analysis. * P

    Techniques Used: Over Expression, Transfection, Real-time Polymerase Chain Reaction, Western Blot, Cell Migration Assay

    ME1 overexpression partially rescues the inhibitory effects of miR-612 expression on bladder cancer cells. (A) mRNA and (B) protein levels of ME1 in T24 cells co-transfected with miR-612 mimic or miR-NC, and with or without ME1 cDNA were measured using reverse transcription-quantitative polymerase chain reaction and western blot analysis, respectively. (C) Cell proliferation, (D) colony formation, (E) Transwell tumor cell migration and (F) Transwell tumor cell invasion assays in T24 cells co-transfected with miR-612 mimic or miR-NC, and with or without ME1 cDNA. * P
    Figure Legend Snippet: ME1 overexpression partially rescues the inhibitory effects of miR-612 expression on bladder cancer cells. (A) mRNA and (B) protein levels of ME1 in T24 cells co-transfected with miR-612 mimic or miR-NC, and with or without ME1 cDNA were measured using reverse transcription-quantitative polymerase chain reaction and western blot analysis, respectively. (C) Cell proliferation, (D) colony formation, (E) Transwell tumor cell migration and (F) Transwell tumor cell invasion assays in T24 cells co-transfected with miR-612 mimic or miR-NC, and with or without ME1 cDNA. * P

    Techniques Used: Over Expression, Expressing, Transfection, Real-time Polymerase Chain Reaction, Western Blot, Migration

    ME1 is a direct target of miR-612. (A) Bioinformatic analysis indicated that ME1 is the target of miR-612. The presumable miR-612-binding sites in the 3′-UTR of ME1 cDNA and the mutant binding sites are shown. (B) The relative luciferase activity was determined in T24 cells following co-transfection with a luciferase reporter plasmid (WT or MUT 3′-UTR ME1 cDNA), and miR-612 mimic or miR-NC. (C) mRNA and (D) protein levels of ME1 in T24 cells following transfection with miR-612 mimic or miR-NC were analyzed using RT-qPCR and western blot analysis, respectively. GAPDH and β-actin served as the internal controls, respectively. (E) The levels of ME1 mRNA were measured using RT-qPCR in 46 paired bladder cancer and ANT specimens. GAPDH was used as an internal control. (F) The correlation between ME1 and miR-612 expression in bladder cancer tissue samples (n=46) was analyzed using Spearman's correlation test. ** P
    Figure Legend Snippet: ME1 is a direct target of miR-612. (A) Bioinformatic analysis indicated that ME1 is the target of miR-612. The presumable miR-612-binding sites in the 3′-UTR of ME1 cDNA and the mutant binding sites are shown. (B) The relative luciferase activity was determined in T24 cells following co-transfection with a luciferase reporter plasmid (WT or MUT 3′-UTR ME1 cDNA), and miR-612 mimic or miR-NC. (C) mRNA and (D) protein levels of ME1 in T24 cells following transfection with miR-612 mimic or miR-NC were analyzed using RT-qPCR and western blot analysis, respectively. GAPDH and β-actin served as the internal controls, respectively. (E) The levels of ME1 mRNA were measured using RT-qPCR in 46 paired bladder cancer and ANT specimens. GAPDH was used as an internal control. (F) The correlation between ME1 and miR-612 expression in bladder cancer tissue samples (n=46) was analyzed using Spearman's correlation test. ** P

    Techniques Used: Binding Assay, Mutagenesis, Luciferase, Activity Assay, Cotransfection, Plasmid Preparation, Transfection, Quantitative RT-PCR, Western Blot, Expressing

    39) Product Images from "MiR-532-5p suppresses renal cancer cell proliferation by disrupting the ETS1-mediated positive feedback loop with the KRAS-NAP1L1/P-ERK axis"

    Article Title: MiR-532-5p suppresses renal cancer cell proliferation by disrupting the ETS1-mediated positive feedback loop with the KRAS-NAP1L1/P-ERK axis

    Journal: British Journal of Cancer

    doi: 10.1038/s41416-018-0196-5

    miR-532-5p is downregulated in RCC tissues and cell lines. a Heat map of joint analysis of differentially expressed miRNAs from two RCC miRNA arrays and miRNAs differentially expressed in both miRNA arrays. b The overlap of top ten downregulated miRNAs from each miRNA array. c miR-532-5p expression in RCC and normal samples from the TCGA RCC data set. d The expression of miR-532-5p was determined in tumour tissues and their adjacent non-cancer tissues by qRT-PCR. U6 was used as an internal control. e Relative miR-532-5p expression levels in RCC tumour are presented as fold change = 2 (ΔCt normal–ΔCt tumour) of tumour versus matched normal tissues. The 0.5-fold change threshold was defined as differentially expressed. f The expression of miR-532-5p was determined in several human RCC cell lines (786-O, OSRC-2, A498, and SN12-PM6) and human normal renal tubular epithelial cell line HK-2. g Kaplan–Meier analyses of the correlations between miR-532-5p expression and overall survival of 871 RCC patients from the TCGA RCC data set. Log-rank test was used to calculate p values
    Figure Legend Snippet: miR-532-5p is downregulated in RCC tissues and cell lines. a Heat map of joint analysis of differentially expressed miRNAs from two RCC miRNA arrays and miRNAs differentially expressed in both miRNA arrays. b The overlap of top ten downregulated miRNAs from each miRNA array. c miR-532-5p expression in RCC and normal samples from the TCGA RCC data set. d The expression of miR-532-5p was determined in tumour tissues and their adjacent non-cancer tissues by qRT-PCR. U6 was used as an internal control. e Relative miR-532-5p expression levels in RCC tumour are presented as fold change = 2 (ΔCt normal–ΔCt tumour) of tumour versus matched normal tissues. The 0.5-fold change threshold was defined as differentially expressed. f The expression of miR-532-5p was determined in several human RCC cell lines (786-O, OSRC-2, A498, and SN12-PM6) and human normal renal tubular epithelial cell line HK-2. g Kaplan–Meier analyses of the correlations between miR-532-5p expression and overall survival of 871 RCC patients from the TCGA RCC data set. Log-rank test was used to calculate p values

    Techniques Used: Expressing, Quantitative RT-PCR

    miR-532-5p inhibits renal cancer cell proliferation in vitro. a The expression of miR-532-5p was determined by qRT-PCR in SN12-PM6 and 786-O cells after transfection with the miR-532-5p mimic or miR-NC. b Cell proliferation was analyzed by CCK8 assays in SN12-PM6 and 786-O cells after transfection with the miR-532-5p mimic or miR-NC. c Colony formation was determined in SN12-PM6 and 786-O cells after transfection with the miR-532-5p mimic or miR-NC. The results were averaged from three experiments; error bars indicate ± 1 SD, * p
    Figure Legend Snippet: miR-532-5p inhibits renal cancer cell proliferation in vitro. a The expression of miR-532-5p was determined by qRT-PCR in SN12-PM6 and 786-O cells after transfection with the miR-532-5p mimic or miR-NC. b Cell proliferation was analyzed by CCK8 assays in SN12-PM6 and 786-O cells after transfection with the miR-532-5p mimic or miR-NC. c Colony formation was determined in SN12-PM6 and 786-O cells after transfection with the miR-532-5p mimic or miR-NC. The results were averaged from three experiments; error bars indicate ± 1 SD, * p

    Techniques Used: In Vitro, Expressing, Quantitative RT-PCR, Transfection

    40) Product Images from "Kaempferol as a flavonoid induces osteoblastic differentiation via estrogen receptor signaling"

    Article Title: Kaempferol as a flavonoid induces osteoblastic differentiation via estrogen receptor signaling

    Journal: Chinese Medicine

    doi: 10.1186/1749-8546-7-10

    Kaempferol-induced osteogenic differentiation is mediated by ER signaling cultured osteoblasts . A: Application of 17β-estradiol (100 nM) or kaempferol (30 - 300 μM) in cultured osteoblasts for 3 days increased ALP activity in a dose-dependent manner. The stimulatory effect was abolished upon pre-treatment with ICI 182,780 (100 nM) for 1 hour. The ALP activities detected after the pre-treatment of ICI 182, 780 were compared with the ALP activities detected without the pre-treatment. The statistically significant results include the blocking effects of 17β-estradiol ( P = 0.0412), kaempferol at 30 μM ( P = 0.0485), 100 μM ( P = 0.0081) and 300 μM ( P = 0.0086). B: Cultured osteoblats were treated with 17β-estradiol (100 nM) or kaempferol (10 μM) for 2 days, with or without pre-treatment with ICI 182,780 (100 nM) for 1 hour. Total RNAs were extracted from the cultures to perform quantitative PCR for osteogenesis-associated genes, including type I collagen ( COL1A1 ), osteonectin, osteocalcin, osterix and Runx2 mRNAs. The mRNA amounts of osteogenesis-associated genes detected after the pre-treatment of ICI 182, 780 were compared with the mRNA amounts detected without the pre-treatment. The statistically significant results include the blocking effects of 17β-estradiol ( P = 0.0012 for COL1A1; P = 0.0070 for osteonectin; P = 0.0033 for osteocalcin; P = 0.0441 for osterix and P = 0.0023 for Runx2) and kaempferol ( P = 0.0065 for COL1A1; P = 0.0063 for osteonectin; P = 0.0072 for osteocalcin; P = 0.0068 for osterix and P = 0.0064 for Runx2). C: Cultured osteoblasts underwent mineralization upon the addition of 17β-estradiol (100 nM) or kaempferol (10 μM) in the presence of β-glycerophosphate (5 mM). After 21 days of treatment, nodules were found, as shown by Alizarin Red staining. The mineralization process was hindered by pre-treatment with ICI 182,780 (100 nM). D: From the cultures of (C), Alizarin Red staining was quantified using a solution of 20% methanol and 10% acetic acid in water, and the reading was done on a spectrophotometer at 450 nm. The normalized alizarin red amounts detected after the pre-treatment of ICI 182, 780 were compared with the amount detected without the pre-treatment. The statistically significant results include the blocking effects of 17β-estradiol ( P = 0.0093) and kaempferol ( P = 0.0085). Values in all panels are expressed as the fold increase from the basal reading (control culture; 0.02% DMSO); mean ± SD, n = 5, each with triplicate samples.
    Figure Legend Snippet: Kaempferol-induced osteogenic differentiation is mediated by ER signaling cultured osteoblasts . A: Application of 17β-estradiol (100 nM) or kaempferol (30 - 300 μM) in cultured osteoblasts for 3 days increased ALP activity in a dose-dependent manner. The stimulatory effect was abolished upon pre-treatment with ICI 182,780 (100 nM) for 1 hour. The ALP activities detected after the pre-treatment of ICI 182, 780 were compared with the ALP activities detected without the pre-treatment. The statistically significant results include the blocking effects of 17β-estradiol ( P = 0.0412), kaempferol at 30 μM ( P = 0.0485), 100 μM ( P = 0.0081) and 300 μM ( P = 0.0086). B: Cultured osteoblats were treated with 17β-estradiol (100 nM) or kaempferol (10 μM) for 2 days, with or without pre-treatment with ICI 182,780 (100 nM) for 1 hour. Total RNAs were extracted from the cultures to perform quantitative PCR for osteogenesis-associated genes, including type I collagen ( COL1A1 ), osteonectin, osteocalcin, osterix and Runx2 mRNAs. The mRNA amounts of osteogenesis-associated genes detected after the pre-treatment of ICI 182, 780 were compared with the mRNA amounts detected without the pre-treatment. The statistically significant results include the blocking effects of 17β-estradiol ( P = 0.0012 for COL1A1; P = 0.0070 for osteonectin; P = 0.0033 for osteocalcin; P = 0.0441 for osterix and P = 0.0023 for Runx2) and kaempferol ( P = 0.0065 for COL1A1; P = 0.0063 for osteonectin; P = 0.0072 for osteocalcin; P = 0.0068 for osterix and P = 0.0064 for Runx2). C: Cultured osteoblasts underwent mineralization upon the addition of 17β-estradiol (100 nM) or kaempferol (10 μM) in the presence of β-glycerophosphate (5 mM). After 21 days of treatment, nodules were found, as shown by Alizarin Red staining. The mineralization process was hindered by pre-treatment with ICI 182,780 (100 nM). D: From the cultures of (C), Alizarin Red staining was quantified using a solution of 20% methanol and 10% acetic acid in water, and the reading was done on a spectrophotometer at 450 nm. The normalized alizarin red amounts detected after the pre-treatment of ICI 182, 780 were compared with the amount detected without the pre-treatment. The statistically significant results include the blocking effects of 17β-estradiol ( P = 0.0093) and kaempferol ( P = 0.0085). Values in all panels are expressed as the fold increase from the basal reading (control culture; 0.02% DMSO); mean ± SD, n = 5, each with triplicate samples.

    Techniques Used: Cell Culture, Activity Assay, Blocking Assay, Real-time Polymerase Chain Reaction, Staining, Spectrophotometry

    Kaempferol-stimulated osteogenic effect is not mediated by activation of the Wnt/β catenin pathway . A: Cultured osteoblasts were treated with kaempferol (10 μM) for 2 days, with or without DKK-1 pre-treatment (0.2 mg/mL) for 1 hour. Total mRNA was extracted from the cultures to perform quantitative PCR for osteogenesis-associated genes, including type I collagen ( COL1A1 ), osteonectin, osteocalcin, Runx2 and osterix. B: A reporter construct corresponding to pWRE-Luc (upper panel) was used as described previously. pWRE-Luc was transfected into cultured osteoblasts for 2 days before addition of Wnt3a (200 ng/mL; the ligand of Wnt/β-catenin pathway) or kaempferol (10 μM). Forty-eight hours later, luciferase activity was assayed. Values are expressed as the fold increase from the basal reading (control culture; 0.02% DMSO); mean ± SD, n = 5, each with triplicate samples.
    Figure Legend Snippet: Kaempferol-stimulated osteogenic effect is not mediated by activation of the Wnt/β catenin pathway . A: Cultured osteoblasts were treated with kaempferol (10 μM) for 2 days, with or without DKK-1 pre-treatment (0.2 mg/mL) for 1 hour. Total mRNA was extracted from the cultures to perform quantitative PCR for osteogenesis-associated genes, including type I collagen ( COL1A1 ), osteonectin, osteocalcin, Runx2 and osterix. B: A reporter construct corresponding to pWRE-Luc (upper panel) was used as described previously. pWRE-Luc was transfected into cultured osteoblasts for 2 days before addition of Wnt3a (200 ng/mL; the ligand of Wnt/β-catenin pathway) or kaempferol (10 μM). Forty-eight hours later, luciferase activity was assayed. Values are expressed as the fold increase from the basal reading (control culture; 0.02% DMSO); mean ± SD, n = 5, each with triplicate samples.

    Techniques Used: Activation Assay, Cell Culture, Real-time Polymerase Chain Reaction, Construct, Transfection, Luciferase, Activity Assay

    Related Articles

    Real-time Polymerase Chain Reaction:

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    Article Title: MicroRNA-429 decreases the invasion ability of gastric cancer cell line BGC-823 by downregulating the expression of heparanase
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    Article Title: Podocalyxin enhances breast tumor growth and metastasis and is a target for monoclonal antibody therapy
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    Article Title: Combinatorial treatment increases IKAP levels in human cells generated from Familial Dysautonomia patients
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    Article Title: VraR Binding to the Promoter Region of agr Inhibits Its Function in Vancomycin-Intermediate Staphylococcus aureus (VISA) and Heterogeneous VISA
    Article Snippet: .. Transcript levels of vraR , hla , hlb , coa , RNAIII, agrA , and saeR were quantified by quantitative real-time PCR (qRT-PCR) using a Kapa SYBR qPCR kit (Kapa Biosystems) in a LightCycler (LC-32; Roche, USA). ..

    Article Title: DZNep inhibits Hif-1α and Wnt signalling molecules to attenuate the proliferation and invasion of BGC-823 gastric cancer cells
    Article Snippet: .. The primers used were as follows: Hif-1α forward, 5′-GAAAGCGCAAGTCTTCAAAG-3′ and reverse, 5′-TGGGTAGGAGATGGAGATGC-3′ (amplified fragment, 167 bp; Sangon Biotech Co., Ltd.); β-actin forward, 5′-ACACTGTGCCCATCTACG-3′ and reverse, 5′-TGTCACGCACGATTTCC-3′ (amplified fragment, 152 bp). qPCR was performed using a SYBR® Green PCR kit (cat. no. KM4101; Kapa Biosystems; Roche Diagnostics). .. The reactions were incubated in a 96-well optical plate at 95°C for 3 min, 95°C for 5 sec, 56°C for 10 sec, 72°C for 25 sec, and 39 cycles of 65°C for 5 sec and 95°C for 50 sec.

    Article Title: Role of NeuroD1 on the negative regulation of Pomc expression by glucocorticoid
    Article Snippet: .. Immunoprecipitated DNA was analyzed by qPCR using KAPA SYBR FAST Universal 2x qPCR Master Mix (KAPA Biosystems) together with 1% of the input chromatin. .. Specific primer pairs were designed to amplify the E-box region of mouse the Pomc gene promoter for qPCR.

    Amplification:

    Article Title: DZNep inhibits Hif-1α and Wnt signalling molecules to attenuate the proliferation and invasion of BGC-823 gastric cancer cells
    Article Snippet: .. The primers used were as follows: Hif-1α forward, 5′-GAAAGCGCAAGTCTTCAAAG-3′ and reverse, 5′-TGGGTAGGAGATGGAGATGC-3′ (amplified fragment, 167 bp; Sangon Biotech Co., Ltd.); β-actin forward, 5′-ACACTGTGCCCATCTACG-3′ and reverse, 5′-TGTCACGCACGATTTCC-3′ (amplified fragment, 152 bp). qPCR was performed using a SYBR® Green PCR kit (cat. no. KM4101; Kapa Biosystems; Roche Diagnostics). .. The reactions were incubated in a 96-well optical plate at 95°C for 3 min, 95°C for 5 sec, 56°C for 10 sec, 72°C for 25 sec, and 39 cycles of 65°C for 5 sec and 95°C for 50 sec.

    Quantitative RT-PCR:

    Article Title: Epstein–Barr Virus BALF0 and BALF1 Modulate Autophagy
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    Article Snippet: .. Transcript levels of vraR , hla , hlb , coa , RNAIII, agrA , and saeR were quantified by quantitative real-time PCR (qRT-PCR) using a Kapa SYBR qPCR kit (Kapa Biosystems) in a LightCycler (LC-32; Roche, USA). ..

    SYBR Green Assay:

    Article Title: MicroRNA-429 decreases the invasion ability of gastric cancer cell line BGC-823 by downregulating the expression of heparanase
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    Article Title: DZNep inhibits Hif-1α and Wnt signalling molecules to attenuate the proliferation and invasion of BGC-823 gastric cancer cells
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    Immunoprecipitation:

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    Expressing:

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    Article Title: Combinatorial treatment increases IKAP levels in human cells generated from Familial Dysautonomia patients
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    Polymerase Chain Reaction:

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