superscript iii kit  (Thermo Fisher)


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    Name:
    SuperScript III First Strand Synthesis System
    Description:
    The SuperScript III First Strand Synthesis System for RT PCR is optimized to synthesize first strand cDNA from purified poly A or total RNA RNA targets from 100 bp to 12 kb can be detected with this system The amount of starting material can vary from 1 pg 5 µg of total RNA SuperScript III Reverse Transcriptase is a version of M MLV RT that has been engineered to reduce RNase H activity and provide increased thermal stability The enzyme is used to synthesize cDNA at a temperature range of 42 55°C providing increased specificity higher yields of cDNA and more full length product than other reverse transcriptases Because SuperScript III RT is not significantly inhibited by ribosomal and transfer RNA it may be used to synthesize first strand cDNA from a total RNA preparation Using the SuperScript III First Strand SystemcDNA synthesis is performed in the first step using either total RNA or poly A selected RNA primed with oligo dT random primers or a gene specific primer In the second step PCR is performed in a separate tube using primers specific for the gene of interest For the PCR reaction we recommend one of the following DNA polymerases Platinum Taq DNA Polymerase provides automatic hot start conditions for increased specificity up to 4 kb Platinum Taq DNA Polymerase High Fidelity provides increased yield and high fidelity for targets up to 15 kb and Platinum Pfx DNA Polymerase provides maximum fidelity for targets up to 12 kb
    Catalog Number:
    18080051
    Price:
    None
    Applications:
    PCR & Real-Time PCR|Reverse Transcription
    Category:
    Kits and Assays
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    Structured Review

    Thermo Fisher superscript iii kit
    Effect of glaucocalyxin-A (GLA) on cyclooxygenase-2 (COX-2) and prostaglandin E 2 (PGE 2 ) expression in lipopolysaccharide (LPS)-stimulated microglia. Cells were pre-treated with the indicated concentrations of GLA for 1 h before incubating with LPS (25 ng/mL or 100 ng/mL) for 18 h A: Primary Microglia, B: BV-2 cells. Results are expressed as a ratio of COX-2 to β-actin. Representative quantification data was shown in the lower panel. C: Cells were pre-treated with the indicated concentrations of GLA for 1 h and then stimulated with LPS (100 ng/mL) for 6 h. Total <t>RNA</t> was prepared and COX-2 mRNA level was determined by RT-PCR. Results are expressed as the ratio of COX-2 to GAPDH. Quantification data are shown in the lower panel. D: PGE 2 levels were analyzed with an enzyme immunoassay kit. Absorbance was measured at 420 nm spectrophotometrically. Data are mean ± S.E.M. (n = 3) for <t>three</t> independent experiments. # P
    The SuperScript III First Strand Synthesis System for RT PCR is optimized to synthesize first strand cDNA from purified poly A or total RNA RNA targets from 100 bp to 12 kb can be detected with this system The amount of starting material can vary from 1 pg 5 µg of total RNA SuperScript III Reverse Transcriptase is a version of M MLV RT that has been engineered to reduce RNase H activity and provide increased thermal stability The enzyme is used to synthesize cDNA at a temperature range of 42 55°C providing increased specificity higher yields of cDNA and more full length product than other reverse transcriptases Because SuperScript III RT is not significantly inhibited by ribosomal and transfer RNA it may be used to synthesize first strand cDNA from a total RNA preparation Using the SuperScript III First Strand SystemcDNA synthesis is performed in the first step using either total RNA or poly A selected RNA primed with oligo dT random primers or a gene specific primer In the second step PCR is performed in a separate tube using primers specific for the gene of interest For the PCR reaction we recommend one of the following DNA polymerases Platinum Taq DNA Polymerase provides automatic hot start conditions for increased specificity up to 4 kb Platinum Taq DNA Polymerase High Fidelity provides increased yield and high fidelity for targets up to 15 kb and Platinum Pfx DNA Polymerase provides maximum fidelity for targets up to 12 kb
    https://www.bioz.com/result/superscript iii kit/product/Thermo Fisher
    Average 99 stars, based on 19 article reviews
    Price from $9.99 to $1999.99
    superscript iii kit - by Bioz Stars, 2020-08
    99/100 stars

    Images

    1) Product Images from "Regulation of Microglia Activity by Glaucocalyxin-A: Attenuation of Lipopolysaccharide-Stimulated Neuroinflammation through NF-?B and p38 MAPK Signaling Pathways"

    Article Title: Regulation of Microglia Activity by Glaucocalyxin-A: Attenuation of Lipopolysaccharide-Stimulated Neuroinflammation through NF-?B and p38 MAPK Signaling Pathways

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0055792

    Effect of glaucocalyxin-A (GLA) on cyclooxygenase-2 (COX-2) and prostaglandin E 2 (PGE 2 ) expression in lipopolysaccharide (LPS)-stimulated microglia. Cells were pre-treated with the indicated concentrations of GLA for 1 h before incubating with LPS (25 ng/mL or 100 ng/mL) for 18 h A: Primary Microglia, B: BV-2 cells. Results are expressed as a ratio of COX-2 to β-actin. Representative quantification data was shown in the lower panel. C: Cells were pre-treated with the indicated concentrations of GLA for 1 h and then stimulated with LPS (100 ng/mL) for 6 h. Total RNA was prepared and COX-2 mRNA level was determined by RT-PCR. Results are expressed as the ratio of COX-2 to GAPDH. Quantification data are shown in the lower panel. D: PGE 2 levels were analyzed with an enzyme immunoassay kit. Absorbance was measured at 420 nm spectrophotometrically. Data are mean ± S.E.M. (n = 3) for three independent experiments. # P
    Figure Legend Snippet: Effect of glaucocalyxin-A (GLA) on cyclooxygenase-2 (COX-2) and prostaglandin E 2 (PGE 2 ) expression in lipopolysaccharide (LPS)-stimulated microglia. Cells were pre-treated with the indicated concentrations of GLA for 1 h before incubating with LPS (25 ng/mL or 100 ng/mL) for 18 h A: Primary Microglia, B: BV-2 cells. Results are expressed as a ratio of COX-2 to β-actin. Representative quantification data was shown in the lower panel. C: Cells were pre-treated with the indicated concentrations of GLA for 1 h and then stimulated with LPS (100 ng/mL) for 6 h. Total RNA was prepared and COX-2 mRNA level was determined by RT-PCR. Results are expressed as the ratio of COX-2 to GAPDH. Quantification data are shown in the lower panel. D: PGE 2 levels were analyzed with an enzyme immunoassay kit. Absorbance was measured at 420 nm spectrophotometrically. Data are mean ± S.E.M. (n = 3) for three independent experiments. # P

    Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay

    Effect of glaucocalyxin-A (GLA) on inducible nitric oxide synthase (iNOS) expression and enzyme activity in lipopolysaccharide (LPS)-stimulated microglia. Cells were pre-treated with the indicated concentrations of GLA for 1 h before incubating with LPS (25 ng/mL or 100 ng/mL) for 18 h A: Primary microglia, B: BV-2 microglia. Lysates were analyzed by immunoblotting with an anti-iNOS antibody. Representative quantification data were shown in the lower panel. Results are expressed as a ratio of iNOS to β-actin. C: BV-2 cells were pre-treated with the indicated concentrations of GLA for 1 h before incubating with LPS (100 ng/mL) for 6 h. The total RNA was isolated and iNOS mRNA level was determined by RT-PCR. Quantification data are shown in the lower panel. Results are expressed as a ratio of iNOS to GAPDH. D: BV-2 microglial cells were pre-treated with the indicated concentrations of GLA for 1 h before incubating with LPS (100 ng/mL) for 18 h. iNOS activity was analyzed with enzyme immunoassay kit. Data are mean ± S.E.M. (n = 3) for three independent experiments. # P
    Figure Legend Snippet: Effect of glaucocalyxin-A (GLA) on inducible nitric oxide synthase (iNOS) expression and enzyme activity in lipopolysaccharide (LPS)-stimulated microglia. Cells were pre-treated with the indicated concentrations of GLA for 1 h before incubating with LPS (25 ng/mL or 100 ng/mL) for 18 h A: Primary microglia, B: BV-2 microglia. Lysates were analyzed by immunoblotting with an anti-iNOS antibody. Representative quantification data were shown in the lower panel. Results are expressed as a ratio of iNOS to β-actin. C: BV-2 cells were pre-treated with the indicated concentrations of GLA for 1 h before incubating with LPS (100 ng/mL) for 6 h. The total RNA was isolated and iNOS mRNA level was determined by RT-PCR. Quantification data are shown in the lower panel. Results are expressed as a ratio of iNOS to GAPDH. D: BV-2 microglial cells were pre-treated with the indicated concentrations of GLA for 1 h before incubating with LPS (100 ng/mL) for 18 h. iNOS activity was analyzed with enzyme immunoassay kit. Data are mean ± S.E.M. (n = 3) for three independent experiments. # P

    Techniques Used: Expressing, Activity Assay, Isolation, Reverse Transcription Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay

    2) Product Images from "A Novel Stress-Induced Sugarcane Gene Confers Tolerance to Drought, Salt and Oxidative Stress in Transgenic Tobacco Plants"

    Article Title: A Novel Stress-Induced Sugarcane Gene Confers Tolerance to Drought, Salt and Oxidative Stress in Transgenic Tobacco Plants

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0044697

    Schematic representation of the pCAMBIA2301::Scdr1 construct and PCR confirmation of plant transgene content. (A) The Scdr1 coding region was cloned between the constitutive CaMV 35S promoter (P35S) and the NOS polyadenylation signal (Nos-t) using pCambia2301 as the backbone. The nptII (kanamycin resistance) gene is also driven by the p35S promoter. LB and RB correspond to the left and right borders of the T-DNA, respectively. The positions of some restriction sites are indicated. (B) Expression of Scdr1 in WT and three T3-generation transgenic lines. Total RNA was extracted from two-week-old seedlings and then analyzed using semi-quantitative RT-PCR. The actin gene was used as an internal standard. (C) Densitometric analysis of the semi-quantitative RT-PCR.
    Figure Legend Snippet: Schematic representation of the pCAMBIA2301::Scdr1 construct and PCR confirmation of plant transgene content. (A) The Scdr1 coding region was cloned between the constitutive CaMV 35S promoter (P35S) and the NOS polyadenylation signal (Nos-t) using pCambia2301 as the backbone. The nptII (kanamycin resistance) gene is also driven by the p35S promoter. LB and RB correspond to the left and right borders of the T-DNA, respectively. The positions of some restriction sites are indicated. (B) Expression of Scdr1 in WT and three T3-generation transgenic lines. Total RNA was extracted from two-week-old seedlings and then analyzed using semi-quantitative RT-PCR. The actin gene was used as an internal standard. (C) Densitometric analysis of the semi-quantitative RT-PCR.

    Techniques Used: Construct, Polymerase Chain Reaction, Clone Assay, Expressing, Transgenic Assay, Quantitative RT-PCR

    3) Product Images from "The Alternative Splicing of Cytoplasmic Polyadenylation Element Binding Protein 2 Drives Anoikis Resistance and the Metastasis of Triple Negative Breast Cancer *"

    Article Title: The Alternative Splicing of Cytoplasmic Polyadenylation Element Binding Protein 2 Drives Anoikis Resistance and the Metastasis of Triple Negative Breast Cancer *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M115.671206

    CPEB2 splicing is altered in AnR TNBC cells and in human breast cancer tissues. A , schematic representation of the experimentally confirmed CPEB2 splice variants associated with AnR in TNBC cells. B , RNA was isolated from the cell lines indicated, then first-strand cDNA synthesis was performed. cDNA samples were subjected to quantitative competitive RT-PCR and electrophoresed on a 5% acrylamide gel to determine the CPEB2A/B ratio (the presented gel is a representative of at least three different experiments; # indicates a statistically significant difference between parental and AnR cell lines (MDA-MB-468 and MDA-MB-231, t test, p ≤ 0.05)). C , human breast tumor ( Tr ) and patient-matched normal control ( Nl or Ctr ) tissue was harvested, and RNA was isolated by the TDAAC. cDNA was synthesized, and samples ( n = 15 for both control and tumor) were subjected to first strand cDNA synthesis and then quantitative PCR ( top graph; Sqrt Ratio A/B , the square root of the ratio of the A isoform divided by the B isoform). A subset of three patient samples with matched controls was subjected to competitive RT-PCR (bottom gel). D , confirmed human TNBC tumor tissues ( n = 45) obtained by the TDAAC were compared with the control patient tissues described in C using quantitative reverse transcriptase-PCR. E , transcript RPKM data for control samples ( n = 4), HER2-overexpressing samples ( n = 94), and TNBC samples ( n = 78) were downloaded from the ATLAS cancer genome archive. The ratio of CPEB2A/CPEB2B was determined as described under “Experimental Procedures.” Outliers and samples for which CPEB2A and -B mRNA levels were both 0 were not considered. CPEB2A/B ratios from competitive RT-PCR experiments were determined using ImageJ. * indicates a statistically significant difference from non-tumor cells (Mann-Whitney, p ≤ 0.05).
    Figure Legend Snippet: CPEB2 splicing is altered in AnR TNBC cells and in human breast cancer tissues. A , schematic representation of the experimentally confirmed CPEB2 splice variants associated with AnR in TNBC cells. B , RNA was isolated from the cell lines indicated, then first-strand cDNA synthesis was performed. cDNA samples were subjected to quantitative competitive RT-PCR and electrophoresed on a 5% acrylamide gel to determine the CPEB2A/B ratio (the presented gel is a representative of at least three different experiments; # indicates a statistically significant difference between parental and AnR cell lines (MDA-MB-468 and MDA-MB-231, t test, p ≤ 0.05)). C , human breast tumor ( Tr ) and patient-matched normal control ( Nl or Ctr ) tissue was harvested, and RNA was isolated by the TDAAC. cDNA was synthesized, and samples ( n = 15 for both control and tumor) were subjected to first strand cDNA synthesis and then quantitative PCR ( top graph; Sqrt Ratio A/B , the square root of the ratio of the A isoform divided by the B isoform). A subset of three patient samples with matched controls was subjected to competitive RT-PCR (bottom gel). D , confirmed human TNBC tumor tissues ( n = 45) obtained by the TDAAC were compared with the control patient tissues described in C using quantitative reverse transcriptase-PCR. E , transcript RPKM data for control samples ( n = 4), HER2-overexpressing samples ( n = 94), and TNBC samples ( n = 78) were downloaded from the ATLAS cancer genome archive. The ratio of CPEB2A/CPEB2B was determined as described under “Experimental Procedures.” Outliers and samples for which CPEB2A and -B mRNA levels were both 0 were not considered. CPEB2A/B ratios from competitive RT-PCR experiments were determined using ImageJ. * indicates a statistically significant difference from non-tumor cells (Mann-Whitney, p ≤ 0.05).

    Techniques Used: Isolation, Reverse Transcription Polymerase Chain Reaction, Acrylamide Gel Assay, Multiple Displacement Amplification, Synthesized, Real-time Polymerase Chain Reaction, Polymerase Chain Reaction, MANN-WHITNEY

    4) Product Images from "Rat NaV1.7 loss-of-function genetic model: Deficient nociceptive and neuropathic pain behavior with retained olfactory function and intra-epidermal nerve fibers"

    Article Title: Rat NaV1.7 loss-of-function genetic model: Deficient nociceptive and neuropathic pain behavior with retained olfactory function and intra-epidermal nerve fibers

    Journal: Molecular Pain

    doi: 10.1177/1744806919881846

    Na V 1.7 Immunostaining in DRG and sciatic nerve. (A and A′) Na V 1.7 immunoreactivity in DRG from WT but not HOM-KI rats. (B and B′). Corresponding isotype control sections. (C and C′) Na V 1.7 immunoreactivity in sciatic nerve from WT but not HOM-KI rats. (D and D′) Corresponding isotype control sections. Scale bars 200 µm. Three rats of each genotype and gender were evaluated. HOM-KI: rats homozygous for the knock-in allele; WT: wild type.
    Figure Legend Snippet: Na V 1.7 Immunostaining in DRG and sciatic nerve. (A and A′) Na V 1.7 immunoreactivity in DRG from WT but not HOM-KI rats. (B and B′). Corresponding isotype control sections. (C and C′) Na V 1.7 immunoreactivity in sciatic nerve from WT but not HOM-KI rats. (D and D′) Corresponding isotype control sections. Scale bars 200 µm. Three rats of each genotype and gender were evaluated. HOM-KI: rats homozygous for the knock-in allele; WT: wild type.

    Techniques Used: Immunostaining, Knock-In

    5) Product Images from "Differential ratio amplicons (Ramp) for the evaluation of RNA integrity extracted from complex environmental samples"

    Article Title: Differential ratio amplicons (Ramp) for the evaluation of RNA integrity extracted from complex environmental samples

    Journal: Environmental Microbiology

    doi: 10.1111/1462-2920.14516

    Effect of UV degradation on RNA integrity measured via the RIN (A), with RT‐Q‐PCR (B) and RIN versus R amp (C). For RIN, RNA integrity visualized in virtual gels (A; left) and electropherogram (A; right) are displayed against incubation period under UV. RNA ladder shows size in nucleotides (nt). B. Effect of degradation on transcript quantification; Amp 1–3: average Ct ( n = 3) of one of the three possible glnA amplicons; amoA : average amoA Ct ( n = 3) of the Bacterial amoA transcript; 16S rRNA : average 16S rRNA Ct ( n = 3) of the bacterial 16S rRNA transcript. Effect of RNA degradation on R amp index is presented in figure C; for comparison, RIN values were also plotted. Greek Letters indicate the result of TukeyHSD tests (points with different letters had values significantly different from each other using 0.05 as threshold for the p value).
    Figure Legend Snippet: Effect of UV degradation on RNA integrity measured via the RIN (A), with RT‐Q‐PCR (B) and RIN versus R amp (C). For RIN, RNA integrity visualized in virtual gels (A; left) and electropherogram (A; right) are displayed against incubation period under UV. RNA ladder shows size in nucleotides (nt). B. Effect of degradation on transcript quantification; Amp 1–3: average Ct ( n = 3) of one of the three possible glnA amplicons; amoA : average amoA Ct ( n = 3) of the Bacterial amoA transcript; 16S rRNA : average 16S rRNA Ct ( n = 3) of the bacterial 16S rRNA transcript. Effect of RNA degradation on R amp index is presented in figure C; for comparison, RIN values were also plotted. Greek Letters indicate the result of TukeyHSD tests (points with different letters had values significantly different from each other using 0.05 as threshold for the p value).

    Techniques Used: Polymerase Chain Reaction, Incubation

    Effect of heat degradation on RNA integrity measured via the RIN (A), with RT‐Q‐PCR (B) and RIN versus R amp (C). For RIN, RNA integrity visualized in virtual gels (A; left) and electropherogram (A; right) are displayed against incubation period at 90°C. RNA ladder shows size in nucleotides (nt). B. Effect of degradation on transcript quantification; Amp 1–3: average Ct ( n = 3) of one of the three possible glnA amplicons; amoA : average amoA Ct ( n = 3) of the Bacterial amoA transcript; 16S rRNA : average 16S rRNA Ct ( n = 3) of the bacterial 16S rRNA transcript. Effect of RNA degradation on R amp index is presented in figure C; for comparison, RIN values were also plotted. Greek Letters indicate the result of TukeyHSD tests (points with different letters had values significantly different from each other using 0.05 as threshold for the p value).
    Figure Legend Snippet: Effect of heat degradation on RNA integrity measured via the RIN (A), with RT‐Q‐PCR (B) and RIN versus R amp (C). For RIN, RNA integrity visualized in virtual gels (A; left) and electropherogram (A; right) are displayed against incubation period at 90°C. RNA ladder shows size in nucleotides (nt). B. Effect of degradation on transcript quantification; Amp 1–3: average Ct ( n = 3) of one of the three possible glnA amplicons; amoA : average amoA Ct ( n = 3) of the Bacterial amoA transcript; 16S rRNA : average 16S rRNA Ct ( n = 3) of the bacterial 16S rRNA transcript. Effect of RNA degradation on R amp index is presented in figure C; for comparison, RIN values were also plotted. Greek Letters indicate the result of TukeyHSD tests (points with different letters had values significantly different from each other using 0.05 as threshold for the p value).

    Techniques Used: Polymerase Chain Reaction, Incubation

    Effect of RNase I degradation on RNA integrity measured via the RIN (A), with RT‐Q‐PCR (B) and RIN versus R amp (C). For RIN, RNA integrity visualized in virtual gels (A; left) and electropherogram (A; right) are displayed against incubation period with RNase I . RNA ladder shows size in nucleotides (nt). B. Effect of degradation on transcript quantification; Amp 1–3: average Ct ( n = 3) of one of the three possible glnA amplicons; amoA : average amoA Ct ( n = 3) of the Bacterial amoA transcript; 16S rRNA : average 16S rRNA Ct ( n = 3) of the bacterial 16S rRNA transcript. Effect of RNA degradation on R amp index is presented in figure C; for comparison, RIN values were also plotted. Greek letters indicate the result of TukeyHSD tests (points with different letters had values significantly different from each other using 0.05 as threshold for the p value).
    Figure Legend Snippet: Effect of RNase I degradation on RNA integrity measured via the RIN (A), with RT‐Q‐PCR (B) and RIN versus R amp (C). For RIN, RNA integrity visualized in virtual gels (A; left) and electropherogram (A; right) are displayed against incubation period with RNase I . RNA ladder shows size in nucleotides (nt). B. Effect of degradation on transcript quantification; Amp 1–3: average Ct ( n = 3) of one of the three possible glnA amplicons; amoA : average amoA Ct ( n = 3) of the Bacterial amoA transcript; 16S rRNA : average 16S rRNA Ct ( n = 3) of the bacterial 16S rRNA transcript. Effect of RNA degradation on R amp index is presented in figure C; for comparison, RIN values were also plotted. Greek letters indicate the result of TukeyHSD tests (points with different letters had values significantly different from each other using 0.05 as threshold for the p value).

    Techniques Used: Polymerase Chain Reaction, Incubation

    6) Product Images from "Lipid transfer from plants to arbuscular mycorrhiza fungi"

    Article Title: Lipid transfer from plants to arbuscular mycorrhiza fungi

    Journal: eLife

    doi: 10.7554/eLife.29107

    Transcript accumulation of KASI and RAM2 genes. ( A ) Transcript accumulation of DIS , DIS-LIKE, KASI and RAM2 in control (mock) and R. irregularis colonized (AM) roots and in different organs of L. japonicus assessed by qRT-PCR. Expression values were normalized to those of the constitutively expressed gene EF1α ( DIS , DIS-LIKE, KASI) and Ubiquitin10 ( RAM2) . Black circles represent three biological replicates. Different letters indicate significant differences (ANOVA; posthoc Tukey; n = 15; p≤0.05, F 4,14 ( KASI ) = 1.191, F 4,14 ( DIS ) = 8.412, F 4,14 ( DIS-LIKE ) = 4.563; p≤0.001, F 4,14 = 67.41 ( RAM2) ). AM plants were inoculated with R. irregularis. Control and AM plants were harvested 5 wpi. ( B ) Arbuscule phenotype in wild type and dis-like-5 mutant roots after 5 wpi with R. irregularis as indicated by acid ink staining. White arrow heads indicate arbuscules. DOI: http://dx.doi.org/10.7554/eLife.29107.018
    Figure Legend Snippet: Transcript accumulation of KASI and RAM2 genes. ( A ) Transcript accumulation of DIS , DIS-LIKE, KASI and RAM2 in control (mock) and R. irregularis colonized (AM) roots and in different organs of L. japonicus assessed by qRT-PCR. Expression values were normalized to those of the constitutively expressed gene EF1α ( DIS , DIS-LIKE, KASI) and Ubiquitin10 ( RAM2) . Black circles represent three biological replicates. Different letters indicate significant differences (ANOVA; posthoc Tukey; n = 15; p≤0.05, F 4,14 ( KASI ) = 1.191, F 4,14 ( DIS ) = 8.412, F 4,14 ( DIS-LIKE ) = 4.563; p≤0.001, F 4,14 = 67.41 ( RAM2) ). AM plants were inoculated with R. irregularis. Control and AM plants were harvested 5 wpi. ( B ) Arbuscule phenotype in wild type and dis-like-5 mutant roots after 5 wpi with R. irregularis as indicated by acid ink staining. White arrow heads indicate arbuscules. DOI: http://dx.doi.org/10.7554/eLife.29107.018

    Techniques Used: Quantitative RT-PCR, Expressing, Mutagenesis, Staining

    7) Product Images from "Junctophilin-4, a component of the endoplasmic reticulum–plasma membrane junctions, regulates Ca2+ dynamics in T cells"

    Article Title: Junctophilin-4, a component of the endoplasmic reticulum–plasma membrane junctions, regulates Ca2+ dynamics in T cells

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    doi: 10.1073/pnas.1524229113

    STIM1 depletion reduces ER Ca 2+ content in T cells. ( A ) Expression of STIM1 ( Left ) and Orai1 ( Right ) in control and JP4-depleted Jurkat cells detected by immunoblotting and quantitative RT-PCR, respectively. The transcript data show mean ± SEM of triplicates. ( B ) STIM1 expression in control (Scr) or STIM1-depleted (STIM1 KD) Jurkat cells determined by immunoblotting. β-Actin was used as a loading control. ( C ) SOCE and ER Ca 2+ content measurements in control (Scr) or STIM1-depleted (STIM1 KD) Jurkat cells after passive store depletion with thapsigargin (TG) (1 μM) in Ca 2+ -free solution, and addition of 2 mM Ca 2+ -containing solution ( Left ). Traces show averaged (±SEM) responses from 30 to 50 Jurkat T cells. Bar graphs show change in ER Ca 2+ content and SOCE (±SEM) from three independent experiments. Control (Scr) and STIM1-depleted (KD) Jurkat cells were treated with ionomycin (1 μM, iono) in Ca 2+ -free solution to measure the ER Ca 2+ content ( Right two panels). Traces show averaged (±SEM) responses from 30 to 50 cells, and bar graph shows change in ER Ca 2+ content (±SEM) from three independent experiments. * P
    Figure Legend Snippet: STIM1 depletion reduces ER Ca 2+ content in T cells. ( A ) Expression of STIM1 ( Left ) and Orai1 ( Right ) in control and JP4-depleted Jurkat cells detected by immunoblotting and quantitative RT-PCR, respectively. The transcript data show mean ± SEM of triplicates. ( B ) STIM1 expression in control (Scr) or STIM1-depleted (STIM1 KD) Jurkat cells determined by immunoblotting. β-Actin was used as a loading control. ( C ) SOCE and ER Ca 2+ content measurements in control (Scr) or STIM1-depleted (STIM1 KD) Jurkat cells after passive store depletion with thapsigargin (TG) (1 μM) in Ca 2+ -free solution, and addition of 2 mM Ca 2+ -containing solution ( Left ). Traces show averaged (±SEM) responses from 30 to 50 Jurkat T cells. Bar graphs show change in ER Ca 2+ content and SOCE (±SEM) from three independent experiments. Control (Scr) and STIM1-depleted (KD) Jurkat cells were treated with ionomycin (1 μM, iono) in Ca 2+ -free solution to measure the ER Ca 2+ content ( Right two panels). Traces show averaged (±SEM) responses from 30 to 50 cells, and bar graph shows change in ER Ca 2+ content (±SEM) from three independent experiments. * P

    Techniques Used: Expressing, Quantitative RT-PCR

    8) Product Images from "Retromer and TBC1D5 maintain late endosomal RAB7 domains to enable amino acid–induced mTORC1 signaling"

    Article Title: Retromer and TBC1D5 maintain late endosomal RAB7 domains to enable amino acid–induced mTORC1 signaling

    Journal: The Journal of Cell Biology

    doi: 10.1083/jcb.201812110

    Hyperactivated RAB7 disrupts amino acid signaling. (A) Parental HeLa cells, VPS29 KO cells, and VPS29 KOs infected with a lentivirus encoding GFP-RAB7 and dominant-negative GFP-RAB7-T22N were starved in amino acid–free DMEM and stimulated with amino acids for 45 min. The level of phosphorylated S6K1 was then determined and quantified by Western blotting over three independent experiments. (B) Parental HeLa cells, VPS29 KO cells, and VPS29 KO cells transfected with Cas9 and a pool of three gRNAs against the RAB7-activating GEF protein CCZ1 were starved in amino acid–free DMEM and stimulated with amino acids for 45 min. The level of phosphorylated S6K1 was then determined and quantified by Western blotting over three independent experiments. (C) Parental HeLa cells, VPS29 KO cells, and VPS29/RAB7a double-KO cells were starved in amino acid–free DMEM and stimulated with amino acids for 45 min. The level of phosphorylated S6K1 was then determined and quantified by Western blotting over three independent experiments. (D) Parental HeLa cells, VPS29 KO cells, and VPS29 KOs treated with siRNA against endogenous RAB7 were starved in amino acid–free DMEM and stimulated with amino acids for 45 min. The level of phosphorylated S6K1 was then determined and quantified by Western blotting over three independent experiments. (E) Parental HeLa cells and VPS35 KO cells were fixed and stained for endogenous RAB7 (Alexa Fluor 488, green) and endogenous LAMP2 (Alexa Fluor 594, red). Images were acquired by confocal microscopy. (F) HeLa WT cells were infected with a lentivirus expressing GFP-RAB7a at low levels. The cells were then fixed in PFA and stained for endogenous LAMTOR1 (Alexa Fluor 594, red) and LAMP1 (Alexa Fluor 405, blue). Z-stacked images were acquired on a Zeiss confocal microscope equipped with Airyscan detectors to achieve higher resolution. The images display a 3D reconstruction after Airyscan processing. Scale bars represent 1 µm (F). Error bars = SD; P values stem from a two-tailed, unpaired t test between the indicated conditions.
    Figure Legend Snippet: Hyperactivated RAB7 disrupts amino acid signaling. (A) Parental HeLa cells, VPS29 KO cells, and VPS29 KOs infected with a lentivirus encoding GFP-RAB7 and dominant-negative GFP-RAB7-T22N were starved in amino acid–free DMEM and stimulated with amino acids for 45 min. The level of phosphorylated S6K1 was then determined and quantified by Western blotting over three independent experiments. (B) Parental HeLa cells, VPS29 KO cells, and VPS29 KO cells transfected with Cas9 and a pool of three gRNAs against the RAB7-activating GEF protein CCZ1 were starved in amino acid–free DMEM and stimulated with amino acids for 45 min. The level of phosphorylated S6K1 was then determined and quantified by Western blotting over three independent experiments. (C) Parental HeLa cells, VPS29 KO cells, and VPS29/RAB7a double-KO cells were starved in amino acid–free DMEM and stimulated with amino acids for 45 min. The level of phosphorylated S6K1 was then determined and quantified by Western blotting over three independent experiments. (D) Parental HeLa cells, VPS29 KO cells, and VPS29 KOs treated with siRNA against endogenous RAB7 were starved in amino acid–free DMEM and stimulated with amino acids for 45 min. The level of phosphorylated S6K1 was then determined and quantified by Western blotting over three independent experiments. (E) Parental HeLa cells and VPS35 KO cells were fixed and stained for endogenous RAB7 (Alexa Fluor 488, green) and endogenous LAMP2 (Alexa Fluor 594, red). Images were acquired by confocal microscopy. (F) HeLa WT cells were infected with a lentivirus expressing GFP-RAB7a at low levels. The cells were then fixed in PFA and stained for endogenous LAMTOR1 (Alexa Fluor 594, red) and LAMP1 (Alexa Fluor 405, blue). Z-stacked images were acquired on a Zeiss confocal microscope equipped with Airyscan detectors to achieve higher resolution. The images display a 3D reconstruction after Airyscan processing. Scale bars represent 1 µm (F). Error bars = SD; P values stem from a two-tailed, unpaired t test between the indicated conditions.

    Techniques Used: Infection, Dominant Negative Mutation, Western Blot, Transfection, Staining, Confocal Microscopy, Expressing, Microscopy, Two Tailed Test

    mTORC1 regulation through growth factors, TSC2, and SLC38A9 localization in retromer-deficient cells. (A) TFEB-GFP was transiently expressed in parental HeLa cells and in VPS35 and VPS29 KO cells. Parental and clonal VPS35 deficient U2OS cells were serum starved overnight and treated with dialyzed FBS for the indicated time points. Activation of mTORC1 was tested through Western blotting of phosphorylated mTORC1 substrates S6K1 and S6. (B) Parental U2OS and two clonal VPS35 KO cell lines were fixed and stained for endogenous TSC2 (Alexa Fluor 488, green) and LAMP2 (Alexa Fluor 594, red). (C) FLAG-SLC38A9 (Alexa Fluor 488, green) was lentivirally expressed in parental and VPS29 or VPS35 KO HeLa cells and costained with the lysosome marker LAMP1 (Alexa Fluor 594, red). (D) GFP-mLST8 was lentivirally expressed in WT and VPS35-deficient cells and precipitated through GFP-trap beads. The precipitates were analyzed for mTORC1 assembly by Western blotting for endogenous RAPTOR and mTOR. (E) Parental and VPS35 KO U2OS cells were serum starved overnight and stimulated with dialyzed FBS for the indicated times. mTORC1 substrate phosphorylation was assessed through Western blotting. Quantifications were done across three independent experiments. Scale bars represent 10 µm. Error bars = SD.
    Figure Legend Snippet: mTORC1 regulation through growth factors, TSC2, and SLC38A9 localization in retromer-deficient cells. (A) TFEB-GFP was transiently expressed in parental HeLa cells and in VPS35 and VPS29 KO cells. Parental and clonal VPS35 deficient U2OS cells were serum starved overnight and treated with dialyzed FBS for the indicated time points. Activation of mTORC1 was tested through Western blotting of phosphorylated mTORC1 substrates S6K1 and S6. (B) Parental U2OS and two clonal VPS35 KO cell lines were fixed and stained for endogenous TSC2 (Alexa Fluor 488, green) and LAMP2 (Alexa Fluor 594, red). (C) FLAG-SLC38A9 (Alexa Fluor 488, green) was lentivirally expressed in parental and VPS29 or VPS35 KO HeLa cells and costained with the lysosome marker LAMP1 (Alexa Fluor 594, red). (D) GFP-mLST8 was lentivirally expressed in WT and VPS35-deficient cells and precipitated through GFP-trap beads. The precipitates were analyzed for mTORC1 assembly by Western blotting for endogenous RAPTOR and mTOR. (E) Parental and VPS35 KO U2OS cells were serum starved overnight and stimulated with dialyzed FBS for the indicated times. mTORC1 substrate phosphorylation was assessed through Western blotting. Quantifications were done across three independent experiments. Scale bars represent 10 µm. Error bars = SD.

    Techniques Used: Activation Assay, Western Blot, Staining, Marker

    CRISPR/Cas9 screening identifies TBC1D5 as the retromer component that maintains amino acid signaling. (A) HeLa cells were transfected with Cas9, a pool of three gRNAs targeting the indicated retromer component, and a puromycin resistance construct. Transfectants were selected with puromycin and incubated for 5 d, followed by starvation in serum- and amino acid–free medium for 3 h and acute (45-min) stimulation with a mixture of amino acids (MEM recipe and glutamine). mTORC1 activity upon amino acid stimulation was assessed by Western blotting of pS6K1 and pS6. The quantification was done across six independent experiments. (B) HeLa cells were transfected with a pool of gRNAs targeting the TBC1D5 gene, starved in serum-free DMEM, and stimulated with a mixture of amino acids. mTORC1 substrate phosphorylation was assessed by Western blotting. (C) HeLa cells were transfected with a mix of gRNAs targeting the indicated RAB7 GAP proteins. The cells were then starved in serum-free DMEM and stimulated with amino acids for 45 min, and mTORC1 substrate phosphorylation was analyzed by Western blotting. (D) HeLa cells were transfected with a mix of gRNAs targeting TBC1D5. 5 d after transfection, the KO cells were infected with a lentivirus encoding GFP-tagged TBC1D5 and the retromer-binding mutant TBC1D5-L142E. The cells were then starved in serum-free DMEM and stimulated with amino acids for 45 min, and mTORC1 substrate phosphorylation was analyzed by Western blotting ( n = 4). (E) Clonal VPS29 KO cells were infected with a lentivirus encoding WT and TBC1D5 binding deficient VPS29-L152E, starved in amino acid–free DMEM, and stimulated with amino acids. mTORC1 substrate phosphorylation was assessed by Western blotting in three independent experiments. (F) The cells were treated as in E, fixed, and stained for endogenous mTOR (Alexa Fluor 488, green) and LAMP2 (Alexa Fluor 594, red). Colocalization was quantified across 12 images from two independent experiments. Error bars = SD. Scale bars represent 10 µm; P values stem from a two-tailed, unpaired t test between the indicated conditions.
    Figure Legend Snippet: CRISPR/Cas9 screening identifies TBC1D5 as the retromer component that maintains amino acid signaling. (A) HeLa cells were transfected with Cas9, a pool of three gRNAs targeting the indicated retromer component, and a puromycin resistance construct. Transfectants were selected with puromycin and incubated for 5 d, followed by starvation in serum- and amino acid–free medium for 3 h and acute (45-min) stimulation with a mixture of amino acids (MEM recipe and glutamine). mTORC1 activity upon amino acid stimulation was assessed by Western blotting of pS6K1 and pS6. The quantification was done across six independent experiments. (B) HeLa cells were transfected with a pool of gRNAs targeting the TBC1D5 gene, starved in serum-free DMEM, and stimulated with a mixture of amino acids. mTORC1 substrate phosphorylation was assessed by Western blotting. (C) HeLa cells were transfected with a mix of gRNAs targeting the indicated RAB7 GAP proteins. The cells were then starved in serum-free DMEM and stimulated with amino acids for 45 min, and mTORC1 substrate phosphorylation was analyzed by Western blotting. (D) HeLa cells were transfected with a mix of gRNAs targeting TBC1D5. 5 d after transfection, the KO cells were infected with a lentivirus encoding GFP-tagged TBC1D5 and the retromer-binding mutant TBC1D5-L142E. The cells were then starved in serum-free DMEM and stimulated with amino acids for 45 min, and mTORC1 substrate phosphorylation was analyzed by Western blotting ( n = 4). (E) Clonal VPS29 KO cells were infected with a lentivirus encoding WT and TBC1D5 binding deficient VPS29-L152E, starved in amino acid–free DMEM, and stimulated with amino acids. mTORC1 substrate phosphorylation was assessed by Western blotting in three independent experiments. (F) The cells were treated as in E, fixed, and stained for endogenous mTOR (Alexa Fluor 488, green) and LAMP2 (Alexa Fluor 594, red). Colocalization was quantified across 12 images from two independent experiments. Error bars = SD. Scale bars represent 10 µm; P values stem from a two-tailed, unpaired t test between the indicated conditions.

    Techniques Used: CRISPR, Transfection, Construct, Incubation, Activity Assay, Western Blot, Infection, Binding Assay, Mutagenesis, Staining, Two Tailed Test

    Loss of VPS35 perturbs amino acid signaling and mTOR recruitment in U2OS osteosarcoma cells. (A) Parental U2OS cells and five clonal VPS35 KO cell lines were starved in EBSS supplemented with glucose and stimulated with amino acids (serum-free DMEM) for 45 min. mTORC1 activation was assessed by Western blotting of phosphorylated mTORC1 substrates S6K1 and S6. The quantification was performed across three independent experiments. (B) The experiment described in A was performed with cells that had been treated with siRNA against VPS35. The quantification was done over three independent experiments. (C) Parental U2OS cells and a clonal VPS35 KO cell line were starved in EBSS and stimulated with serum-free DMEM for the indicated time points. mTORC1 activation was assayed by Western blotting of phosphorylated mTORC1 substrates S6K1 and S6. The quantification was done over three independent experiments. (D) The experiment described in C was performed in cells treated with siRNA against VPS35 ( n = 3). (E) Parental U2OS cells and a clonal VPS35 KO cell line were starved in EBSS and stimulated with serum free DMEM for 45 min. The cells were fixed in cold PFA and stained for endogenous mTOR (Alexa Fluor 594, red) and LAMP1 (Alexa Fluor 488, green) and DNA (DAPI, blue). The quantification was done across 20 images from two independent experiments. (F) Two clonal VPS35 KO cell lines were infected with a lentivirus encoding only puromycin resistance (empty control virus expressing puromycin resistance only) and with a lentivirus encoding HA-VPS35. These cells were then starved in EBSS and stimulated with serum-free DMEM for the indicated times. mTORC1 activation was assessed by Western blotting of phosphorylated mTORC1 substrates. The quantifications were done over three independent experiments. Error bars = SD. Scale bars represent 10 µm; *, P
    Figure Legend Snippet: Loss of VPS35 perturbs amino acid signaling and mTOR recruitment in U2OS osteosarcoma cells. (A) Parental U2OS cells and five clonal VPS35 KO cell lines were starved in EBSS supplemented with glucose and stimulated with amino acids (serum-free DMEM) for 45 min. mTORC1 activation was assessed by Western blotting of phosphorylated mTORC1 substrates S6K1 and S6. The quantification was performed across three independent experiments. (B) The experiment described in A was performed with cells that had been treated with siRNA against VPS35. The quantification was done over three independent experiments. (C) Parental U2OS cells and a clonal VPS35 KO cell line were starved in EBSS and stimulated with serum-free DMEM for the indicated time points. mTORC1 activation was assayed by Western blotting of phosphorylated mTORC1 substrates S6K1 and S6. The quantification was done over three independent experiments. (D) The experiment described in C was performed in cells treated with siRNA against VPS35 ( n = 3). (E) Parental U2OS cells and a clonal VPS35 KO cell line were starved in EBSS and stimulated with serum free DMEM for 45 min. The cells were fixed in cold PFA and stained for endogenous mTOR (Alexa Fluor 594, red) and LAMP1 (Alexa Fluor 488, green) and DNA (DAPI, blue). The quantification was done across 20 images from two independent experiments. (F) Two clonal VPS35 KO cell lines were infected with a lentivirus encoding only puromycin resistance (empty control virus expressing puromycin resistance only) and with a lentivirus encoding HA-VPS35. These cells were then starved in EBSS and stimulated with serum-free DMEM for the indicated times. mTORC1 activation was assessed by Western blotting of phosphorylated mTORC1 substrates. The quantifications were done over three independent experiments. Error bars = SD. Scale bars represent 10 µm; *, P

    Techniques Used: Activation Assay, Western Blot, Staining, Infection, Expressing

    Amino acid signaling and mTOR recruitment to lysosomes are defective in VPS29-deficient HeLa cells. (A) Parental HeLa and four VPS35 KO cell lines generated with CRISPR/Cas9 were starved in serum-free DMEM without amino acids and stimulated with a mixture of amino acids (MEM recipe) and glutamine for 45 min. mTORC1 activation was assessed by Western blotting of phosphorylated mTORC1 substrates S6K1 and S6. (B) Parental HeLa cells, VPS29 KO cells, and VPS29 KO cells with lentivirally reexpressed VPS29-myc were starved in serum- and amino acid–free medium and stimulated with amino acids for 45 min. The quantification was done over three independent experiments. (C) Parental HeLa cells and clonal VPS29 KO cells were cultured in either full growth medium (FM) or serum- and amino acid–free DMEM (AA starve) or were acutely stimulated with amino acids after 1 h of starvation. mTORC1 activation was assessed by Western blotting of phosphorylated mTORC1 substrates S6K1. (D) Parental HeLa cells and VPS29 KO cells were starved in serum- and amino acid–free DMEM and stimulated with amino acids for the indicated time points. mTORC1 activation was assessed by Western blotting of phosphorylated mTORC1 substrates S6K1 and S6. The quantification was done over three independent experiments. (E) Parental HeLa cells and VPS29 KO cells were starved in serum- and amino acid–free DMEM and stimulated with the indicated individual amino acids. mTORC1 activation was assessed by Western blotting of phosphorylated mTORC1 substrate S6K1. (F) Parental HeLa cells and clonal VPS29 KO cells were starved in serum- and amino acid–free DMEM for 3 h and stimulated with amino acids for 45 min. Following stimulation, cells were fixed and stained for endogenous mTOR (Alexa Fluor 488, green) and endogenous LAMP2 (Alexa Fluor 594, red) and DNA (DAPI, blue). The colocalization between mTOR and LAMP2 was quantified across 20 images from two independent experiments. (G) Parental HeLa cells (top panel) and VPS29 KO cells (bottom panel) were starved in EBSS for the indicated time points and stimulated with amino acids for 5 and 15 min. The cells were then fixed in PFA and stained for endogenous mTOR (lower row) and endogenous LAMP2 (upper row). Error bars = SD. Scale bars represent 10 µm; *, P
    Figure Legend Snippet: Amino acid signaling and mTOR recruitment to lysosomes are defective in VPS29-deficient HeLa cells. (A) Parental HeLa and four VPS35 KO cell lines generated with CRISPR/Cas9 were starved in serum-free DMEM without amino acids and stimulated with a mixture of amino acids (MEM recipe) and glutamine for 45 min. mTORC1 activation was assessed by Western blotting of phosphorylated mTORC1 substrates S6K1 and S6. (B) Parental HeLa cells, VPS29 KO cells, and VPS29 KO cells with lentivirally reexpressed VPS29-myc were starved in serum- and amino acid–free medium and stimulated with amino acids for 45 min. The quantification was done over three independent experiments. (C) Parental HeLa cells and clonal VPS29 KO cells were cultured in either full growth medium (FM) or serum- and amino acid–free DMEM (AA starve) or were acutely stimulated with amino acids after 1 h of starvation. mTORC1 activation was assessed by Western blotting of phosphorylated mTORC1 substrates S6K1. (D) Parental HeLa cells and VPS29 KO cells were starved in serum- and amino acid–free DMEM and stimulated with amino acids for the indicated time points. mTORC1 activation was assessed by Western blotting of phosphorylated mTORC1 substrates S6K1 and S6. The quantification was done over three independent experiments. (E) Parental HeLa cells and VPS29 KO cells were starved in serum- and amino acid–free DMEM and stimulated with the indicated individual amino acids. mTORC1 activation was assessed by Western blotting of phosphorylated mTORC1 substrate S6K1. (F) Parental HeLa cells and clonal VPS29 KO cells were starved in serum- and amino acid–free DMEM for 3 h and stimulated with amino acids for 45 min. Following stimulation, cells were fixed and stained for endogenous mTOR (Alexa Fluor 488, green) and endogenous LAMP2 (Alexa Fluor 594, red) and DNA (DAPI, blue). The colocalization between mTOR and LAMP2 was quantified across 20 images from two independent experiments. (G) Parental HeLa cells (top panel) and VPS29 KO cells (bottom panel) were starved in EBSS for the indicated time points and stimulated with amino acids for 5 and 15 min. The cells were then fixed in PFA and stained for endogenous mTOR (lower row) and endogenous LAMP2 (upper row). Error bars = SD. Scale bars represent 10 µm; *, P

    Techniques Used: Generated, CRISPR, Activation Assay, Western Blot, Cell Culture, Staining

    9) Product Images from "Modeling Dengue Virus-Hepatic Cell Interactions Using Human Pluripotent Stem Cell-Derived Hepatocyte-like Cells"

    Article Title: Modeling Dengue Virus-Hepatic Cell Interactions Using Human Pluripotent Stem Cell-Derived Hepatocyte-like Cells

    Journal: Stem Cell Reports

    doi: 10.1016/j.stemcr.2016.07.012

    HLCs Supported Persistent and Productive Infection of DENV (A) DENV2 infection of HLCs in a differentiation stage-dependent manner. The hESCs (day 0) and differentiated cells at various differentiation stages from day 1 to day 13 were inoculated with DENV2 (strain 16681) at an MOI of 2 for 48 hr, and cell lysates were collected for western blotting. (B) Immunostaining analysis of DENV2-infected HLCs. hESCs (day 0), definitive endoderm (day 3), hepatic progenitors (day 6), and HLCs (day 10) were infected by DENV2 as described above. Cell were fixed and stained with anti-NS3 antibody. Scale bar, 50 μm. (C) DENV2 infection was blocked in HLCs by inhibitors. HLCs at day 15 after differentiation were pretreated with IFN-α2a (1,000 U/mL), MPA (1 μM), CQ (5 μg/mL), or DMSO (0.1%, v/v) for 8 hr, then subjected to DENV2 infection as described above. (D) qRT-PCR analysis of DENV RNA levels. Cells were treated as described above. The relative DENV RNA level in each sample was normalized to DMSO-treated samples. (E) Persistent replication of DENV2 in HLCs. Day-15 HLCs were exposed to DENV2 for 6 hr before the cells were stringently washed. NS1 protein in the supernatant collected every 24 hr after infection was measured by ELISA assay. (F) DENV2 particles released from HLCs efficiently infected C6/36 cells. HLCs at day 15 were infected with DENV2 at an MOI of 2 for 6 hr. The inoculum was then removed and the cells washed stringently with PBS five times before the fresh medium was added. The supernatants were collected every 24 hr from day 16 to day 20. After each change of the spent medium, cells were washed stringently as described above. Each medium supernatant was added to naive C6/36 cells and incubated for 24 hr. Cells were then lysed and subjected to western blotting analysis. DENV2 derived from C6/36 cells was used as a positive control. (G) Virus titration. Virus titers were calculated in the above supernatants collected at 24, 48, and 72 hr post infection. (H) Upregulation of DENV entry factors during the differentiation process. Total RNA from differentiated cells at the indicated days were extracted and subjected to real-time qRT-PCR to measure the cellular expressions of AXL , HAVCR1 , PROS , and CLDN1 . GAPDH was used as the reference and its expression was set as 1. All the genes were normalized to GAPDH to obtain their relative gene-expression ratio. Three independent experiments were performed. Data are presented as mean ± SD. p Values were calculated by Student's t test: ∗ p
    Figure Legend Snippet: HLCs Supported Persistent and Productive Infection of DENV (A) DENV2 infection of HLCs in a differentiation stage-dependent manner. The hESCs (day 0) and differentiated cells at various differentiation stages from day 1 to day 13 were inoculated with DENV2 (strain 16681) at an MOI of 2 for 48 hr, and cell lysates were collected for western blotting. (B) Immunostaining analysis of DENV2-infected HLCs. hESCs (day 0), definitive endoderm (day 3), hepatic progenitors (day 6), and HLCs (day 10) were infected by DENV2 as described above. Cell were fixed and stained with anti-NS3 antibody. Scale bar, 50 μm. (C) DENV2 infection was blocked in HLCs by inhibitors. HLCs at day 15 after differentiation were pretreated with IFN-α2a (1,000 U/mL), MPA (1 μM), CQ (5 μg/mL), or DMSO (0.1%, v/v) for 8 hr, then subjected to DENV2 infection as described above. (D) qRT-PCR analysis of DENV RNA levels. Cells were treated as described above. The relative DENV RNA level in each sample was normalized to DMSO-treated samples. (E) Persistent replication of DENV2 in HLCs. Day-15 HLCs were exposed to DENV2 for 6 hr before the cells were stringently washed. NS1 protein in the supernatant collected every 24 hr after infection was measured by ELISA assay. (F) DENV2 particles released from HLCs efficiently infected C6/36 cells. HLCs at day 15 were infected with DENV2 at an MOI of 2 for 6 hr. The inoculum was then removed and the cells washed stringently with PBS five times before the fresh medium was added. The supernatants were collected every 24 hr from day 16 to day 20. After each change of the spent medium, cells were washed stringently as described above. Each medium supernatant was added to naive C6/36 cells and incubated for 24 hr. Cells were then lysed and subjected to western blotting analysis. DENV2 derived from C6/36 cells was used as a positive control. (G) Virus titration. Virus titers were calculated in the above supernatants collected at 24, 48, and 72 hr post infection. (H) Upregulation of DENV entry factors during the differentiation process. Total RNA from differentiated cells at the indicated days were extracted and subjected to real-time qRT-PCR to measure the cellular expressions of AXL , HAVCR1 , PROS , and CLDN1 . GAPDH was used as the reference and its expression was set as 1. All the genes were normalized to GAPDH to obtain their relative gene-expression ratio. Three independent experiments were performed. Data are presented as mean ± SD. p Values were calculated by Student's t test: ∗ p

    Techniques Used: Infection, Western Blot, Immunostaining, Staining, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Incubation, Derivative Assay, Positive Control, Titration, Expressing

    DENV Infection Inhibited the Expression of ALB and F5 (A) Bar plot of log 2 (DENV2-treated/untreated) ratios of RNA-seq expression values for the 291 RefSeq genes preferentially expressed in liver ( Liu et al., 2008 ). RNA-seq data were analyzed in the same way as described in Figure 4 . (B) Immunostaining of DENV2-infected HLCs. HLCs at day 15 were infected by DENV2 at an MOI of 2 for 72 hr. Cells were fixed for immunofluorescence analysis. Scale bar, 50 μm. (C) ALB and F5 expression were downregulated in both the HLCs and PHHs after DENV2 infection. PHHs and HLCs were infected by DENV2 at an MOI of 5 for 72 hr and then subjected to western blotting. (D) Other three serotypes of DENV reduced F5 and ALB expression in the HLCs and PHHs. HLCs and PHHs were infected by DENV type 1-Hawaii, DENV type 3-MK-594-87, DENV type 4-H241, and DENV type 4-D85-019 as described above. Cell lysates were collected for western blotting. See also Figure S5 and Table S2 .
    Figure Legend Snippet: DENV Infection Inhibited the Expression of ALB and F5 (A) Bar plot of log 2 (DENV2-treated/untreated) ratios of RNA-seq expression values for the 291 RefSeq genes preferentially expressed in liver ( Liu et al., 2008 ). RNA-seq data were analyzed in the same way as described in Figure 4 . (B) Immunostaining of DENV2-infected HLCs. HLCs at day 15 were infected by DENV2 at an MOI of 2 for 72 hr. Cells were fixed for immunofluorescence analysis. Scale bar, 50 μm. (C) ALB and F5 expression were downregulated in both the HLCs and PHHs after DENV2 infection. PHHs and HLCs were infected by DENV2 at an MOI of 5 for 72 hr and then subjected to western blotting. (D) Other three serotypes of DENV reduced F5 and ALB expression in the HLCs and PHHs. HLCs and PHHs were infected by DENV type 1-Hawaii, DENV type 3-MK-594-87, DENV type 4-H241, and DENV type 4-D85-019 as described above. Cell lysates were collected for western blotting. See also Figure S5 and Table S2 .

    Techniques Used: Infection, Expressing, RNA Sequencing Assay, Immunostaining, Immunofluorescence, Western Blot

    10) Product Images from "Overexpression of an evolutionarily conserved drought-responsive sugarcane gene enhances salinity and drought resilience"

    Article Title: Overexpression of an evolutionarily conserved drought-responsive sugarcane gene enhances salinity and drought resilience

    Journal: Annals of Botany

    doi: 10.1093/aob/mcz044

    Cloning strategy used for Scdr2 overexpression in tobacco plants. (A) The complete coding sequence of Scdr2 was cloned under the control of the constitutive Cauliflower mosaic virus (CaMV) 35S promoter (p35S) and with the NOS polyadenylation signal (Nos-t) using pCambia2301 as the backbone. nptII (kanamycin resistance) gene expression was also driven by the p35S promoter. LB and RB correspond to the T-DNA left and right borders, respectively. Positions of some of the restriction sites are indicated. (B) Expression of Scdr2 in three independent T 3 generation transgenic lines and the WT. Total RNA was extracted from 2-week-old seedlings and then analysed using semi-quantitative RT–PCR. The Scdr2 gene product was obtained after 21 cycles, while the product of the tobacco actin gene, which was used as an internal standard, was obtained after 28 cycles ( n = 3). (C) Densitometric analysis of Scdr2 expression that was obtained from the semi-quantitative RT–PCR shown in (B).
    Figure Legend Snippet: Cloning strategy used for Scdr2 overexpression in tobacco plants. (A) The complete coding sequence of Scdr2 was cloned under the control of the constitutive Cauliflower mosaic virus (CaMV) 35S promoter (p35S) and with the NOS polyadenylation signal (Nos-t) using pCambia2301 as the backbone. nptII (kanamycin resistance) gene expression was also driven by the p35S promoter. LB and RB correspond to the T-DNA left and right borders, respectively. Positions of some of the restriction sites are indicated. (B) Expression of Scdr2 in three independent T 3 generation transgenic lines and the WT. Total RNA was extracted from 2-week-old seedlings and then analysed using semi-quantitative RT–PCR. The Scdr2 gene product was obtained after 21 cycles, while the product of the tobacco actin gene, which was used as an internal standard, was obtained after 28 cycles ( n = 3). (C) Densitometric analysis of Scdr2 expression that was obtained from the semi-quantitative RT–PCR shown in (B).

    Techniques Used: Clone Assay, Over Expression, Sequencing, Expressing, Transgenic Assay, Quantitative RT-PCR

    11) Product Images from "First Report of a Fatal Case Associated with EV-D68 Infection in Hong Kong and Emergence of an Interclade Recombinant in China Revealed by Genome Analysis"

    Article Title: First Report of a Fatal Case Associated with EV-D68 Infection in Hong Kong and Emergence of an Interclade Recombinant in China Revealed by Genome Analysis

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms18051065

    Phylogenetic analysis using various gene sequences of EV-D68 strains. The trees were constructed using the ML method, with bootstrap values calculated from 1000 trees. Sequences for 207 nucleotide positions in each VP4 region ( A ), 744 nucleotide positions in each VP2 region ( B ), 705 nucleotide pos itions in each VP3 region ( C ), 441 nucleotide positions in each 2A region ( D ), 297 nucleotide positions in each 2B region ( E ), 990 nucleotide positions in each 2C region ( F ), 333 nucleotide positions in each 3A–3B region ( G ), 549 nucleotide positions in each 3C region ( H ) and 1371 nucleotide positions in each 3D region ( I ) were included in the analysis. Bootstrap values expressed as percentages are shown at the nodes and the scale reflects the number of nucleotide substitutions per site along the branches. Only bootstrap values > 70% are shown. Black squares indicate the three EV-D68 strains subjected to complete genome sequencing in the present study. The interclade recombinants from mainland China are highlighted in grey.
    Figure Legend Snippet: Phylogenetic analysis using various gene sequences of EV-D68 strains. The trees were constructed using the ML method, with bootstrap values calculated from 1000 trees. Sequences for 207 nucleotide positions in each VP4 region ( A ), 744 nucleotide positions in each VP2 region ( B ), 705 nucleotide pos itions in each VP3 region ( C ), 441 nucleotide positions in each 2A region ( D ), 297 nucleotide positions in each 2B region ( E ), 990 nucleotide positions in each 2C region ( F ), 333 nucleotide positions in each 3A–3B region ( G ), 549 nucleotide positions in each 3C region ( H ) and 1371 nucleotide positions in each 3D region ( I ) were included in the analysis. Bootstrap values expressed as percentages are shown at the nodes and the scale reflects the number of nucleotide substitutions per site along the branches. Only bootstrap values > 70% are shown. Black squares indicate the three EV-D68 strains subjected to complete genome sequencing in the present study. The interclade recombinants from mainland China are highlighted in grey.

    Techniques Used: Construct, Sequencing

    12) Product Images from "A Petunia Homeodomain-Leucine Zipper Protein, PhHD-Zip, Plays an Important Role in Flower Senescence"

    Article Title: A Petunia Homeodomain-Leucine Zipper Protein, PhHD-Zip, Plays an Important Role in Flower Senescence

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0088320

    Expression of PhHD-Zip in petunia flowers in response to ethylene and 1-MCP treatments. “ Ethylene ” Flowers harvested at anthesis and treated continuously with ethylene (3 ppm), “ 1-MCP/Ethylene” Flowers harvested at anthesis and treated with 1-MCP (50 nL/L) for 4 hours before a continuous ethylene treatment. A. A representative gel image from semi-quantitative PCR of RNA isolated from corollas harvested at intervals. 26S RNA: the internal control. Samples were analyzed after 30 cycles for PhHD-Zip and after 24 cycles for 26S RNA . B. Relative expression levels of PhHD-Zip (quantification of the gel pictures; error bars show SE of the means of three biological replicates).
    Figure Legend Snippet: Expression of PhHD-Zip in petunia flowers in response to ethylene and 1-MCP treatments. “ Ethylene ” Flowers harvested at anthesis and treated continuously with ethylene (3 ppm), “ 1-MCP/Ethylene” Flowers harvested at anthesis and treated with 1-MCP (50 nL/L) for 4 hours before a continuous ethylene treatment. A. A representative gel image from semi-quantitative PCR of RNA isolated from corollas harvested at intervals. 26S RNA: the internal control. Samples were analyzed after 30 cycles for PhHD-Zip and after 24 cycles for 26S RNA . B. Relative expression levels of PhHD-Zip (quantification of the gel pictures; error bars show SE of the means of three biological replicates).

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction, Isolation

    Expression of PhHD-Zip in different tissues of petunia. A. A representative gel image from semi-quantitative PCR of RNA isolated from different tissues. 26S RNA: the internal control. Samples were analyzed after 33 cycles for PhHD-Zip , and after 24 cycles for 26S RNA . B. Relative expression levels of PhHD-Zip in different tissues (quantification of the gel pictures; error bars show SE of the means of three biological replicates; different letters denote significant differences using Duncan’s test at P
    Figure Legend Snippet: Expression of PhHD-Zip in different tissues of petunia. A. A representative gel image from semi-quantitative PCR of RNA isolated from different tissues. 26S RNA: the internal control. Samples were analyzed after 33 cycles for PhHD-Zip , and after 24 cycles for 26S RNA . B. Relative expression levels of PhHD-Zip in different tissues (quantification of the gel pictures; error bars show SE of the means of three biological replicates; different letters denote significant differences using Duncan’s test at P

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction, Isolation

    Expression of senescence-related genes in D7 petunia flowers. Abundance of transcripts of genes associated with senescence were determined at D7 in purple control flowers (WT), in white flowers of plants inoculated with the CHS /TRV reporter construct (VW), and in white flowers of plant inoculated with the PhHD-Zip / CHS /TRV silencing construct. A. A representative gel image from semi-quantitative PCR of RNA isolated from corollas. 26S RNA: the internal control. Samples were analyzed after 33 cycles for ACS, after 30 cycles for other genes, and after 24 cycles for 26S RNA , respectively. B. Relative expression levels of different genes (quantification of the gel pictures; error bars show SE of the means of three biological replicates; different letters denote significant differences using Duncan’s test at P
    Figure Legend Snippet: Expression of senescence-related genes in D7 petunia flowers. Abundance of transcripts of genes associated with senescence were determined at D7 in purple control flowers (WT), in white flowers of plants inoculated with the CHS /TRV reporter construct (VW), and in white flowers of plant inoculated with the PhHD-Zip / CHS /TRV silencing construct. A. A representative gel image from semi-quantitative PCR of RNA isolated from corollas. 26S RNA: the internal control. Samples were analyzed after 33 cycles for ACS, after 30 cycles for other genes, and after 24 cycles for 26S RNA , respectively. B. Relative expression levels of different genes (quantification of the gel pictures; error bars show SE of the means of three biological replicates; different letters denote significant differences using Duncan’s test at P

    Techniques Used: Expressing, Construct, Real-time Polymerase Chain Reaction, Isolation

    Expression of PhHD-Zip in petunia flower under abiotic stress. Petunia flowers harvested at anthesis were placed in tubes with water, without water, with 50°C. A. A representative gel image from semi-quantitative PCR of RNA isolated from corollas harvested at intervals. 26S RNA: the internal control. Samples were analyzed after 30 cycles for PhHD-Zip, and after 24 cycles for 26S RNA. B. Relative expression levels of PhHD-Zip (quantification of the gel pictures; error bars show SE of the means of three biological replicates). C. Relative expression levels of PhHD-Zip determined using the same RNA samples, but using real-time quantitative PCR (error bars correspond to SE of the means of three biological replicates).
    Figure Legend Snippet: Expression of PhHD-Zip in petunia flower under abiotic stress. Petunia flowers harvested at anthesis were placed in tubes with water, without water, with 50°C. A. A representative gel image from semi-quantitative PCR of RNA isolated from corollas harvested at intervals. 26S RNA: the internal control. Samples were analyzed after 30 cycles for PhHD-Zip, and after 24 cycles for 26S RNA. B. Relative expression levels of PhHD-Zip (quantification of the gel pictures; error bars show SE of the means of three biological replicates). C. Relative expression levels of PhHD-Zip determined using the same RNA samples, but using real-time quantitative PCR (error bars correspond to SE of the means of three biological replicates).

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction, Isolation

    Expression of PhHD-Zip in petunia corollas during flower senescence. A. A representative gel image from semi-quantitative PCR of RNA isolated from corollas harvested at intervals after anthesis. D0: at anthesis; D2, D4, D7: 2, 4, and 7 days after anthesis, respectively. 26S RNA: the internal control. Samples were analyzed after 30 cycles of amplification for PhHD-Zip , and after 24 cycles of amplification for 26S RNA . B. Relative expression levels of PhHD-Zip (quantification of the gel pictures; error bars show SE of the means of three biological replicates).
    Figure Legend Snippet: Expression of PhHD-Zip in petunia corollas during flower senescence. A. A representative gel image from semi-quantitative PCR of RNA isolated from corollas harvested at intervals after anthesis. D0: at anthesis; D2, D4, D7: 2, 4, and 7 days after anthesis, respectively. 26S RNA: the internal control. Samples were analyzed after 30 cycles of amplification for PhHD-Zip , and after 24 cycles of amplification for 26S RNA . B. Relative expression levels of PhHD-Zip (quantification of the gel pictures; error bars show SE of the means of three biological replicates).

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction, Isolation, Amplification

    Effect of transgenic over-expression of PhHD-Zip on abundance of transcripts of PhHD-Zip and of ethylene biosynthesis genes in petunia corollas. WT: wild type flower; #3, #7: two transgenic lines of 35S:: PhHD-Zip . A. A representative gel image from semi-quantitative PCR of RNA isolated from harvested corollas. 26S RNA: the internal control. Samples were analyzed after 30 cycles for PhHD-Zip, ACO1 and ACO4 ; after 33 cycles for ACS ; and after 24 cycles for 26S RNA . B. Relative expression level of PhHD-Zip and ethylene biosynthesis genes (quantification of the gel pictures; error bars show SE of the means of three biological replicates; different letters denote significant differences using Duncan’s test at P
    Figure Legend Snippet: Effect of transgenic over-expression of PhHD-Zip on abundance of transcripts of PhHD-Zip and of ethylene biosynthesis genes in petunia corollas. WT: wild type flower; #3, #7: two transgenic lines of 35S:: PhHD-Zip . A. A representative gel image from semi-quantitative PCR of RNA isolated from harvested corollas. 26S RNA: the internal control. Samples were analyzed after 30 cycles for PhHD-Zip, ACO1 and ACO4 ; after 33 cycles for ACS ; and after 24 cycles for 26S RNA . B. Relative expression level of PhHD-Zip and ethylene biosynthesis genes (quantification of the gel pictures; error bars show SE of the means of three biological replicates; different letters denote significant differences using Duncan’s test at P

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

    13) Product Images from "Protocol Dependence of Sequencing-Based Gene Expression Measurements"

    Article Title: Protocol Dependence of Sequencing-Based Gene Expression Measurements

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0019287

    Analysis of expression using genomic bins. Three different genomic regions (A, B, C) are compared for expression with RiboMinus depleted RNA from liver and K562 samples. The vertical bars of varying heights show the expression level in each bin and the constant height bars designate those bins which are over-expressed at least 3× in either sample. Known annotated genes are labeled. In C, there is a 550 kb region with no annotated genes which is highly expressed in K562 but not liver and corresponds to a very long intergenic region (vlinc) on chromosome 4 [28] . The corresponding chromosomes and the coordinates of the regions (hg18 version of the genome) are shown.
    Figure Legend Snippet: Analysis of expression using genomic bins. Three different genomic regions (A, B, C) are compared for expression with RiboMinus depleted RNA from liver and K562 samples. The vertical bars of varying heights show the expression level in each bin and the constant height bars designate those bins which are over-expressed at least 3× in either sample. Known annotated genes are labeled. In C, there is a 550 kb region with no annotated genes which is highly expressed in K562 but not liver and corresponds to a very long intergenic region (vlinc) on chromosome 4 [28] . The corresponding chromosomes and the coordinates of the regions (hg18 version of the genome) are shown.

    Techniques Used: Expressing, Labeling

    14) Product Images from "Activation of hippocampal microglia in a murine model of cancer-induced pain"

    Article Title: Activation of hippocampal microglia in a murine model of cancer-induced pain

    Journal: Journal of Pain Research

    doi: 10.2147/JPR.S191860

    IF tumors increase hippocampal microglial markers. Notes: ( A–C ) qPCR confirmed the dramatic increase of hippocampal microglia in tumor-bearing mice, as demonstrated by microglial markers allograft inflammatory factor 1/ionized calcium-binding adapter molecule 1 (AIF1/Iba1), CD68, and CD11b; treatment with the CSF1R inhibitor Pexidartinib dramatically reduced hippocampal microglia across IF and SC tumor-bearing models. IF tumor-bearing mice demonstrated significantly higher levels of hippocampal microglial mRNA relative to SC tumor-bearing mice. ( D–F ) Factors associated with microglial activation were also affected by peripheral tumor, including TLR2, IL-1β, and the immediate-early gene c-Fos. Bars represent the mean of three or four biological replicates as a fold change relative to sham (n=1), with error bars indicating SEM. * P
    Figure Legend Snippet: IF tumors increase hippocampal microglial markers. Notes: ( A–C ) qPCR confirmed the dramatic increase of hippocampal microglia in tumor-bearing mice, as demonstrated by microglial markers allograft inflammatory factor 1/ionized calcium-binding adapter molecule 1 (AIF1/Iba1), CD68, and CD11b; treatment with the CSF1R inhibitor Pexidartinib dramatically reduced hippocampal microglia across IF and SC tumor-bearing models. IF tumor-bearing mice demonstrated significantly higher levels of hippocampal microglial mRNA relative to SC tumor-bearing mice. ( D–F ) Factors associated with microglial activation were also affected by peripheral tumor, including TLR2, IL-1β, and the immediate-early gene c-Fos. Bars represent the mean of three or four biological replicates as a fold change relative to sham (n=1), with error bars indicating SEM. * P

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

    15) Product Images from "Conservation of tRNA and rRNA 5-methylcytosine in the kingdom Plantae"

    Article Title: Conservation of tRNA and rRNA 5-methylcytosine in the kingdom Plantae

    Journal: BMC Plant Biology

    doi: 10.1186/s12870-015-0580-8

    NSUN5 methylates C2268 in Arabidopsis nuclear LSU 25S rRNA. a Genomic origins of methylated and non-methylated rRNA species. Methylated rRNAs were detected from all three genomes (3 biological replicates). b Left: Heatmap showing percentage methylation of cytosines in nuclear (N), chloroplast (C) and mitochondrial (M) rRNA sequences in wild type and mutants nop2a-2 , nsun5-1 , nop2b-1 and nop2c-1 . Cytosine positions are indicated next to rRNAs (3 biological replicates). Right: Partial secondary structure of 25S nuclear LSU rRNA helix 70 (domain IV) showing the cytosine position 2268 in red, which is methylated by NSUN5. c Genomic structure of nop2b , nop2c and nsun5 mutants showing T-DNA insertions (triangles) in exons (filled boxes). d Analysis of RNA methylation by NSUN5 at position C2268 on BS treated nuclear LSU 25S rRNA template. Above: Restriction maps of dCAPS amplified products showing the expected digest patterns of methylated and non-methylated template. The 25S_rRNA_F dCAPS primer contains a G mismatch at position four to generate a HinfI restriction site when C2268 is methylated. Below: Cleavage of PCR amplified product by HinfI confirms C2268 methylation in wild type as opposed to non-methylated C2268 in nsun5-1 results in loss of HinfI restriction site. Loading control is undigested PCR product
    Figure Legend Snippet: NSUN5 methylates C2268 in Arabidopsis nuclear LSU 25S rRNA. a Genomic origins of methylated and non-methylated rRNA species. Methylated rRNAs were detected from all three genomes (3 biological replicates). b Left: Heatmap showing percentage methylation of cytosines in nuclear (N), chloroplast (C) and mitochondrial (M) rRNA sequences in wild type and mutants nop2a-2 , nsun5-1 , nop2b-1 and nop2c-1 . Cytosine positions are indicated next to rRNAs (3 biological replicates). Right: Partial secondary structure of 25S nuclear LSU rRNA helix 70 (domain IV) showing the cytosine position 2268 in red, which is methylated by NSUN5. c Genomic structure of nop2b , nop2c and nsun5 mutants showing T-DNA insertions (triangles) in exons (filled boxes). d Analysis of RNA methylation by NSUN5 at position C2268 on BS treated nuclear LSU 25S rRNA template. Above: Restriction maps of dCAPS amplified products showing the expected digest patterns of methylated and non-methylated template. The 25S_rRNA_F dCAPS primer contains a G mismatch at position four to generate a HinfI restriction site when C2268 is methylated. Below: Cleavage of PCR amplified product by HinfI confirms C2268 methylation in wild type as opposed to non-methylated C2268 in nsun5-1 results in loss of HinfI restriction site. Loading control is undigested PCR product

    Techniques Used: Methylation, Amplification, Polymerase Chain Reaction

    16) Product Images from "A preliminary analysis of hepatitis C virus in pancreatic islet cells"

    Article Title: A preliminary analysis of hepatitis C virus in pancreatic islet cells

    Journal: Virology Journal

    doi: 10.1186/s12985-017-0905-3

    Total RNA from 3 different human islets donors was isolated using TRIzol reagent in combination with the RNeasy Mini kit followed by DNase treatment. 500 ng of total RNA were retrotranscribed using the Superscript III kit. The cDNAs obtained after retrotranscription were used as templates for quantitative real-time RT-PCR for mRNAs corresponding to the HCV entry factors CD81, occludin, claudin-1, and SR-B1. The relative amount of specific mRNA was normalized to Glyceraldehyde 3-phosphate dehydrogenase ( GAPDH ). mRNA levels in Huh7.5 cells and human islets are relative to those in 293 T cells. Bars represent means ± SEM from three independent experiments
    Figure Legend Snippet: Total RNA from 3 different human islets donors was isolated using TRIzol reagent in combination with the RNeasy Mini kit followed by DNase treatment. 500 ng of total RNA were retrotranscribed using the Superscript III kit. The cDNAs obtained after retrotranscription were used as templates for quantitative real-time RT-PCR for mRNAs corresponding to the HCV entry factors CD81, occludin, claudin-1, and SR-B1. The relative amount of specific mRNA was normalized to Glyceraldehyde 3-phosphate dehydrogenase ( GAPDH ). mRNA levels in Huh7.5 cells and human islets are relative to those in 293 T cells. Bars represent means ± SEM from three independent experiments

    Techniques Used: Isolation, Quantitative RT-PCR

    17) Product Images from "Flap endonuclease 1 is involved in cccDNA formation in the hepatitis B virus"

    Article Title: Flap endonuclease 1 is involved in cccDNA formation in the hepatitis B virus

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1007124

    The FEN1 inhibitor, PTPD, reduces cccDNA production. Effect of FEN1 inhibition on HBV-replicating cells. Hep38.7-Tet cells were treated with dimethylsulfoxide (DMSO) as a vehicle control, PTPD (5 μM), or 3TC (50 μM) in the absence of tetracycline for 5 days. At day 5, levels of HBV DNA, HBV RNA (pgRNA normalized by HPRT) and pre-C mRNA were analyzed. qPCR analysis of HBV DNA in (A) culture supernatant, (B) cytoplasmic NC-DNA, (C) cccDNA, and (D) pgRNA. Each result represents the mean ± SEM of three independent experiments. Asterisks indicate statistically significant differences; * P
    Figure Legend Snippet: The FEN1 inhibitor, PTPD, reduces cccDNA production. Effect of FEN1 inhibition on HBV-replicating cells. Hep38.7-Tet cells were treated with dimethylsulfoxide (DMSO) as a vehicle control, PTPD (5 μM), or 3TC (50 μM) in the absence of tetracycline for 5 days. At day 5, levels of HBV DNA, HBV RNA (pgRNA normalized by HPRT) and pre-C mRNA were analyzed. qPCR analysis of HBV DNA in (A) culture supernatant, (B) cytoplasmic NC-DNA, (C) cccDNA, and (D) pgRNA. Each result represents the mean ± SEM of three independent experiments. Asterisks indicate statistically significant differences; * P

    Techniques Used: Inhibition, Real-time Polymerase Chain Reaction

    Requirement of nuclease activity and the C-terminus of FEN1 for cccDNA production. (A) Schematic presentation of FEN1 protein. D181A: nuclease-deficient mutant. ΔC: deletion mutant unable to bind WRN protein. (B) FEN assay. Flap endonuclease activity of immunoprecipitated FEN1 protein (wt, D181A, or ΔC) was determined as in Fig 1A . (C) pResQ lentiviral vectors carrying FEN1 shRNA and the FEN1 transgene (wt, D181A, or ΔC, see also S8 Fig ) were transduced into Hep38.7-Tet cells. After puromycin selection, NC-DNA was analyzed by Southern blotting and qPCR, respectively. Each result represents the mean ± SEM of three independent experiments. Asterisks indicate statistically significant differences; **** P
    Figure Legend Snippet: Requirement of nuclease activity and the C-terminus of FEN1 for cccDNA production. (A) Schematic presentation of FEN1 protein. D181A: nuclease-deficient mutant. ΔC: deletion mutant unable to bind WRN protein. (B) FEN assay. Flap endonuclease activity of immunoprecipitated FEN1 protein (wt, D181A, or ΔC) was determined as in Fig 1A . (C) pResQ lentiviral vectors carrying FEN1 shRNA and the FEN1 transgene (wt, D181A, or ΔC, see also S8 Fig ) were transduced into Hep38.7-Tet cells. After puromycin selection, NC-DNA was analyzed by Southern blotting and qPCR, respectively. Each result represents the mean ± SEM of three independent experiments. Asterisks indicate statistically significant differences; **** P

    Techniques Used: Activity Assay, Mutagenesis, Immunoprecipitation, shRNA, Selection, Southern Blot, Real-time Polymerase Chain Reaction

    Deletion of the C-terminus disrupts the nuclear localization and reduces HBV DNA association of FEN1 protein. (A) Expression vectors of FEN1-GFP, FEN1ΔC-GFP, or mock vector (pcDNA4/TO) were transfected into Hep38.7-Tet cells. The nucleus was visualized by co-transfection of the nuclear localization signal (NLS)-tagged DsRed vector. (B–C) Myc-FEN1 or Myc-FEN1ΔC vector was transfected into Hep38.7-Tet cells. (B) Myc-tagged protein expression (before cross linkage) shown by Western blot. Two blots with different protein loadings are shown. (C) Myc-FEN1-transfected Hep38.7-Tet cells were cross-linked, and the lysates were immunoprecipitated with either control IgG or anti-Myc antibody. The immunoprecipitants were subjected to qPCR analysis using a primer pair to detect the core region of HBV. Each result represents the mean ± SEM of three independent experiments. Asterisks indicate statistically significant differences; *** P
    Figure Legend Snippet: Deletion of the C-terminus disrupts the nuclear localization and reduces HBV DNA association of FEN1 protein. (A) Expression vectors of FEN1-GFP, FEN1ΔC-GFP, or mock vector (pcDNA4/TO) were transfected into Hep38.7-Tet cells. The nucleus was visualized by co-transfection of the nuclear localization signal (NLS)-tagged DsRed vector. (B–C) Myc-FEN1 or Myc-FEN1ΔC vector was transfected into Hep38.7-Tet cells. (B) Myc-tagged protein expression (before cross linkage) shown by Western blot. Two blots with different protein loadings are shown. (C) Myc-FEN1-transfected Hep38.7-Tet cells were cross-linked, and the lysates were immunoprecipitated with either control IgG or anti-Myc antibody. The immunoprecipitants were subjected to qPCR analysis using a primer pair to detect the core region of HBV. Each result represents the mean ± SEM of three independent experiments. Asterisks indicate statistically significant differences; *** P

    Techniques Used: Expressing, Plasmid Preparation, Transfection, Cotransfection, Western Blot, Immunoprecipitation, Real-time Polymerase Chain Reaction

    FEN1 protein facilitates cccDNA formation in vitro . (A) Schematic presentation of in vitro cccDNA formation assay. Purified NC-DNA (10 8 copies) was incubated with recombinant FEN1, Bst DNA polymerase, and Taq DNA ligase. Following incubation, the DNA was purified and analyzed (B–F). Regions for qPCR amplification (E and F) were indicated as p. The 5.4-kb PstI fragment in HBV plasmid (Control) has a partial HBV sequence but does not have core and intact P genes. (B) cccDNA-selective qPCR. Each result represents the mean ± SEM of three independent experiments. Asterisks indicate statistically significant differences; *** P
    Figure Legend Snippet: FEN1 protein facilitates cccDNA formation in vitro . (A) Schematic presentation of in vitro cccDNA formation assay. Purified NC-DNA (10 8 copies) was incubated with recombinant FEN1, Bst DNA polymerase, and Taq DNA ligase. Following incubation, the DNA was purified and analyzed (B–F). Regions for qPCR amplification (E and F) were indicated as p. The 5.4-kb PstI fragment in HBV plasmid (Control) has a partial HBV sequence but does not have core and intact P genes. (B) cccDNA-selective qPCR. Each result represents the mean ± SEM of three independent experiments. Asterisks indicate statistically significant differences; *** P

    Techniques Used: In Vitro, Tube Formation Assay, Purification, Incubation, Recombinant, Real-time Polymerase Chain Reaction, Amplification, Plasmid Preparation, Sequencing

    FEN1 siRNA knockdown and CRISPR/Cas9-mediated gene editing reduce cccDNA production. (A–D) Hep38.7-Tet cells were transfected with FEN1 -specific siRNA (siFEN1 #1 or #2) or control (siCtrl), and cultured without tetracycline. Four days after transfection, FEN1 mRNA/protein and HBV DNA were analyzed. (A) FEN1 mRNA quantified by RT-qPCR (normalized by HPRT) (upper panel) and Western blotting of FEN1 protein (lower panel). GAPDH expression is shown as a loading control. FEN1 protein expression gives rise to two bands in our study, which may be due to post-translational modification. (B–C) Levels of HBV DNA in cytoplasmic nucleocapsid (NC) (B) and cccDNA (C). (D) Efficiency of cccDNA formation (cccDNA levels normalized by cytoplasmic NC-DNA) was calculated. Each result represents the mean ± SEM of three independent experiments. Asterisks indicate statistically significant differences; * P
    Figure Legend Snippet: FEN1 siRNA knockdown and CRISPR/Cas9-mediated gene editing reduce cccDNA production. (A–D) Hep38.7-Tet cells were transfected with FEN1 -specific siRNA (siFEN1 #1 or #2) or control (siCtrl), and cultured without tetracycline. Four days after transfection, FEN1 mRNA/protein and HBV DNA were analyzed. (A) FEN1 mRNA quantified by RT-qPCR (normalized by HPRT) (upper panel) and Western blotting of FEN1 protein (lower panel). GAPDH expression is shown as a loading control. FEN1 protein expression gives rise to two bands in our study, which may be due to post-translational modification. (B–C) Levels of HBV DNA in cytoplasmic nucleocapsid (NC) (B) and cccDNA (C). (D) Efficiency of cccDNA formation (cccDNA levels normalized by cytoplasmic NC-DNA) was calculated. Each result represents the mean ± SEM of three independent experiments. Asterisks indicate statistically significant differences; * P

    Techniques Used: CRISPR, Transfection, Cell Culture, Quantitative RT-PCR, Western Blot, Expressing, Modification

    18) Product Images from "A BCR-ABL Mutant Lacking Direct Binding Sites for the GRB2, CBL and CRKL Adapter Proteins Fails to Induce Leukemia in Mice"

    Article Title: A BCR-ABL Mutant Lacking Direct Binding Sites for the GRB2, CBL and CRKL Adapter Proteins Fails to Induce Leukemia in Mice

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0007439

    BCR-ABL triple mutant fails to induce leukemia in mice. Murine bone marrow transplantation studies were performed, transplanting bone marrow infected with retroviral supernatant from MIG-BCR-ABL-wild type, MIG- BCR-ABL-triple mutant or MIG vector alone, into lethally irradiated recipients. Mice were monitored post transplant for signs of disease onset. (A) Survival curves for the three recipient populations. (B) Green fluorescent protein (GFP, upper panel) or glyceraldehyde-3-phosphate dehydrogenase (GAPDH, lower panel) was amplified by RT-PCR from RNA purified from spleens of triple mutant, wild type or vector only recipients and visualized on agarose gels. (C) H E stain from the liver (a–c), spleen (d–f) and Wright/Giemsa stain from peripheral blood smear (g–i) of representative mice at harvest; vector only (upper panels), triple mutant (center panels) and wild type (lower panels).
    Figure Legend Snippet: BCR-ABL triple mutant fails to induce leukemia in mice. Murine bone marrow transplantation studies were performed, transplanting bone marrow infected with retroviral supernatant from MIG-BCR-ABL-wild type, MIG- BCR-ABL-triple mutant or MIG vector alone, into lethally irradiated recipients. Mice were monitored post transplant for signs of disease onset. (A) Survival curves for the three recipient populations. (B) Green fluorescent protein (GFP, upper panel) or glyceraldehyde-3-phosphate dehydrogenase (GAPDH, lower panel) was amplified by RT-PCR from RNA purified from spleens of triple mutant, wild type or vector only recipients and visualized on agarose gels. (C) H E stain from the liver (a–c), spleen (d–f) and Wright/Giemsa stain from peripheral blood smear (g–i) of representative mice at harvest; vector only (upper panels), triple mutant (center panels) and wild type (lower panels).

    Techniques Used: Mutagenesis, Mouse Assay, Transplantation Assay, Infection, Plasmid Preparation, Irradiation, Amplification, Reverse Transcription Polymerase Chain Reaction, Purification, Staining, Giemsa Stain

    19) Product Images from "RAB38 is a potential prognostic factor for tumor recurrence in non-small cell lung cancer"

    Article Title: RAB38 is a potential prognostic factor for tumor recurrence in non-small cell lung cancer

    Journal: Oncology Letters

    doi: 10.3892/ol.2019.10547

    Upregulation of RAB38 in non-small cell lung cancer with an active epidermal growth factor receptor mutation. (A) Expression of RAB38 in A549 and HCC827 cells was detected using RT-PCR analysis. HCC827 cells were transfected with shRNA targeting RAB38 or empty vector. (B) Knockdown efficiency was determined by RT-PCR and western blot analyses. (C) In vitro invasiveness of gene-modified HCC827 cells was performed with a Matrigel invasion chamber. Original magnification, ×100. (D) RAB38 knockdown significantly inhibited tumor Matrigel invasiveness compared with that in the negative, empty vector controls. A total of three independent experiments were performed. RAB38, Ras-related protein Rab-38; RT-PCR, reverse transcription-polymerase chain reaction; shRNA, short hairpin RNA.
    Figure Legend Snippet: Upregulation of RAB38 in non-small cell lung cancer with an active epidermal growth factor receptor mutation. (A) Expression of RAB38 in A549 and HCC827 cells was detected using RT-PCR analysis. HCC827 cells were transfected with shRNA targeting RAB38 or empty vector. (B) Knockdown efficiency was determined by RT-PCR and western blot analyses. (C) In vitro invasiveness of gene-modified HCC827 cells was performed with a Matrigel invasion chamber. Original magnification, ×100. (D) RAB38 knockdown significantly inhibited tumor Matrigel invasiveness compared with that in the negative, empty vector controls. A total of three independent experiments were performed. RAB38, Ras-related protein Rab-38; RT-PCR, reverse transcription-polymerase chain reaction; shRNA, short hairpin RNA.

    Techniques Used: Mutagenesis, Expressing, Reverse Transcription Polymerase Chain Reaction, Transfection, shRNA, Plasmid Preparation, Western Blot, In Vitro, Modification

    20) Product Images from "TREM2 and ?-catenin regulate bone homeostasis by controlling the rate of osteoclastogenesis"

    Article Title: TREM2 and ?-catenin regulate bone homeostasis by controlling the rate of osteoclastogenesis

    Journal: Journal of immunology (Baltimore, Md. : 1950)

    doi: 10.4049/jimmunol.1102836

    TREM2-deficient osteoclast precursors exhibit accelerated osteoclastogenesis. OcP generated from WT and TREM2 −/− mice were cultured in vitro with 10 ng/ml M-CSF and 100 ng/ml RANKL to generate multinuclear osteoclasts. (A and B) Development of osteoclasts was monitored at different timepoints by TRAP staining. (A) Representative TRAP-stained images of the cultures. (B) The number of TRAP-positive cells containing three or more nuclei was scored (total). In addition, the nuclei in each osteoclast was enumerated as follows: 3–10; 6–10 and > 10 nuclei per TRAP-positive cell. (C) Effect of TREM2 deficiency on the expression of the osteoclastic differentiation markers Nfatc1 (encoding NFATc1), Acp5 (encoding TRAP), Ctsk (encoding cathepsin K), and Calcr (encoding calcitonin receptor) measured by Q-PCR. (D) Quantification of TRAP activity in WT and TREM2 −/− OcP and OC obtained by culturing OcP for 3 days with M-CSF and RANKL. (E) Apoptosis of osteoclast cultures as determined by quantification of DNA fragments by ELISA. (F, G and H) Osteoclast differentiation induced on bone slices. (F) After 4 and 7 days of differentiation cultures were fixed and stained with Phalloidin-FITC to visualize the osteoclast actin rings. (G) Bone resorption pits were revealed by lectin staining of the bone slices (brown reaction product). (H) Supernatants of the cultures were collected at day 4 and 6 of culture and bone resorption was assessed by measuring the degradation products of the C-terminal telopeptides of Type I collagen by ELISA. *, P
    Figure Legend Snippet: TREM2-deficient osteoclast precursors exhibit accelerated osteoclastogenesis. OcP generated from WT and TREM2 −/− mice were cultured in vitro with 10 ng/ml M-CSF and 100 ng/ml RANKL to generate multinuclear osteoclasts. (A and B) Development of osteoclasts was monitored at different timepoints by TRAP staining. (A) Representative TRAP-stained images of the cultures. (B) The number of TRAP-positive cells containing three or more nuclei was scored (total). In addition, the nuclei in each osteoclast was enumerated as follows: 3–10; 6–10 and > 10 nuclei per TRAP-positive cell. (C) Effect of TREM2 deficiency on the expression of the osteoclastic differentiation markers Nfatc1 (encoding NFATc1), Acp5 (encoding TRAP), Ctsk (encoding cathepsin K), and Calcr (encoding calcitonin receptor) measured by Q-PCR. (D) Quantification of TRAP activity in WT and TREM2 −/− OcP and OC obtained by culturing OcP for 3 days with M-CSF and RANKL. (E) Apoptosis of osteoclast cultures as determined by quantification of DNA fragments by ELISA. (F, G and H) Osteoclast differentiation induced on bone slices. (F) After 4 and 7 days of differentiation cultures were fixed and stained with Phalloidin-FITC to visualize the osteoclast actin rings. (G) Bone resorption pits were revealed by lectin staining of the bone slices (brown reaction product). (H) Supernatants of the cultures were collected at day 4 and 6 of culture and bone resorption was assessed by measuring the degradation products of the C-terminal telopeptides of Type I collagen by ELISA. *, P

    Techniques Used: Generated, Mouse Assay, Cell Culture, In Vitro, Staining, Expressing, Polymerase Chain Reaction, Activity Assay, Enzyme-linked Immunosorbent Assay

    β-catenin-deficient osteoclast precursors proliferate less to M-CSF and exhibit accelerated osteoclastogenesis. (A) Immunoblot analysis of β-catenin and actin (loading control) in OcP total protein lysates from control LysM-Cre +/+ β-catenin +/+ (β-cat +/+ ) and LysM-Cre +/+ β-catenin fl/fl (β-cat Δ ̃ Δ mice. (B) Defect of M-CSF-induced proliferation in β-catenin-deficient OcP. Proliferation of β-cat +/+ and β-cat Δ ̃ Δ OcP was measured 12 h after incubation with BrdU in the presence of 100 ng/ml M-CSF. (C and D) OcP from β-cat +/+ and β-cat Δ ̃ Δ mice were cultured with 10 ng/ml M-CSF and 100 ng/ml RANKL to generate multinuclear osteoclasts. Development of osteoclasts was monitored at different timepoints by TRAP staining. (C) Representative images of TRAP-stained cultures. (D) The number of TRAP-positive cells containing three or more nuclei was scored (total). In addition, the nuclei in each osteoclast was enumerated as follows: 3–10; 6–10 and > 10 nuclei per TRAP-positive cell. (E) Effect of β-catenin deficiency on the expression of the osteoclastic differentiation markers Nfatc1 (encoding NFATc1), Acp5 (encoding TRAP), Ctsk (encoding cathepsin K), and Calcr (encoding calcitonin receptor) by Q-PCR. (F and G) Osteoclast differentiation induced on bone slices. (F) After 4 and 7 days of differentiation cultures were fixed and stained with Phalloidin-FITC to visualize the osteoclast actin rings. (G) Bone resorption pits were revealed by lectin staining of the bone slices (brown reaction product). *, P
    Figure Legend Snippet: β-catenin-deficient osteoclast precursors proliferate less to M-CSF and exhibit accelerated osteoclastogenesis. (A) Immunoblot analysis of β-catenin and actin (loading control) in OcP total protein lysates from control LysM-Cre +/+ β-catenin +/+ (β-cat +/+ ) and LysM-Cre +/+ β-catenin fl/fl (β-cat Δ ̃ Δ mice. (B) Defect of M-CSF-induced proliferation in β-catenin-deficient OcP. Proliferation of β-cat +/+ and β-cat Δ ̃ Δ OcP was measured 12 h after incubation with BrdU in the presence of 100 ng/ml M-CSF. (C and D) OcP from β-cat +/+ and β-cat Δ ̃ Δ mice were cultured with 10 ng/ml M-CSF and 100 ng/ml RANKL to generate multinuclear osteoclasts. Development of osteoclasts was monitored at different timepoints by TRAP staining. (C) Representative images of TRAP-stained cultures. (D) The number of TRAP-positive cells containing three or more nuclei was scored (total). In addition, the nuclei in each osteoclast was enumerated as follows: 3–10; 6–10 and > 10 nuclei per TRAP-positive cell. (E) Effect of β-catenin deficiency on the expression of the osteoclastic differentiation markers Nfatc1 (encoding NFATc1), Acp5 (encoding TRAP), Ctsk (encoding cathepsin K), and Calcr (encoding calcitonin receptor) by Q-PCR. (F and G) Osteoclast differentiation induced on bone slices. (F) After 4 and 7 days of differentiation cultures were fixed and stained with Phalloidin-FITC to visualize the osteoclast actin rings. (G) Bone resorption pits were revealed by lectin staining of the bone slices (brown reaction product). *, P

    Techniques Used: Mouse Assay, Incubation, Cell Culture, Staining, Expressing, Polymerase Chain Reaction

    21) Product Images from "Extragenic Accumulation of RNA Polymerase II Enhances Transcription by RNA Polymerase III"

    Article Title: Extragenic Accumulation of RNA Polymerase II Enhances Transcription by RNA Polymerase III

    Journal: PLoS Genetics

    doi: 10.1371/journal.pgen.0030212

    Low α-Amanitin Concentrations Inhibit U6 snRNA Maxigene Expression HeLa cells were transiently transfected with 1 μg U6–1 , U6–8 , and U6–9 maxigenes carrying a 9 bp insertion and treated simultaneously with 50 nM α-amanitin oleate for 20 h or left untreated. Expression of U6 maxigenes, LDHA, 28S rRNA, 5S rRNA and pre-tRNA Tyr was measured by gene-specific reverse transcription, followed by conventional PCR and agarose gel electrophoresis (A) or qPCR (B). qPCR values were normalized to 28S rRNA and expression levels are expressed relative to untreated controls. Error bars represent the SEM. The data represents the average of at least three independent experiments.
    Figure Legend Snippet: Low α-Amanitin Concentrations Inhibit U6 snRNA Maxigene Expression HeLa cells were transiently transfected with 1 μg U6–1 , U6–8 , and U6–9 maxigenes carrying a 9 bp insertion and treated simultaneously with 50 nM α-amanitin oleate for 20 h or left untreated. Expression of U6 maxigenes, LDHA, 28S rRNA, 5S rRNA and pre-tRNA Tyr was measured by gene-specific reverse transcription, followed by conventional PCR and agarose gel electrophoresis (A) or qPCR (B). qPCR values were normalized to 28S rRNA and expression levels are expressed relative to untreated controls. Error bars represent the SEM. The data represents the average of at least three independent experiments.

    Techniques Used: Expressing, Transfection, Polymerase Chain Reaction, Agarose Gel Electrophoresis, Real-time Polymerase Chain Reaction

    Pol III Transcribes U6–1 Maxigene Primer extension analysis of RNA from HeLa cells cotransfected with GFP expression plasmid and U6–1 maxigene. (A) Design of the two U6–1 maxigenes with insertions at +66 and +87 bp (white boxes) to allow for maxigene-specific reverse transcription. Construct “U6–1 maxiT” harbors five thymidine residues directly downstream of the linker insertion; “U6–1 MaxiC” harbors the same primer binding site but lacks the T's. The cross-hatched box represents the reverse primer specific for the downstream insertion, yielding extension products of either 114 bp (U6–1 maxiT) or 109 bp (U6–1 maxiC). Grey filled box and black filled box represent reverse primers specific for upstream insertion with or without T residues, respectively, leading to extension products of 83 bp (U6–1 maxiT) or 79 bp (U6–1 maxiC). Primer extension of mRNA derived from cotransfected GFP plasmid yields a 158 bp product. Primer extension products were separated on a denaturing polyacrylamide gel and exposed on a PhosphorImager. (B) A representative gel is shown.
    Figure Legend Snippet: Pol III Transcribes U6–1 Maxigene Primer extension analysis of RNA from HeLa cells cotransfected with GFP expression plasmid and U6–1 maxigene. (A) Design of the two U6–1 maxigenes with insertions at +66 and +87 bp (white boxes) to allow for maxigene-specific reverse transcription. Construct “U6–1 maxiT” harbors five thymidine residues directly downstream of the linker insertion; “U6–1 MaxiC” harbors the same primer binding site but lacks the T's. The cross-hatched box represents the reverse primer specific for the downstream insertion, yielding extension products of either 114 bp (U6–1 maxiT) or 109 bp (U6–1 maxiC). Grey filled box and black filled box represent reverse primers specific for upstream insertion with or without T residues, respectively, leading to extension products of 83 bp (U6–1 maxiT) or 79 bp (U6–1 maxiC). Primer extension of mRNA derived from cotransfected GFP plasmid yields a 158 bp product. Primer extension products were separated on a denaturing polyacrylamide gel and exposed on a PhosphorImager. (B) A representative gel is shown.

    Techniques Used: Expressing, Plasmid Preparation, Construct, Binding Assay, Derivative Assay

    α-Amanitin Reduces Endogenous U6 snRNA Expression HeLa cells were transfected with a tRNA Arg maxigene and 16 h later treated with 10 μg/ml α-amanitin or left untreated. After 3 h, 250 μCi [ 32 P] orthophosphate was added to the medium for 6 h. Total RNA was collected and 5S rRNA along with tRNA Arg , U2 and U6 snRNAs were hybrid selected with biotinylated complementary oligos. Hybrid-selected RNA was separated on a denaturating polyacrylamide gel and exposed to PhosphorImager plates. The 5S rRNA bands served as a loading control. Three biological replicates were analyzed and a representative gel is shown.
    Figure Legend Snippet: α-Amanitin Reduces Endogenous U6 snRNA Expression HeLa cells were transfected with a tRNA Arg maxigene and 16 h later treated with 10 μg/ml α-amanitin or left untreated. After 3 h, 250 μCi [ 32 P] orthophosphate was added to the medium for 6 h. Total RNA was collected and 5S rRNA along with tRNA Arg , U2 and U6 snRNAs were hybrid selected with biotinylated complementary oligos. Hybrid-selected RNA was separated on a denaturating polyacrylamide gel and exposed to PhosphorImager plates. The 5S rRNA bands served as a loading control. Three biological replicates were analyzed and a representative gel is shown.

    Techniques Used: Expressing, Transfection

    22) Product Images from "Human SETMAR is a DNA sequence-specific histone-methylase with a broad effect on the transcriptome"

    Article Title: Human SETMAR is a DNA sequence-specific histone-methylase with a broad effect on the transcriptome

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gky937

    Hsmar1 remnants in the human genome and expression of ectopic SETMAR. ( A ) The SETMAR domain and exon structure are illustrated together with the helix-turn-helix (HTH) DNA binding motifs and key active site residues in the methylase and transposase active sites. The third D residue (aspartate) that coordinates the catalytic metal ion is an N (asparagine) in SETMAR. All 260 full-length copies of Hsmar1 have inactivating point mutations and indels. Made1 elements comprise of six bp flanked by a pair of ITRs. ( B and C ) Distribution of the 6,334 ITRs in the human genome. ( D ) A Western blot for the FLAG-tagged codon optimized SETMAR in the U2OS, SMF and SMFN cell lines. SMFN has the N210A substitution of an essential residue in the SETMAR methylase domain active site. ( E ) qRT-PCR of the endogenous and the transgenic SETMAR in the SMF and SMFN cell lines. Figures above rightmost columns are the ratios of expression derived from the RNA-seq experiments presented in Figure 3 . ( F ) Western blot of the indicated cell lines using an H3K36me2 antibody and a histone H3 antibody as a loading control. The lanes probed for H3 and H3K36me2, respectively, were from a single gel loaded with the same amount of protein. The same result was obtained from three biological replicates (R1 to R3) performed on different batches of cells.
    Figure Legend Snippet: Hsmar1 remnants in the human genome and expression of ectopic SETMAR. ( A ) The SETMAR domain and exon structure are illustrated together with the helix-turn-helix (HTH) DNA binding motifs and key active site residues in the methylase and transposase active sites. The third D residue (aspartate) that coordinates the catalytic metal ion is an N (asparagine) in SETMAR. All 260 full-length copies of Hsmar1 have inactivating point mutations and indels. Made1 elements comprise of six bp flanked by a pair of ITRs. ( B and C ) Distribution of the 6,334 ITRs in the human genome. ( D ) A Western blot for the FLAG-tagged codon optimized SETMAR in the U2OS, SMF and SMFN cell lines. SMFN has the N210A substitution of an essential residue in the SETMAR methylase domain active site. ( E ) qRT-PCR of the endogenous and the transgenic SETMAR in the SMF and SMFN cell lines. Figures above rightmost columns are the ratios of expression derived from the RNA-seq experiments presented in Figure 3 . ( F ) Western blot of the indicated cell lines using an H3K36me2 antibody and a histone H3 antibody as a loading control. The lanes probed for H3 and H3K36me2, respectively, were from a single gel loaded with the same amount of protein. The same result was obtained from three biological replicates (R1 to R3) performed on different batches of cells.

    Techniques Used: Expressing, Binding Assay, Western Blot, Quantitative RT-PCR, Transgenic Assay, Derivative Assay, RNA Sequencing Assay

    23) Product Images from "Evaluation of diversity among common beans (Phaseolus vulgaris L.) from two centers of domestication using 'omics' technologies"

    Article Title: Evaluation of diversity among common beans (Phaseolus vulgaris L.) from two centers of domestication using 'omics' technologies

    Journal: BMC Genomics

    doi: 10.1186/1471-2164-11-686

    Transcriptomic analysis of navy bean and white kidney bean derived from microarray analysis of gene expression . Principle components analysis of navy (red) and white kidney (blue) harvested 2 wk after plant flowering. Three biological replicates of bean cDNA were hybridized to spotted soybean microarrays.
    Figure Legend Snippet: Transcriptomic analysis of navy bean and white kidney bean derived from microarray analysis of gene expression . Principle components analysis of navy (red) and white kidney (blue) harvested 2 wk after plant flowering. Three biological replicates of bean cDNA were hybridized to spotted soybean microarrays.

    Techniques Used: Derivative Assay, Microarray, Expressing

    24) Product Images from "Towards an understanding of C9orf82 protein/CAAP1 function"

    Article Title: Towards an understanding of C9orf82 protein/CAAP1 function

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0210526

    CRISPR/Cas9 mediated inactivation of C9orf82 in the mouse germline. (A) Schematic presentation of the C9orf82 locus in the mouse and its CRISPR/Cas9 based inactivation. Cas9 activity targeted by the two gRNAs at the C9orf82 locus flanking exon 2 and 4 is indicated (blue flashes). The resulting deletion of a 7 kb DNA fragment in the targeted allele can be detected and distinguished from wild type (WT) are indicated by the three primers (black arrows). (B) Genotype PCR to distinguish WT (459 bps) from C9orf82 ko/ko (367 bps) allele. As expected, mice heterozygous for the targeted allele show both bands. (C) Validation of the designed mutation by qRT-PCR. Using qRT-PCR the lack of exons 2,3, and 4 in the mutant cDNA of C9orf82 was confirming using the primers indicated as black arrows. Stability of truncated C9orf82 mRNA checked by qRT-PCR using the indicated primers (red arrows) targeting exon 6. (D) In silico analysis shows that after exon 2–4 deletion, the remaining transcript either as a result of splicing from exon 1 to exon 5 or exon 1 to 6 contain pre-mature translational stop codons. As a result, after the deletion only the first 82 amino-terminal sequence remains in frame. (E) C9orf82 ko/ko mice were born at Mendelian frequencies. Depicted are the expected and observed frequencies of WT, heterozygous (HET) and homozygous (ko) out of 131 pups born from five breeding pairs (HET x HET).
    Figure Legend Snippet: CRISPR/Cas9 mediated inactivation of C9orf82 in the mouse germline. (A) Schematic presentation of the C9orf82 locus in the mouse and its CRISPR/Cas9 based inactivation. Cas9 activity targeted by the two gRNAs at the C9orf82 locus flanking exon 2 and 4 is indicated (blue flashes). The resulting deletion of a 7 kb DNA fragment in the targeted allele can be detected and distinguished from wild type (WT) are indicated by the three primers (black arrows). (B) Genotype PCR to distinguish WT (459 bps) from C9orf82 ko/ko (367 bps) allele. As expected, mice heterozygous for the targeted allele show both bands. (C) Validation of the designed mutation by qRT-PCR. Using qRT-PCR the lack of exons 2,3, and 4 in the mutant cDNA of C9orf82 was confirming using the primers indicated as black arrows. Stability of truncated C9orf82 mRNA checked by qRT-PCR using the indicated primers (red arrows) targeting exon 6. (D) In silico analysis shows that after exon 2–4 deletion, the remaining transcript either as a result of splicing from exon 1 to exon 5 or exon 1 to 6 contain pre-mature translational stop codons. As a result, after the deletion only the first 82 amino-terminal sequence remains in frame. (E) C9orf82 ko/ko mice were born at Mendelian frequencies. Depicted are the expected and observed frequencies of WT, heterozygous (HET) and homozygous (ko) out of 131 pups born from five breeding pairs (HET x HET).

    Techniques Used: CRISPR, Activity Assay, Polymerase Chain Reaction, Mouse Assay, Mutagenesis, Quantitative RT-PCR, In Silico, Sequencing

    25) Product Images from "Serpine2/PN-1 Is Required for Proliferative Expansion of Pre-Neoplastic Lesions and Malignant Progression to Medulloblastoma"

    Article Title: Serpine2/PN-1 Is Required for Proliferative Expansion of Pre-Neoplastic Lesions and Malignant Progression to Medulloblastoma

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0124870

    Genetic reduction of Serpine2/ Pn-1 interferes with transcriptional silencing of the wild-type Ptch1 allele in Ptch1 Δ/+ mice resulting in reduced cell proliferation. (A) Quantitative analysis of transcripts from the wild-type Ptch1 allele. Expression of the wild-type Ptch1 allele is lost from all Ptch1 Δ/+ PNLs by six weeks after birth (n = 8). In contrast, the wild-type Ptch1 allele remains expressed in 5 of 8 Ptch1 Δ/+ Pn-1 Δ/+ samples (p≤0.001). (B, C) Comparative analysis of the cerebella of wild-type (Wt), Ptch1 Δ/+ and Ptch1 Δ/+ Pn-1 Δ/+ mice at 6 weeks by RNA in situ hybridization and qPCR analysis (p≤0.001). As Gli1 and Ptch1 are direct transcriptional targets of Hedgehog signaling, their expression serves to sense signal transduction. Note that the RNA in situ probe detects Ptch1 transcripts from both the mutant and wild-type Ptch1 allele. (D) Most Ptch1 Δ/+ PNL cells are Ki67-positive (brown staining), i.e. are actively proliferating. Fewer Ki67-positive cells are detected in Ptch1 Δ/+ Pn-1 Δ/+ PNLs. (E) Cdk6 expression is up-regulated in PNLs of Ptch1 Δ/+ mice, while it is reduced in PNLs of Ptch1 Δ/+ Pn-1 Δ/+ mice. (F) Mycn is overexpressed in PNLs of Ptch1 Δ/+ mice, while its expression is significantly lower in PNLs of Ptch1 Δ/+ Pn-1 Δ/+ mice. qPCR analysis shows that Mycn , Cdk6 and Ki-67 are not expressed by the IGL of wild-type mice at 6 weeks of age. Therefore, the relative expression of these three genes in PNLs of Ptch1 Δ/+ mice is set to 100% to allow comparison with the expression in Ptch1 Δ/+ Pn-1 Δ/+ PNLs (p≤0.001 for all results). IGL: internal granular layer; ML: molecular layer; PNL: pre-neoplastic lesion. Scale bar: 100μm (representative for all low magnifications shown).
    Figure Legend Snippet: Genetic reduction of Serpine2/ Pn-1 interferes with transcriptional silencing of the wild-type Ptch1 allele in Ptch1 Δ/+ mice resulting in reduced cell proliferation. (A) Quantitative analysis of transcripts from the wild-type Ptch1 allele. Expression of the wild-type Ptch1 allele is lost from all Ptch1 Δ/+ PNLs by six weeks after birth (n = 8). In contrast, the wild-type Ptch1 allele remains expressed in 5 of 8 Ptch1 Δ/+ Pn-1 Δ/+ samples (p≤0.001). (B, C) Comparative analysis of the cerebella of wild-type (Wt), Ptch1 Δ/+ and Ptch1 Δ/+ Pn-1 Δ/+ mice at 6 weeks by RNA in situ hybridization and qPCR analysis (p≤0.001). As Gli1 and Ptch1 are direct transcriptional targets of Hedgehog signaling, their expression serves to sense signal transduction. Note that the RNA in situ probe detects Ptch1 transcripts from both the mutant and wild-type Ptch1 allele. (D) Most Ptch1 Δ/+ PNL cells are Ki67-positive (brown staining), i.e. are actively proliferating. Fewer Ki67-positive cells are detected in Ptch1 Δ/+ Pn-1 Δ/+ PNLs. (E) Cdk6 expression is up-regulated in PNLs of Ptch1 Δ/+ mice, while it is reduced in PNLs of Ptch1 Δ/+ Pn-1 Δ/+ mice. (F) Mycn is overexpressed in PNLs of Ptch1 Δ/+ mice, while its expression is significantly lower in PNLs of Ptch1 Δ/+ Pn-1 Δ/+ mice. qPCR analysis shows that Mycn , Cdk6 and Ki-67 are not expressed by the IGL of wild-type mice at 6 weeks of age. Therefore, the relative expression of these three genes in PNLs of Ptch1 Δ/+ mice is set to 100% to allow comparison with the expression in Ptch1 Δ/+ Pn-1 Δ/+ PNLs (p≤0.001 for all results). IGL: internal granular layer; ML: molecular layer; PNL: pre-neoplastic lesion. Scale bar: 100μm (representative for all low magnifications shown).

    Techniques Used: Mouse Assay, Expressing, RNA In Situ Hybridization, Real-time Polymerase Chain Reaction, Transduction, In Situ, Mutagenesis, Staining

    26) Product Images from "An Engineered Mouse to Identify Proliferating Cells and Their Derivatives"

    Article Title: An Engineered Mouse to Identify Proliferating Cells and Their Derivatives

    Journal: Frontiers in Cell and Developmental Biology

    doi: 10.3389/fcell.2020.00388

    Aurkb transcription is coupled with cell proliferation. (A) MEFs were cultured for 48 h with normal media (control), or treated with hydroxyurea (2 mM), or mimosine (1 mM). The transcriptional levels of Aurkb were quantified by qRT-PCR. Gapdh was used as a cDNA loading control. Three independent biological samples were used in each condition. * P
    Figure Legend Snippet: Aurkb transcription is coupled with cell proliferation. (A) MEFs were cultured for 48 h with normal media (control), or treated with hydroxyurea (2 mM), or mimosine (1 mM). The transcriptional levels of Aurkb were quantified by qRT-PCR. Gapdh was used as a cDNA loading control. Three independent biological samples were used in each condition. * P

    Techniques Used: Cell Culture, Quantitative RT-PCR

    27) Product Images from "Topological Organization of Multi-chromosomal Regions by Firre"

    Article Title: Topological Organization of Multi-chromosomal Regions by Firre

    Journal: Nature structural & molecular biology

    doi: 10.1038/nsmb.2764

    The trans-chromosomal contacts of Firre are mediated via its interaction with the nuclear matrix protein hnRNPU (a) RNA-FISH co-localization of Firre introns (green) with Ypel4 or Slc25a12 introns (red) in the absence of hnRNPU. (b) RNA-FISH co-localization of the trans-interacting loci Ppp1r10 and Ypel4 in the absence of Firre expression in ΔFirre male mESCs compared to the wild-type mESCs (rows 1, 2). (c) RNA-FISH co-localization of trans-sites in mLFs (row 3). Scale bars, 40 μm. (d) A model for Firre as a ‘regional organization factor.’ Firre transcripts accumulate at the site of their transcription. hnRNPU binds to the RRD of Firre and facilitates interactions with trans-chromosomal regions through one of several possible mechanisms: I) Tertiary interactions with nuclear matrix components, II) Direct binding of hnRNPU to matrix attachment regions in trans , or III) As yet undetermined interactions with other protein complexes to facilitate indirect binding to DNA.
    Figure Legend Snippet: The trans-chromosomal contacts of Firre are mediated via its interaction with the nuclear matrix protein hnRNPU (a) RNA-FISH co-localization of Firre introns (green) with Ypel4 or Slc25a12 introns (red) in the absence of hnRNPU. (b) RNA-FISH co-localization of the trans-interacting loci Ppp1r10 and Ypel4 in the absence of Firre expression in ΔFirre male mESCs compared to the wild-type mESCs (rows 1, 2). (c) RNA-FISH co-localization of trans-sites in mLFs (row 3). Scale bars, 40 μm. (d) A model for Firre as a ‘regional organization factor.’ Firre transcripts accumulate at the site of their transcription. hnRNPU binds to the RRD of Firre and facilitates interactions with trans-chromosomal regions through one of several possible mechanisms: I) Tertiary interactions with nuclear matrix components, II) Direct binding of hnRNPU to matrix attachment regions in trans , or III) As yet undetermined interactions with other protein complexes to facilitate indirect binding to DNA.

    Techniques Used: Fluorescence In Situ Hybridization, Expressing, Binding Assay

    28) Product Images from "Inhibition of endocytic lipid antigen presentation by common lipophilic environmental pollutants"

    Article Title: Inhibition of endocytic lipid antigen presentation by common lipophilic environmental pollutants

    Journal: Scientific Reports

    doi: 10.1038/s41598-017-02229-7

    Altered gene expression profiles upon BaP exposure. Human monocyte-derived DCs from each donor (n = 3) were incubated with BaP and sorted for labeled conventional DCs, which were further used for RNA extraction and transcriptomic analysis. A volcano plot shows the number of differentially expressed genes (vertical lines = two-fold intensity difference, horizontal line = 0.05 false discovery rate-adjusted p-value) ( a ). The altered genes were clustered by functions ( b ). The expression level of some targeted genes was confirmed using RT-qPCR by normalization to B2M gene expression ( c ). Gene expression in BaP-exposed and non-exposed DCs was compared in three individual donors. The standard errors were calculated from triplicate reactions. The p-values were calculated using Student’s t-tests and are shown as **(p
    Figure Legend Snippet: Altered gene expression profiles upon BaP exposure. Human monocyte-derived DCs from each donor (n = 3) were incubated with BaP and sorted for labeled conventional DCs, which were further used for RNA extraction and transcriptomic analysis. A volcano plot shows the number of differentially expressed genes (vertical lines = two-fold intensity difference, horizontal line = 0.05 false discovery rate-adjusted p-value) ( a ). The altered genes were clustered by functions ( b ). The expression level of some targeted genes was confirmed using RT-qPCR by normalization to B2M gene expression ( c ). Gene expression in BaP-exposed and non-exposed DCs was compared in three individual donors. The standard errors were calculated from triplicate reactions. The p-values were calculated using Student’s t-tests and are shown as **(p

    Techniques Used: Expressing, Derivative Assay, Incubation, Labeling, RNA Extraction, Quantitative RT-PCR

    29) Product Images from "Interferon alpha impairs insulin production in human beta cells via endoplasmic reticulum stress"

    Article Title: Interferon alpha impairs insulin production in human beta cells via endoplasmic reticulum stress

    Journal: Journal of autoimmunity

    doi: 10.1016/j.jaut.2017.02.002

    Effect of IFNα on BiP induction in human islets and EndoC-βH1 cells (A–D) Human islets and EndoC-βH1 cells were pretreated overnight with 2.5 mM PBA or with 1 mM TUDCA or with medium only (negative control) and then cultured in presence of IFNα 1000 U/ml for 48 hours. (A) The expression levels of mRNAs for BiP were determined by real-time RT-PCR analysis of total RNA from three different preparations of human islets or EndoC-βH1 cells treated as above. mRNA levels in treated cells are relative to those in vehicle-treated cells (CTRL). Bars represent means ± SEM from three independent experiments. ***p
    Figure Legend Snippet: Effect of IFNα on BiP induction in human islets and EndoC-βH1 cells (A–D) Human islets and EndoC-βH1 cells were pretreated overnight with 2.5 mM PBA or with 1 mM TUDCA or with medium only (negative control) and then cultured in presence of IFNα 1000 U/ml for 48 hours. (A) The expression levels of mRNAs for BiP were determined by real-time RT-PCR analysis of total RNA from three different preparations of human islets or EndoC-βH1 cells treated as above. mRNA levels in treated cells are relative to those in vehicle-treated cells (CTRL). Bars represent means ± SEM from three independent experiments. ***p

    Techniques Used: Negative Control, Cell Culture, Expressing, Quantitative RT-PCR

    30) Product Images from "Role of HCV Core gene of genotype 1a and 3a and host gene Cox-2 in HCV-induced pathogenesis"

    Article Title: Role of HCV Core gene of genotype 1a and 3a and host gene Cox-2 in HCV-induced pathogenesis

    Journal: Virology Journal

    doi: 10.1186/1743-422X-8-155

    Combined effect of Cox-2 and HCV 3a Core siRNAs on Cox-2, iNOS, VEGF and p-Akt gene expression . (A) Huh-7 cells were transfected with HCV 3a Core expression vector (C) or mock-treated (M) along with or without siRNAs (Csi27 and COXsi) alone or in combination (Csi27+COXsi) for 48 hrs. Total RNA was quantified by Real-Time PCR and is shown as fold induction for Cox-2, iNOS and VEGF genes using their gene specific primers. (B) Western blot analysis of Huh-7 cells treated with and without Core, Cox-2 and combined siRNA was carried out using specific antibodies. Three independent experiments with triplicate determinations were performed. Error bars indicate mean S.D, * p
    Figure Legend Snippet: Combined effect of Cox-2 and HCV 3a Core siRNAs on Cox-2, iNOS, VEGF and p-Akt gene expression . (A) Huh-7 cells were transfected with HCV 3a Core expression vector (C) or mock-treated (M) along with or without siRNAs (Csi27 and COXsi) alone or in combination (Csi27+COXsi) for 48 hrs. Total RNA was quantified by Real-Time PCR and is shown as fold induction for Cox-2, iNOS and VEGF genes using their gene specific primers. (B) Western blot analysis of Huh-7 cells treated with and without Core, Cox-2 and combined siRNA was carried out using specific antibodies. Three independent experiments with triplicate determinations were performed. Error bars indicate mean S.D, * p

    Techniques Used: Expressing, Transfection, Plasmid Preparation, Real-time Polymerase Chain Reaction, Western Blot

    HCV 3a Core-specific siRNA inhibit expression of genes involved in HCV pathogenesis . Huh-7 cells were transfected with HCV 3a Core expression vector or mock along with or without siRNA. (A) Dose dependent mRNA expression of HCV 3a Core gene as a result of 10 μM, 20 μM and 40 μM of siRNAs for 48 hrs. Cells were harvested and relative RNA levels in PCR Csi27 and Csi352 siRNAs tranfected cells were determined using semi-quantitative PCR. Expression levels for mock-transfected (M), HCV 3a Core expression plasmid (C), scramble siRNA (SC) and GAPDH are also shown. (B) Silencing effect of HCV 3a Core gene on the RNA expression levels of cellular genes (Cox-2, iNOS and VEGF) 48 hrs post transfection on Real-Time PCR using gene specific primers in comparison to mock was performed. GAPDH was used as internal control. Three independent experiments were performed having triplicate samples. Error bars indicate, mean S.D, *p
    Figure Legend Snippet: HCV 3a Core-specific siRNA inhibit expression of genes involved in HCV pathogenesis . Huh-7 cells were transfected with HCV 3a Core expression vector or mock along with or without siRNA. (A) Dose dependent mRNA expression of HCV 3a Core gene as a result of 10 μM, 20 μM and 40 μM of siRNAs for 48 hrs. Cells were harvested and relative RNA levels in PCR Csi27 and Csi352 siRNAs tranfected cells were determined using semi-quantitative PCR. Expression levels for mock-transfected (M), HCV 3a Core expression plasmid (C), scramble siRNA (SC) and GAPDH are also shown. (B) Silencing effect of HCV 3a Core gene on the RNA expression levels of cellular genes (Cox-2, iNOS and VEGF) 48 hrs post transfection on Real-Time PCR using gene specific primers in comparison to mock was performed. GAPDH was used as internal control. Three independent experiments were performed having triplicate samples. Error bars indicate, mean S.D, *p

    Techniques Used: Expressing, Transfection, Plasmid Preparation, Polymerase Chain Reaction, Real-time Polymerase Chain Reaction, RNA Expression

    31) Product Images from "The transcription factors TFEB and TFE3 link the FLCN-AMPK signaling axis to innate immune response and pathogen resistance"

    Article Title: The transcription factors TFEB and TFE3 link the FLCN-AMPK signaling axis to innate immune response and pathogen resistance

    Journal: bioRxiv

    doi: 10.1101/463430

    The regulation of TFEB/TFE3 by FLCN is evolutionarily conserved through mTOR independent mechanisms (A) Immunoblot of isolated cytosolic-soluble fractions and nuclear fractions of wild-type and FLCN KO mouse embryonic fibroblasts (MEFs). (B) Relative mRNA levels measured by qRT-PCR of indicated genes in wild-type and FLCN KO MEFs. Data represent the average of three independent experiments done in triplicates ± SEM. Significance was determined using student’s t-test ( ** p
    Figure Legend Snippet: The regulation of TFEB/TFE3 by FLCN is evolutionarily conserved through mTOR independent mechanisms (A) Immunoblot of isolated cytosolic-soluble fractions and nuclear fractions of wild-type and FLCN KO mouse embryonic fibroblasts (MEFs). (B) Relative mRNA levels measured by qRT-PCR of indicated genes in wild-type and FLCN KO MEFs. Data represent the average of three independent experiments done in triplicates ± SEM. Significance was determined using student’s t-test ( ** p

    Techniques Used: Isolation, Quantitative RT-PCR

    AMPK regulates TFEB/TFE3-mediated innate immune response (A) Immunoblot of wild-type or AMPKα1/α2 double knock out (DKO) MEFs stimulated with the AMPK activator; GSK-621 (30 μM) for 1 h. (B) Representative images of TFEB and TFE3 staining in wild-type and AMPK DKO MEFs before and after treatment with GSK-621 (30 μM) for 1 h. Scale bars represent 20 μm. (C) Quantification of the percentage of cells showing TFEB and TFE3 nuclear staining of the conditions described in (B). (D) Relative IL-6 mRNA levels measured by qRT-PCR in wild-type MEFs transfected with EV or shTFEB/TFE3, stimulated with GSK-621 for 2 h. Data represents the average of three independent experiments, each done in triplicates ± SEM. Significance was determined using one-way ANOVA with the application of Bonferroni correction ( * p
    Figure Legend Snippet: AMPK regulates TFEB/TFE3-mediated innate immune response (A) Immunoblot of wild-type or AMPKα1/α2 double knock out (DKO) MEFs stimulated with the AMPK activator; GSK-621 (30 μM) for 1 h. (B) Representative images of TFEB and TFE3 staining in wild-type and AMPK DKO MEFs before and after treatment with GSK-621 (30 μM) for 1 h. Scale bars represent 20 μm. (C) Quantification of the percentage of cells showing TFEB and TFE3 nuclear staining of the conditions described in (B). (D) Relative IL-6 mRNA levels measured by qRT-PCR in wild-type MEFs transfected with EV or shTFEB/TFE3, stimulated with GSK-621 for 2 h. Data represents the average of three independent experiments, each done in triplicates ± SEM. Significance was determined using one-way ANOVA with the application of Bonferroni correction ( * p

    Techniques Used: Knock-Out, Staining, Quantitative RT-PCR, Transfection

    32) Product Images from "Spleen Tyrosine Kinase (Syk)-dependent Calcium Signals Mediate Efficient CpG-induced Exocytosis of Tumor Necrosis Factor ? (TNF?) in Innate Immune Cells *"

    Article Title: Spleen Tyrosine Kinase (Syk)-dependent Calcium Signals Mediate Efficient CpG-induced Exocytosis of Tumor Necrosis Factor ? (TNF?) in Innate Immune Cells *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M113.454405

    Canonical signaling downstream of TLR9 is intact in Syk-deficient cells. A , RNA was extracted from RAW cells stimulated with CpG. Quantitative RT-PCR was performed, and the TLR9 message was normalized to GAPDH message. The bar graph is representative of three independent experiments performed in triplicate. B and C , RAW cells ( top panel ) and BMDCs ( bottom panel ) were stimulated at various times with CpG, and cell lysates were analyzed by Western blot analysis for phosphorylation of the MAP kinases ERK and p38 ( B ) and degradation of IκBα ( C ). D , nuclear extracts were isolated from RAW cells stimulated for 30 min with CpG and analyzed by Western blot analysis for nuclear translocation of NF-κB. Blots were developed on Odyssey and analyzed by Licor software and are representative of at least four independent experiments.
    Figure Legend Snippet: Canonical signaling downstream of TLR9 is intact in Syk-deficient cells. A , RNA was extracted from RAW cells stimulated with CpG. Quantitative RT-PCR was performed, and the TLR9 message was normalized to GAPDH message. The bar graph is representative of three independent experiments performed in triplicate. B and C , RAW cells ( top panel ) and BMDCs ( bottom panel ) were stimulated at various times with CpG, and cell lysates were analyzed by Western blot analysis for phosphorylation of the MAP kinases ERK and p38 ( B ) and degradation of IκBα ( C ). D , nuclear extracts were isolated from RAW cells stimulated for 30 min with CpG and analyzed by Western blot analysis for nuclear translocation of NF-κB. Blots were developed on Odyssey and analyzed by Licor software and are representative of at least four independent experiments.

    Techniques Used: Quantitative RT-PCR, Western Blot, Isolation, Translocation Assay, Software

    CpG-induced TNFα is transcribed and translated normally in Syk-deficient cells. A , RAW cells were stimulated with CpG, and RNA was isolated and analyzed by quantitative RT-PCR. The TNFα message was normalized to the GAPDH message. The bar graph is representative of three independent experiments performed in triplicate. B , RAW cells were stimulated for 3 h with CpG in the presence of brefeldin A, and cells were harvested, permeabilized, and stained for TNFα for analysis by flow cytometry. Histograms are representative of at least five independent experiments performed for each cell type.
    Figure Legend Snippet: CpG-induced TNFα is transcribed and translated normally in Syk-deficient cells. A , RAW cells were stimulated with CpG, and RNA was isolated and analyzed by quantitative RT-PCR. The TNFα message was normalized to the GAPDH message. The bar graph is representative of three independent experiments performed in triplicate. B , RAW cells were stimulated for 3 h with CpG in the presence of brefeldin A, and cells were harvested, permeabilized, and stained for TNFα for analysis by flow cytometry. Histograms are representative of at least five independent experiments performed for each cell type.

    Techniques Used: Isolation, Quantitative RT-PCR, Staining, Flow Cytometry, Cytometry

    33) Product Images from "Enhanced Cardiac Function in Gravin Mutant Mice Involves Alterations in the ?-Adrenergic Receptor Signaling Cascade"

    Article Title: Enhanced Cardiac Function in Gravin Mutant Mice Involves Alterations in the ?-Adrenergic Receptor Signaling Cascade

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0074784

    Phosphorylation of cardiac Myosin Binding Protein C in response to acute ISO stimulation in WT and gravin-t/t mice. ( A ) Representative western blots of the three phosphorylation sites of cardiac myosin binding protein C (cMyBPC) as well as total cMyBPC in heart homogenates isolated from WT and gravin-t/t mice following acute vehicle or ISO infusion (10µg/g/min); (Lane 1: WT VEH; Lane 2: WT ISO; Lane 3: gravin-t/t VEH; Lane 4: gravin-t/t ISO). ( B - D ) The bar graphs show the ratio of phosphorylated to total protein for p273, p282 and p302 respectively normalized to WT vehicle. Data are expressed as the mean ± S.E.M.; n= 4 to 6 samples; *p
    Figure Legend Snippet: Phosphorylation of cardiac Myosin Binding Protein C in response to acute ISO stimulation in WT and gravin-t/t mice. ( A ) Representative western blots of the three phosphorylation sites of cardiac myosin binding protein C (cMyBPC) as well as total cMyBPC in heart homogenates isolated from WT and gravin-t/t mice following acute vehicle or ISO infusion (10µg/g/min); (Lane 1: WT VEH; Lane 2: WT ISO; Lane 3: gravin-t/t VEH; Lane 4: gravin-t/t ISO). ( B - D ) The bar graphs show the ratio of phosphorylated to total protein for p273, p282 and p302 respectively normalized to WT vehicle. Data are expressed as the mean ± S.E.M.; n= 4 to 6 samples; *p

    Techniques Used: Binding Assay, Mouse Assay, Western Blot, Isolation

    34) Product Images from "Activation of NFAT signaling establishes a tumorigenic microenvironment through cell autonomous and non-cell autonomous mechanisms"

    Article Title: Activation of NFAT signaling establishes a tumorigenic microenvironment through cell autonomous and non-cell autonomous mechanisms

    Journal: Oncogene

    doi: 10.1038/onc.2013.132

    Transcript profiling revealed that NFATc1 activation creates a proinflammatory microenvironment for tumor development Gene ontology analysis revealed that the top categories for genes with significant transcriptional changes include cell proliferation, growth, and metabolic processes ( A ). Venn diagram showed 156 genes commonly changed across all three sets ( B ). A number of the secretory factors were among the genes with the most significant changes. These and other significantly changed genes with potential links to NFAT signaling were subjected to further RT-PCR using skin from mutant and control mice treated with Dox for 48 hours ( C ). CT; skin from control mice, MT; skin from mutant mice. Some of these genes were further validated by Q-PCR, which shows similar trend of expression change in mutant skin as suggested by microarray ( D ). H E and PCNA staining showed hyper-proliferative mutant skin epidermis after 2 days of NFATc1 activation compared to control samples ( E - H ). Immunostaining showed more extensive expression of IL1β and OSM in skin from mutant mice compared to their littermate controls ( I - L ).
    Figure Legend Snippet: Transcript profiling revealed that NFATc1 activation creates a proinflammatory microenvironment for tumor development Gene ontology analysis revealed that the top categories for genes with significant transcriptional changes include cell proliferation, growth, and metabolic processes ( A ). Venn diagram showed 156 genes commonly changed across all three sets ( B ). A number of the secretory factors were among the genes with the most significant changes. These and other significantly changed genes with potential links to NFAT signaling were subjected to further RT-PCR using skin from mutant and control mice treated with Dox for 48 hours ( C ). CT; skin from control mice, MT; skin from mutant mice. Some of these genes were further validated by Q-PCR, which shows similar trend of expression change in mutant skin as suggested by microarray ( D ). H E and PCNA staining showed hyper-proliferative mutant skin epidermis after 2 days of NFATc1 activation compared to control samples ( E - H ). Immunostaining showed more extensive expression of IL1β and OSM in skin from mutant mice compared to their littermate controls ( I - L ).

    Techniques Used: Activation Assay, Reverse Transcription Polymerase Chain Reaction, Mutagenesis, Mouse Assay, Polymerase Chain Reaction, Expressing, Microarray, Staining, Immunostaining

    35) Product Images from "Plastic Transcriptomes Stabilize Immunity to Pathogen Diversity: The Jasmonic Acid and Salicylic Acid Networks within the Arabidopsis/Botrytis Pathosystem [OPEN]"

    Article Title: Plastic Transcriptomes Stabilize Immunity to Pathogen Diversity: The Jasmonic Acid and Salicylic Acid Networks within the Arabidopsis/Botrytis Pathosystem [OPEN]

    Journal: The Plant Cell

    doi: 10.1105/tpc.17.00348

    Distribution of Genetic Variation Controlling Defense Responses. Violin plots illustrating the distribution of estimated broad-sense heritability (H 2 ) values for transcripts responding to 96 B. cinerea isolates across Arabidopsis genotypes. Heritability is partitioned across the different sources, 96 pathogen genotypes (Isolate), the Col-0, coi1-1 , and npr1-1 plant genotypes (Host), and the corresponding interaction. The red lines indicate heritability values for lesion area, while blue shows those for camalexin accumulation in the same experiments. Plant defense-related phenotypes of camalexin accumulation and lesion area were measured on leaves from three Arabidopsis genotypes at 72 HPI with 96 diverse B. cinerea isolates. The transcriptomic analysis was conducted by sequencing mRNA extracted from infected Arabidopsis leaves at 16 HPI. (A) Heritability distributions for all 23,898 detected Arabidopsis transcripts in B. cinerea infected plant tissues. (B) Heritability values for Arabidopsis transcripts in B. cinerea infected plant tissues estimated by individually modeling the Col-0, coi1-1 , and npr1-1 genotypes. The Arabidopsis genotypes are presented on the horizontal axis.
    Figure Legend Snippet: Distribution of Genetic Variation Controlling Defense Responses. Violin plots illustrating the distribution of estimated broad-sense heritability (H 2 ) values for transcripts responding to 96 B. cinerea isolates across Arabidopsis genotypes. Heritability is partitioned across the different sources, 96 pathogen genotypes (Isolate), the Col-0, coi1-1 , and npr1-1 plant genotypes (Host), and the corresponding interaction. The red lines indicate heritability values for lesion area, while blue shows those for camalexin accumulation in the same experiments. Plant defense-related phenotypes of camalexin accumulation and lesion area were measured on leaves from three Arabidopsis genotypes at 72 HPI with 96 diverse B. cinerea isolates. The transcriptomic analysis was conducted by sequencing mRNA extracted from infected Arabidopsis leaves at 16 HPI. (A) Heritability distributions for all 23,898 detected Arabidopsis transcripts in B. cinerea infected plant tissues. (B) Heritability values for Arabidopsis transcripts in B. cinerea infected plant tissues estimated by individually modeling the Col-0, coi1-1 , and npr1-1 genotypes. The Arabidopsis genotypes are presented on the horizontal axis.

    Techniques Used: Sequencing, Infection

    36) Product Images from "A Petunia Homeodomain-Leucine Zipper Protein, PhHD-Zip, Plays an Important Role in Flower Senescence"

    Article Title: A Petunia Homeodomain-Leucine Zipper Protein, PhHD-Zip, Plays an Important Role in Flower Senescence

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0088320

    Expression of PhHD-Zip in petunia flowers in response to ethylene and 1-MCP treatments. “ Ethylene ” Flowers harvested at anthesis and treated continuously with ethylene (3 ppm), “ 1-MCP/Ethylene” Flowers harvested at anthesis and treated with 1-MCP (50 nL/L) for 4 hours before a continuous ethylene treatment. A. A representative gel image from semi-quantitative PCR of RNA isolated from corollas harvested at intervals. 26S RNA: the internal control. Samples were analyzed after 30 cycles for PhHD-Zip and after 24 cycles for 26S RNA . B. Relative expression levels of PhHD-Zip (quantification of the gel pictures; error bars show SE of the means of three biological replicates).
    Figure Legend Snippet: Expression of PhHD-Zip in petunia flowers in response to ethylene and 1-MCP treatments. “ Ethylene ” Flowers harvested at anthesis and treated continuously with ethylene (3 ppm), “ 1-MCP/Ethylene” Flowers harvested at anthesis and treated with 1-MCP (50 nL/L) for 4 hours before a continuous ethylene treatment. A. A representative gel image from semi-quantitative PCR of RNA isolated from corollas harvested at intervals. 26S RNA: the internal control. Samples were analyzed after 30 cycles for PhHD-Zip and after 24 cycles for 26S RNA . B. Relative expression levels of PhHD-Zip (quantification of the gel pictures; error bars show SE of the means of three biological replicates).

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction, Isolation

    Expression of PhHD-Zip in different tissues of petunia. A. A representative gel image from semi-quantitative PCR of RNA isolated from different tissues. 26S RNA: the internal control. Samples were analyzed after 33 cycles for PhHD-Zip , and after 24 cycles for 26S RNA . B. Relative expression levels of PhHD-Zip in different tissues (quantification of the gel pictures; error bars show SE of the means of three biological replicates; different letters denote significant differences using Duncan’s test at P
    Figure Legend Snippet: Expression of PhHD-Zip in different tissues of petunia. A. A representative gel image from semi-quantitative PCR of RNA isolated from different tissues. 26S RNA: the internal control. Samples were analyzed after 33 cycles for PhHD-Zip , and after 24 cycles for 26S RNA . B. Relative expression levels of PhHD-Zip in different tissues (quantification of the gel pictures; error bars show SE of the means of three biological replicates; different letters denote significant differences using Duncan’s test at P

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction, Isolation

    Expression of senescence-related genes in D7 petunia flowers. Abundance of transcripts of genes associated with senescence were determined at D7 in purple control flowers (WT), in white flowers of plants inoculated with the CHS /TRV reporter construct (VW), and in white flowers of plant inoculated with the PhHD-Zip / CHS /TRV silencing construct. A. A representative gel image from semi-quantitative PCR of RNA isolated from corollas. 26S RNA: the internal control. Samples were analyzed after 33 cycles for ACS, after 30 cycles for other genes, and after 24 cycles for 26S RNA , respectively. B. Relative expression levels of different genes (quantification of the gel pictures; error bars show SE of the means of three biological replicates; different letters denote significant differences using Duncan’s test at P
    Figure Legend Snippet: Expression of senescence-related genes in D7 petunia flowers. Abundance of transcripts of genes associated with senescence were determined at D7 in purple control flowers (WT), in white flowers of plants inoculated with the CHS /TRV reporter construct (VW), and in white flowers of plant inoculated with the PhHD-Zip / CHS /TRV silencing construct. A. A representative gel image from semi-quantitative PCR of RNA isolated from corollas. 26S RNA: the internal control. Samples were analyzed after 33 cycles for ACS, after 30 cycles for other genes, and after 24 cycles for 26S RNA , respectively. B. Relative expression levels of different genes (quantification of the gel pictures; error bars show SE of the means of three biological replicates; different letters denote significant differences using Duncan’s test at P

    Techniques Used: Expressing, Construct, Real-time Polymerase Chain Reaction, Isolation

    Expression of PhHD-Zip in petunia flower under abiotic stress. Petunia flowers harvested at anthesis were placed in tubes with water, without water, with 50°C. A. A representative gel image from semi-quantitative PCR of RNA isolated from corollas harvested at intervals. 26S RNA: the internal control. Samples were analyzed after 30 cycles for PhHD-Zip, and after 24 cycles for 26S RNA. B. Relative expression levels of PhHD-Zip (quantification of the gel pictures; error bars show SE of the means of three biological replicates). C. Relative expression levels of PhHD-Zip determined using the same RNA samples, but using real-time quantitative PCR (error bars correspond to SE of the means of three biological replicates).
    Figure Legend Snippet: Expression of PhHD-Zip in petunia flower under abiotic stress. Petunia flowers harvested at anthesis were placed in tubes with water, without water, with 50°C. A. A representative gel image from semi-quantitative PCR of RNA isolated from corollas harvested at intervals. 26S RNA: the internal control. Samples were analyzed after 30 cycles for PhHD-Zip, and after 24 cycles for 26S RNA. B. Relative expression levels of PhHD-Zip (quantification of the gel pictures; error bars show SE of the means of three biological replicates). C. Relative expression levels of PhHD-Zip determined using the same RNA samples, but using real-time quantitative PCR (error bars correspond to SE of the means of three biological replicates).

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction, Isolation

    Expression of PhHD-Zip in petunia corollas during flower senescence. A. A representative gel image from semi-quantitative PCR of RNA isolated from corollas harvested at intervals after anthesis. D0: at anthesis; D2, D4, D7: 2, 4, and 7 days after anthesis, respectively. 26S RNA: the internal control. Samples were analyzed after 30 cycles of amplification for PhHD-Zip , and after 24 cycles of amplification for 26S RNA . B. Relative expression levels of PhHD-Zip (quantification of the gel pictures; error bars show SE of the means of three biological replicates).
    Figure Legend Snippet: Expression of PhHD-Zip in petunia corollas during flower senescence. A. A representative gel image from semi-quantitative PCR of RNA isolated from corollas harvested at intervals after anthesis. D0: at anthesis; D2, D4, D7: 2, 4, and 7 days after anthesis, respectively. 26S RNA: the internal control. Samples were analyzed after 30 cycles of amplification for PhHD-Zip , and after 24 cycles of amplification for 26S RNA . B. Relative expression levels of PhHD-Zip (quantification of the gel pictures; error bars show SE of the means of three biological replicates).

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction, Isolation, Amplification

    Effect of transgenic over-expression of PhHD-Zip on abundance of transcripts of PhHD-Zip and of ethylene biosynthesis genes in petunia corollas. WT: wild type flower; #3, #7: two transgenic lines of 35S:: PhHD-Zip . A. A representative gel image from semi-quantitative PCR of RNA isolated from harvested corollas. 26S RNA: the internal control. Samples were analyzed after 30 cycles for PhHD-Zip, ACO1 and ACO4 ; after 33 cycles for ACS ; and after 24 cycles for 26S RNA . B. Relative expression level of PhHD-Zip and ethylene biosynthesis genes (quantification of the gel pictures; error bars show SE of the means of three biological replicates; different letters denote significant differences using Duncan’s test at P
    Figure Legend Snippet: Effect of transgenic over-expression of PhHD-Zip on abundance of transcripts of PhHD-Zip and of ethylene biosynthesis genes in petunia corollas. WT: wild type flower; #3, #7: two transgenic lines of 35S:: PhHD-Zip . A. A representative gel image from semi-quantitative PCR of RNA isolated from harvested corollas. 26S RNA: the internal control. Samples were analyzed after 30 cycles for PhHD-Zip, ACO1 and ACO4 ; after 33 cycles for ACS ; and after 24 cycles for 26S RNA . B. Relative expression level of PhHD-Zip and ethylene biosynthesis genes (quantification of the gel pictures; error bars show SE of the means of three biological replicates; different letters denote significant differences using Duncan’s test at P

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

    37) Product Images from "Large-scale identification of wheat genes resistant to cereal cyst nematode Heterodera avenae using comparative transcriptomic analysis"

    Article Title: Large-scale identification of wheat genes resistant to cereal cyst nematode Heterodera avenae using comparative transcriptomic analysis

    Journal: BMC Genomics

    doi: 10.1186/s12864-015-2037-8

    Production of ROS and induction of ROS-producing genes within VP1620 respond to H. avenae infestation: a ROS contents within infected and uninfected roots of VP1620 at three time points after CCN infection. The ROS content was determined by a luminol-chemiluminescence assay (_0, no CCN inoculation; _CN, CCN inoculated lines; error bars represent the SE ( n = 3)). b Identification of ROS-producing genes in VP1620 significantly induced after H. avenae infestation and relative expression detection assayed by qRT-PCR. The expression level of each ROS-producing gene in I_0 was arbitrarily set to 1. Abbreviations: POX, Class III Peroxidase; LOX, Lipoxygenase. Mean and standard errors were determined using data from three independent replicates
    Figure Legend Snippet: Production of ROS and induction of ROS-producing genes within VP1620 respond to H. avenae infestation: a ROS contents within infected and uninfected roots of VP1620 at three time points after CCN infection. The ROS content was determined by a luminol-chemiluminescence assay (_0, no CCN inoculation; _CN, CCN inoculated lines; error bars represent the SE ( n = 3)). b Identification of ROS-producing genes in VP1620 significantly induced after H. avenae infestation and relative expression detection assayed by qRT-PCR. The expression level of each ROS-producing gene in I_0 was arbitrarily set to 1. Abbreviations: POX, Class III Peroxidase; LOX, Lipoxygenase. Mean and standard errors were determined using data from three independent replicates

    Techniques Used: Infection, Chemiluminescence Immunoassay, Expressing, Quantitative RT-PCR

    38) Product Images from "A novel small-form NEDD4 regulates cell invasiveness and apoptosis to promote tumor metastasis"

    Article Title: A novel small-form NEDD4 regulates cell invasiveness and apoptosis to promote tumor metastasis

    Journal: Oncotarget

    doi:

    Generation of potent metastatic cell lines, SKT and SKM, and identifying metastatic-associated gene, sNEDD4 (A) Schematic diagram of the experimental procedure. (B) The invasive abilities of SK, SKT, and SKM cells were determined with the Transwell invasion assay. (C) Western blot analysis was used to determined NEDD4 protein expression of SK, SKT, and SKM cells. Three (left; upper, middle, and lower) and two (right; upper and lower) major bands were detected using NEDD4 and NEDD4–1 antibodies in SKM cells, respectively. (D) Schematic diagram of NEDD4 mRNA (Upper). The open box represents the coding region of NEDD4 . Classic and predictive ATG sites are labeled with gray bars. The lower schematic diagram represents NEDD4 and sNEDD4 protein structures. (E) NEDD4 mRNA levels were determined via qRT-PCR with primers amplified from two different segments, S1 and S2 (mark in panel D). (F) NEDD4 and sNEDD4 protein levels of SK, SKM and SK cells transiently transfected with empty vector (Vec) or sNEDD4 expression plasmid (sNED). (G) sNEDD4 was detected using Northern blot. SK cells transfected with sNEDD4 expressing plasmid (sNED) serve as a positive control. IB, immunobloting; S1, 366–478 nucleotides; S2, 649–751 nucleotides; *** P
    Figure Legend Snippet: Generation of potent metastatic cell lines, SKT and SKM, and identifying metastatic-associated gene, sNEDD4 (A) Schematic diagram of the experimental procedure. (B) The invasive abilities of SK, SKT, and SKM cells were determined with the Transwell invasion assay. (C) Western blot analysis was used to determined NEDD4 protein expression of SK, SKT, and SKM cells. Three (left; upper, middle, and lower) and two (right; upper and lower) major bands were detected using NEDD4 and NEDD4–1 antibodies in SKM cells, respectively. (D) Schematic diagram of NEDD4 mRNA (Upper). The open box represents the coding region of NEDD4 . Classic and predictive ATG sites are labeled with gray bars. The lower schematic diagram represents NEDD4 and sNEDD4 protein structures. (E) NEDD4 mRNA levels were determined via qRT-PCR with primers amplified from two different segments, S1 and S2 (mark in panel D). (F) NEDD4 and sNEDD4 protein levels of SK, SKM and SK cells transiently transfected with empty vector (Vec) or sNEDD4 expression plasmid (sNED). (G) sNEDD4 was detected using Northern blot. SK cells transfected with sNEDD4 expressing plasmid (sNED) serve as a positive control. IB, immunobloting; S1, 366–478 nucleotides; S2, 649–751 nucleotides; *** P

    Techniques Used: Transwell Invasion Assay, Western Blot, Expressing, Labeling, Quantitative RT-PCR, Amplification, Transfection, Plasmid Preparation, Northern Blot, Positive Control

    39) Product Images from "Phenoloxidase Activity Acts as a Mosquito Innate Immune Response against Infection with Semliki Forest Virus"

    Article Title: Phenoloxidase Activity Acts as a Mosquito Innate Immune Response against Infection with Semliki Forest Virus

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1002977

    Expression of Egf1.0 increases mortality of Ae. aegypti and replication of SFV in vivo . ( A ) Ae. aegypti were fed blood containing SFV4(3H)- FFLuc -Egf1.0F or SFV4(3H)- FFLuc -Egf1.0R. Uninfected blood meals served as a control. Mosquito mortality was then monitored daily post-bloodmeal. Combined survival data from three independent experiments (cohorts of 22–25 infected mosquitoes per virus or control mosquitoes in each experiment) are shown. Error bars show standard deviation. ( B ) SFV genome copy number as determined by real time qPCR. Total RNA was extracted 3 days post-bloodmeal from mosquitoes infected with SFV4(3H)- FFLuc -Egf1.0F or SFV4(3H)- FFLuc -Egf1.0R. Viral genome RNA levels from 10 mosquitoes for each virus are shown. Values at 0 represent uninfected mosquitoes. Horizontal bar indicates average genome copy number from infected mosquitoes. This experiment was repeated three times with similar results.
    Figure Legend Snippet: Expression of Egf1.0 increases mortality of Ae. aegypti and replication of SFV in vivo . ( A ) Ae. aegypti were fed blood containing SFV4(3H)- FFLuc -Egf1.0F or SFV4(3H)- FFLuc -Egf1.0R. Uninfected blood meals served as a control. Mosquito mortality was then monitored daily post-bloodmeal. Combined survival data from three independent experiments (cohorts of 22–25 infected mosquitoes per virus or control mosquitoes in each experiment) are shown. Error bars show standard deviation. ( B ) SFV genome copy number as determined by real time qPCR. Total RNA was extracted 3 days post-bloodmeal from mosquitoes infected with SFV4(3H)- FFLuc -Egf1.0F or SFV4(3H)- FFLuc -Egf1.0R. Viral genome RNA levels from 10 mosquitoes for each virus are shown. Values at 0 represent uninfected mosquitoes. Horizontal bar indicates average genome copy number from infected mosquitoes. This experiment was repeated three times with similar results.

    Techniques Used: Expressing, In Vivo, Infection, Standard Deviation, Real-time Polymerase Chain Reaction

    40) Product Images from "CD55 regulates self-renewal and cisplatin resistance in endometrioid tumors"

    Article Title: CD55 regulates self-renewal and cisplatin resistance in endometrioid tumors

    Journal: The Journal of Experimental Medicine

    doi: 10.1084/jem.20170438

    CD55 is sufficient to drive self-renewal and cisplatin-resistance in endometrioid non-CSCs. (A) Immunoblots of cisplatin-naive non-CSCs with CD55 overexpression (OE) and empty vector controls were probed with CD55, NANOG, SOX2, and OCT4. Actin was used as loading control. Data are representative of two independent experiments. (B) mRNA expression was determined by quantitative real-time PCR and compared between CD55-overexpressing A2780 non-CSCs and empty vector control non-CSCs. Actin was used as a control. Three technical replicates were used. (C) Limiting dilution analysis plots of empty vector control compared with CD55 overexpression in cisplatin-naive non-CSCs. The graph compares the estimates of the percentage of self-renewal frequency in sorted populations with the corresponding p-values. Data are representative of three independent experiments. (D) A2780 non-CSCs transduced with CD55 overexpression and empty vector controls were flowed for GFP signal intensity, which indicates NANOG promoter activity. (E) Tumorsphere from A2780 non-CSCs transduced with CD55 and empty vector control were imaged using a digital immunofluorescence microscope. (F) CD55-overexpressing cisplatin-naive non-CSCs and their empty vector controls were treated with 0–50 µM cisplatin, and percentage of surviving cells was graphed. Data are representative of three independent experiments. (G) Relative caspase 3/7 activity of CD55-overexpressing cisplatin-naive cells and empty vector controls after cisplatin treatment. Relative caspase activities in cisplatin treated groups were calculated after normalizing the corrected readings to untreated controls in each group. Data are representative of two independent experiments, and three technical replicates were used in each. *, P
    Figure Legend Snippet: CD55 is sufficient to drive self-renewal and cisplatin-resistance in endometrioid non-CSCs. (A) Immunoblots of cisplatin-naive non-CSCs with CD55 overexpression (OE) and empty vector controls were probed with CD55, NANOG, SOX2, and OCT4. Actin was used as loading control. Data are representative of two independent experiments. (B) mRNA expression was determined by quantitative real-time PCR and compared between CD55-overexpressing A2780 non-CSCs and empty vector control non-CSCs. Actin was used as a control. Three technical replicates were used. (C) Limiting dilution analysis plots of empty vector control compared with CD55 overexpression in cisplatin-naive non-CSCs. The graph compares the estimates of the percentage of self-renewal frequency in sorted populations with the corresponding p-values. Data are representative of three independent experiments. (D) A2780 non-CSCs transduced with CD55 overexpression and empty vector controls were flowed for GFP signal intensity, which indicates NANOG promoter activity. (E) Tumorsphere from A2780 non-CSCs transduced with CD55 and empty vector control were imaged using a digital immunofluorescence microscope. (F) CD55-overexpressing cisplatin-naive non-CSCs and their empty vector controls were treated with 0–50 µM cisplatin, and percentage of surviving cells was graphed. Data are representative of three independent experiments. (G) Relative caspase 3/7 activity of CD55-overexpressing cisplatin-naive cells and empty vector controls after cisplatin treatment. Relative caspase activities in cisplatin treated groups were calculated after normalizing the corrected readings to untreated controls in each group. Data are representative of two independent experiments, and three technical replicates were used in each. *, P

    Techniques Used: Western Blot, Over Expression, Plasmid Preparation, Expressing, Real-time Polymerase Chain Reaction, Transduction, Activity Assay, Immunofluorescence, Microscopy

    CD55 is highly expressed on endometrioid ovarian and uterine CSCs and cisplatin-resistant cells. (A) A high-throughput flow cytometry screen of 242 different surface CD markers in cisplatin-naive (A2780) and cisplatin-resistant (CP70) ovarian cancer cells was performed to investigate the differential expression of these markers between CSCs versus non-CSCs and cisplatin-naive versus cisplatin-resistant cells. (B) Of 242 markers, CD55 was the most highly and differentially expressed between cisplatin-naive CSCs versus non-CSCs and cisplatin-resistant versus cisplatin-naive cells. (C and D) Cell lysates from cisplatin-naive A2780 reporter, TOV112D, and PDX (EEC-4) cells sorted into CSCs and non-CSCs by GFP expression and CD49f expression, respectively, were probed with anti-CD55, CD59, and CD46 antibodies. Actin was used as a loading control. Data are representative of three independent experiments. (E) Protein and mRNA expression of CD55, CD59, and CD46 were assessed in lysates from cisplatin-naive (A2780) and cisplatin-resistant (CP70) cells. Actin was used as a control. Data are representative of two independent experiments. (F) Limiting dilution analysis of CD55+ compared with CD55− cisplatin-naive cells. The graph represents the estimates in percentage of self-renewal frequency in sorted populations with the corresponding p-values. Data represent two independent experiments. (G) Kaplan-Meier (K-M) progression-free survival curve for endometrioid ovarian cancer patients who had high versus low tumor CD55 expression before therapy was obtained from K-M plotter database ( http://kmplot.com/analysis/ ). *, P
    Figure Legend Snippet: CD55 is highly expressed on endometrioid ovarian and uterine CSCs and cisplatin-resistant cells. (A) A high-throughput flow cytometry screen of 242 different surface CD markers in cisplatin-naive (A2780) and cisplatin-resistant (CP70) ovarian cancer cells was performed to investigate the differential expression of these markers between CSCs versus non-CSCs and cisplatin-naive versus cisplatin-resistant cells. (B) Of 242 markers, CD55 was the most highly and differentially expressed between cisplatin-naive CSCs versus non-CSCs and cisplatin-resistant versus cisplatin-naive cells. (C and D) Cell lysates from cisplatin-naive A2780 reporter, TOV112D, and PDX (EEC-4) cells sorted into CSCs and non-CSCs by GFP expression and CD49f expression, respectively, were probed with anti-CD55, CD59, and CD46 antibodies. Actin was used as a loading control. Data are representative of three independent experiments. (E) Protein and mRNA expression of CD55, CD59, and CD46 were assessed in lysates from cisplatin-naive (A2780) and cisplatin-resistant (CP70) cells. Actin was used as a control. Data are representative of two independent experiments. (F) Limiting dilution analysis of CD55+ compared with CD55− cisplatin-naive cells. The graph represents the estimates in percentage of self-renewal frequency in sorted populations with the corresponding p-values. Data represent two independent experiments. (G) Kaplan-Meier (K-M) progression-free survival curve for endometrioid ovarian cancer patients who had high versus low tumor CD55 expression before therapy was obtained from K-M plotter database ( http://kmplot.com/analysis/ ). *, P

    Techniques Used: High Throughput Screening Assay, Flow Cytometry, Cytometry, Expressing

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    Amplification:

    Article Title: Activity of the Calcium Channel Pore Cch1 Is Dependent on a Modulatory Region of the Subunit Mid1 in Cryptococcus neoformans
    Article Snippet: .. Briefly, the MID1 amplicon was generated from cDNA synthesized by a SuperScript III first-strand synthesis system for reverse transcriptase PCR (RT-PCR) (Invitrogen) using total RNA as the template with the following primers: MID1-cDNA-F and MID1-cDNA-R ( ). .. RNA was isolated from a culture (∼5 × 107 cells) of a wild-type strain of C. neoformans var. grubii (H99, serotype A).

    Synthesized:

    Article Title: Activity of the Calcium Channel Pore Cch1 Is Dependent on a Modulatory Region of the Subunit Mid1 in Cryptococcus neoformans
    Article Snippet: .. Briefly, the MID1 amplicon was generated from cDNA synthesized by a SuperScript III first-strand synthesis system for reverse transcriptase PCR (RT-PCR) (Invitrogen) using total RNA as the template with the following primers: MID1-cDNA-F and MID1-cDNA-R ( ). .. RNA was isolated from a culture (∼5 × 107 cells) of a wild-type strain of C. neoformans var. grubii (H99, serotype A).

    Article Title: Effects of RNA Splicing Inhibitors on Amyloid Precursor Protein Expression
    Article Snippet: .. RT-PCR The RNA was extracted from HEK293T cells using an RNeasy Mini Kit (74104, Qiagen) and synthesized into the first DNA strand by SuperScript III reverse transcriptase (18080-051, Life Technologies). .. PCR was done by Choice Taq Mastermix (CB4070-8, Denville Scientific).

    Real-time Polymerase Chain Reaction:

    Article Title: Identification of Active Loci of a Human Endogenous Retrovirus in Neurons of Patients with Amyotrophic Lateral Sclerosis
    Article Snippet: .. DNase I digested RNA samples were reverse transcribed using random hexamers and a gene-specific HERV-K pol primer (5′-GTTGAAGAGCTCGACCTACAAAA- 3′) using SuperScript III First-Strand Synthesis System for real-time-PCR (Invitrogen). .. Real-time PCR using SYBR Green was used to determine the level of HERV-K pol transcripts.

    Polymerase Chain Reaction:

    Article Title: Activity of the Calcium Channel Pore Cch1 Is Dependent on a Modulatory Region of the Subunit Mid1 in Cryptococcus neoformans
    Article Snippet: .. Briefly, the MID1 amplicon was generated from cDNA synthesized by a SuperScript III first-strand synthesis system for reverse transcriptase PCR (RT-PCR) (Invitrogen) using total RNA as the template with the following primers: MID1-cDNA-F and MID1-cDNA-R ( ). .. RNA was isolated from a culture (∼5 × 107 cells) of a wild-type strain of C. neoformans var. grubii (H99, serotype A).

    Generated:

    Article Title: Activity of the Calcium Channel Pore Cch1 Is Dependent on a Modulatory Region of the Subunit Mid1 in Cryptococcus neoformans
    Article Snippet: .. Briefly, the MID1 amplicon was generated from cDNA synthesized by a SuperScript III first-strand synthesis system for reverse transcriptase PCR (RT-PCR) (Invitrogen) using total RNA as the template with the following primers: MID1-cDNA-F and MID1-cDNA-R ( ). .. RNA was isolated from a culture (∼5 × 107 cells) of a wild-type strain of C. neoformans var. grubii (H99, serotype A).

    Reverse Transcription Polymerase Chain Reaction:

    Article Title: Activity of the Calcium Channel Pore Cch1 Is Dependent on a Modulatory Region of the Subunit Mid1 in Cryptococcus neoformans
    Article Snippet: .. Briefly, the MID1 amplicon was generated from cDNA synthesized by a SuperScript III first-strand synthesis system for reverse transcriptase PCR (RT-PCR) (Invitrogen) using total RNA as the template with the following primers: MID1-cDNA-F and MID1-cDNA-R ( ). .. RNA was isolated from a culture (∼5 × 107 cells) of a wild-type strain of C. neoformans var. grubii (H99, serotype A).

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    Article Snippet: .. Total RNA was extracted from PPAR-γ heterozygous and PPAR-γ null MEFs, SSc and healthy skin fibroblasts with TRIZOL reagent (Invitrogen Corp. Carlsbad, CA), and total RNA (1.0 mg) was reverse transcribed for RT-PCR analysis using Superscript III first Strand Synthesis System (Invitrogen, Carlsbad, CA). .. Real time PCR was performed with SYBR®GREEN Master Mix using ABI Prism 7500 sequence detector and SDS analysis software (PE Applied Biosystems, Forster City, CA).

    Article Title: Telomere maintenance during anterior regeneration and aging in the freshwater annelid Aeolosoma viride
    Article Snippet: .. Reverse transcription was performed with the SuperScript® III First-Strand Synthesis System for RT-PCR (Invitrogen) using either oligo dT or random hexamers. ..

    Article Title: Effects of RNA Splicing Inhibitors on Amyloid Precursor Protein Expression
    Article Snippet: .. RT-PCR The RNA was extracted from HEK293T cells using an RNeasy Mini Kit (74104, Qiagen) and synthesized into the first DNA strand by SuperScript III reverse transcriptase (18080-051, Life Technologies). .. PCR was done by Choice Taq Mastermix (CB4070-8, Denville Scientific).

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    Thermo Fisher superscript iii first strand synthesis system
    Effect of ETR on the expression of iNOS and proinflammatory cytokines in LPS-stimulated RAW264.7 cells. (A) Expression levels of iNOS mRNA (top) and protein (bottom) were determined by <t>RT-PCR</t> and western blotting. GAPDH and β-actin served as internal controls for RT-PCR and western blotting, respectively. (B) Expression levels of TNF-α, IL-1β and IL-6 mRNA were determined by RT-PCR. (C) Concentrations of IL-1β, TNF-α and IL-6 cytokines in cell culture supernatants were determined by ELISA. Data are presented as the mean ± standard deviation of <t>three</t> independent replicates. *P
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    Effect of ETR on the expression of iNOS and proinflammatory cytokines in LPS-stimulated RAW264.7 cells. (A) Expression levels of iNOS mRNA (top) and protein (bottom) were determined by RT-PCR and western blotting. GAPDH and β-actin served as internal controls for RT-PCR and western blotting, respectively. (B) Expression levels of TNF-α, IL-1β and IL-6 mRNA were determined by RT-PCR. (C) Concentrations of IL-1β, TNF-α and IL-6 cytokines in cell culture supernatants were determined by ELISA. Data are presented as the mean ± standard deviation of three independent replicates. *P

    Journal: Molecular Medicine Reports

    Article Title: Regulatory effects and molecular mechanism of Trigonostemon reidioides on lipopolysaccharide-induced inflammatory responses in RAW264.7 cells

    doi: 10.3892/mmr.2017.7297

    Figure Lengend Snippet: Effect of ETR on the expression of iNOS and proinflammatory cytokines in LPS-stimulated RAW264.7 cells. (A) Expression levels of iNOS mRNA (top) and protein (bottom) were determined by RT-PCR and western blotting. GAPDH and β-actin served as internal controls for RT-PCR and western blotting, respectively. (B) Expression levels of TNF-α, IL-1β and IL-6 mRNA were determined by RT-PCR. (C) Concentrations of IL-1β, TNF-α and IL-6 cytokines in cell culture supernatants were determined by ELISA. Data are presented as the mean ± standard deviation of three independent replicates. *P

    Article Snippet: RT-PCR was performed using the SuperScript® III First-Strand Synthesis System (Thermo Fisher Scientific, Inc.) and Taq DNA polymerase (Invitrogen; Thermo Fisher Scientific, Inc.).

    Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot, Cell Culture, Enzyme-linked Immunosorbent Assay, Standard Deviation

    β maj globin gene expression affects β min globin gene activation by a looped enhancer. ( A ) Interaction frequency determined by chromatin conformation capture (3C) between locations across β-globin locus using HS2 enhancer as the anchor (gray bar) observed for induced ΔCore, ΔCore/GATA and ΔCore/GATA/KLF1 MEL cell lines and uninduced and induced control MEL cells. Yellow triangles along bottom of X axis indicate BglII restriction sites. ( B ) Primary transcripts of β maj and β min globin genes were examined by RT-qPCR after deletion of Core, Core/GATA or ΔCore/GATA/KLF1 elements in the β maj globin gene promoter in induced MEL cells. ( C ) RNA PolII occupancy at β maj and β min globin genes in control, ΔCore, ΔCore/GATA and ΔCore/GATA/KLF1 induced MEL cell lines determined by ChIP-qPCR. ( D ) KLF1 occupancy at β maj and β min globin genes in control, ΔCore, ΔCore/GATA and ΔCore/GATA/KLF1 induced MEL cell lines determined by ChIP-qPCR. Error bars indicate SEM of three biological replicates. (*) P

    Journal: Nucleic Acids Research

    Article Title: LDB1-mediated enhancer looping can be established independent of mediator and cohesin

    doi: 10.1093/nar/gkx433

    Figure Lengend Snippet: β maj globin gene expression affects β min globin gene activation by a looped enhancer. ( A ) Interaction frequency determined by chromatin conformation capture (3C) between locations across β-globin locus using HS2 enhancer as the anchor (gray bar) observed for induced ΔCore, ΔCore/GATA and ΔCore/GATA/KLF1 MEL cell lines and uninduced and induced control MEL cells. Yellow triangles along bottom of X axis indicate BglII restriction sites. ( B ) Primary transcripts of β maj and β min globin genes were examined by RT-qPCR after deletion of Core, Core/GATA or ΔCore/GATA/KLF1 elements in the β maj globin gene promoter in induced MEL cells. ( C ) RNA PolII occupancy at β maj and β min globin genes in control, ΔCore, ΔCore/GATA and ΔCore/GATA/KLF1 induced MEL cell lines determined by ChIP-qPCR. ( D ) KLF1 occupancy at β maj and β min globin genes in control, ΔCore, ΔCore/GATA and ΔCore/GATA/KLF1 induced MEL cell lines determined by ChIP-qPCR. Error bars indicate SEM of three biological replicates. (*) P

    Article Snippet: RNA was reverse transcribed using SuperScript® III First-Strand Synthesis System following manufacturer's instructions (Thermo Fisher Scientific) with random hexamers (Life Technologies).

    Techniques: Expressing, Activation Assay, Quantitative RT-PCR, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction

    Expression of fruit bat tetherin is IFN-inducible and required for robust IFN-mediated inhibition of VSV infection. (A) A549 cells and the indicated fruit bat cell lines were incubated with increasing amounts of pan-IFN-α, inoculated with a single-cycle VSV vector bearing VSV-G and encoding luciferase (VSV*ΔG-FLuc), and the luciferase activity in cell lysates was quantified. The luciferase activity measured for cells treated with medium without pan-IFN-α was set as 100%. The results of a representative experiment performed with triplicate samples are shown and were confirmed in a separate experiment. Error bars indicate the SEM. The 50% inhibitory concentration (IC 50 ) was calculated for each cell line and is indicated. (B) Human (A549) and fruit bat (EpoNi/22.1) cells were pan-IFN-α or mock treated, and cellular RNA was extracted, reverse transcribed into cDNA, and analyzed for transcript levels of β-actin, Mx1 (myxovirus resistance protein), and tetherin by qRT-PCR. Shown are combined data (given as the fold change in expression upon IFN treatment, normalized to β-actin) of three independent experiments. Error bars indicate the SEM. (C) Human (A549) and fruit bat (EpoNi/22.1) cells were transfected with the indicated siRNAs (ns, nonsense = control) or left untransfected and then either treated with pan-IFN-α or mock treated, followed by inoculation with replication-competent VSV encoding eGFP. Finally, infected cells (as determined by the expression of eGFP) were detected via fluorescence microscopy. Similar results were obtained in five separate experiments. (D) Viral titers in the cellular supernatants of the untransfected, IFN-α- or mock-treated cells described in panel C were quantified. The relative (x-fold) differences between viral titers in the supernatants of pan-IFN-α- and mock-treated cells were calculated. The average of six independent experiments performed with triplicate samples is shown. Error bars indicate the SEM. (E) Viral titers in the cellular supernatants of the siRNA-transfected and IFN-treated cells described in panel C were quantified. The relative (x-fold) differences between viral titers of pan-IFN-α-treated cells transfected with control or tetherin-specific siRNA were calculated. The average of six independent experiments performed with triplicate samples is shown. Error bars indicate the SEM. (F) Relative tetherin transcript levels in IFN-treated fruit bat cells (EpoNi/22.1), which were previously transfected with either control (ns) or fruit bat tetherin-specific (batTetherin) siRNA, were compared by qRT-PCR (normalized against β-actin transcript levels). Shown are the combined results of three independent experiments performed with triplicate samples, in which tetherin transcript levels of IFN-treated cells that received control siRNA were set as 100%. Error bars indicate the SEM. (G) Human (A549) and fruit bat (EpoNi/22.1) cells were transfected with control or tetherin-specific siRNA prior to treatment with pan-IFN-α. At 48 h posttransfection, the cells were inoculated with single-cycle VSV vector pseudotyped with VSV-G (VSV*ΔG-FLuc). After incubation for 1 h, the cells were washed and further incubated for 8 h before virus-encoded luciferase activity was quantified in the cell lysates. Shown are normalized data from three independent experiments performed with quadruplicate samples in which transduction of cells without prior IFN treatment was set as 100%. Error bars indicate the SEM. The statistical significance of the data presented in panels D and E was analyzed by using a Mann-Whitney U test, while the data presented in panels F and G were analyzed by using a paired, two-tailed Student t test (ns, P > 0.05; **, P ≤ 0.01).

    Journal: Journal of Virology

    Article Title: Tetherin Inhibits Nipah Virus but Not Ebola Virus Replication in Fruit Bat Cells

    doi: 10.1128/JVI.01821-18

    Figure Lengend Snippet: Expression of fruit bat tetherin is IFN-inducible and required for robust IFN-mediated inhibition of VSV infection. (A) A549 cells and the indicated fruit bat cell lines were incubated with increasing amounts of pan-IFN-α, inoculated with a single-cycle VSV vector bearing VSV-G and encoding luciferase (VSV*ΔG-FLuc), and the luciferase activity in cell lysates was quantified. The luciferase activity measured for cells treated with medium without pan-IFN-α was set as 100%. The results of a representative experiment performed with triplicate samples are shown and were confirmed in a separate experiment. Error bars indicate the SEM. The 50% inhibitory concentration (IC 50 ) was calculated for each cell line and is indicated. (B) Human (A549) and fruit bat (EpoNi/22.1) cells were pan-IFN-α or mock treated, and cellular RNA was extracted, reverse transcribed into cDNA, and analyzed for transcript levels of β-actin, Mx1 (myxovirus resistance protein), and tetherin by qRT-PCR. Shown are combined data (given as the fold change in expression upon IFN treatment, normalized to β-actin) of three independent experiments. Error bars indicate the SEM. (C) Human (A549) and fruit bat (EpoNi/22.1) cells were transfected with the indicated siRNAs (ns, nonsense = control) or left untransfected and then either treated with pan-IFN-α or mock treated, followed by inoculation with replication-competent VSV encoding eGFP. Finally, infected cells (as determined by the expression of eGFP) were detected via fluorescence microscopy. Similar results were obtained in five separate experiments. (D) Viral titers in the cellular supernatants of the untransfected, IFN-α- or mock-treated cells described in panel C were quantified. The relative (x-fold) differences between viral titers in the supernatants of pan-IFN-α- and mock-treated cells were calculated. The average of six independent experiments performed with triplicate samples is shown. Error bars indicate the SEM. (E) Viral titers in the cellular supernatants of the siRNA-transfected and IFN-treated cells described in panel C were quantified. The relative (x-fold) differences between viral titers of pan-IFN-α-treated cells transfected with control or tetherin-specific siRNA were calculated. The average of six independent experiments performed with triplicate samples is shown. Error bars indicate the SEM. (F) Relative tetherin transcript levels in IFN-treated fruit bat cells (EpoNi/22.1), which were previously transfected with either control (ns) or fruit bat tetherin-specific (batTetherin) siRNA, were compared by qRT-PCR (normalized against β-actin transcript levels). Shown are the combined results of three independent experiments performed with triplicate samples, in which tetherin transcript levels of IFN-treated cells that received control siRNA were set as 100%. Error bars indicate the SEM. (G) Human (A549) and fruit bat (EpoNi/22.1) cells were transfected with control or tetherin-specific siRNA prior to treatment with pan-IFN-α. At 48 h posttransfection, the cells were inoculated with single-cycle VSV vector pseudotyped with VSV-G (VSV*ΔG-FLuc). After incubation for 1 h, the cells were washed and further incubated for 8 h before virus-encoded luciferase activity was quantified in the cell lysates. Shown are normalized data from three independent experiments performed with quadruplicate samples in which transduction of cells without prior IFN treatment was set as 100%. Error bars indicate the SEM. The statistical significance of the data presented in panels D and E was analyzed by using a Mann-Whitney U test, while the data presented in panels F and G were analyzed by using a paired, two-tailed Student t test (ns, P > 0.05; **, P ≤ 0.01).

    Article Snippet: Next, 1 µg of RNA was used as a template for cDNA synthesis using the SuperScript III first-strand synthesis system (Thermo Fisher Scientific) according to the manufacturer’s protocol (for random hexamers).

    Techniques: Expressing, Inhibition, Infection, Incubation, Plasmid Preparation, Luciferase, Activity Assay, Concentration Assay, Quantitative RT-PCR, Transfection, Fluorescence, Microscopy, Transduction, MANN-WHITNEY, Two Tailed Test

    Inhibition of mTOR-related genes and mTORC1 downstream effectors by RAD001. Notes: qPCR detection of mTOR-related genes, including ( A ) mTOR complexes, ( B ) mTORC1-positive regulation, and ( C ) mTORC2-positive regulation in T24 cells treated with 1 or 5 µM RAD001. Cells were seeded 24 hours before RAD001 treatment. Total RNA samples were extracted at 24 hours posttreatment, reverse transcribed, and subjected to the detection of genes involved in mTOR signaling. The values are shown as the mean ± SD of three independent experiments; * P

    Journal: Drug Design, Development and Therapy

    Article Title: Autophagy inhibition enhances RAD001-induced cytotoxicity in human bladder cancer cells

    doi: 10.2147/DDDT.S95900

    Figure Lengend Snippet: Inhibition of mTOR-related genes and mTORC1 downstream effectors by RAD001. Notes: qPCR detection of mTOR-related genes, including ( A ) mTOR complexes, ( B ) mTORC1-positive regulation, and ( C ) mTORC2-positive regulation in T24 cells treated with 1 or 5 µM RAD001. Cells were seeded 24 hours before RAD001 treatment. Total RNA samples were extracted at 24 hours posttreatment, reverse transcribed, and subjected to the detection of genes involved in mTOR signaling. The values are shown as the mean ± SD of three independent experiments; * P

    Article Snippet: Two micrograms of total RNA was subjected to reverse transcription using SuperScript III First-Strand Synthesis System (Thermo Fisher Scientific). qPCR was performed as previously described.

    Techniques: Inhibition, Real-time Polymerase Chain Reaction