Structured Review

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Gene Ontology enrichment analysis for the differentially expressed genes. a Venn diagrams for numbers of DEGs in the <t>dcl2b</t> mutant. The left panel shows up- (Cluster 1) and downregulated (Cluster 2) genes in virus-free dcl2b . The right panel shows up- (Cluster 3) and downregulated (Cluster 4) genes in virus-infected dcl2b after excluding the overlapped 916 transcripts. b , d , f , h GO enrichment analysis of Cluster 1-Cluster 4 genes. c , e , g , i Heat map of genes enriched during the mitochondrial <t>RNA</t> metabolic process, hormone signaling, response to stimulus, and lipid and fatty acid metabolism pathways, respectively
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Images

1) Product Images from "Tomato DCL2b is required for the biosynthesis of 22-nt small RNAs, the resulting secondary siRNAs, and the host defense against ToMV"

Article Title: Tomato DCL2b is required for the biosynthesis of 22-nt small RNAs, the resulting secondary siRNAs, and the host defense against ToMV

Journal: Horticulture Research

doi: 10.1038/s41438-018-0073-7

Gene Ontology enrichment analysis for the differentially expressed genes. a Venn diagrams for numbers of DEGs in the dcl2b mutant. The left panel shows up- (Cluster 1) and downregulated (Cluster 2) genes in virus-free dcl2b . The right panel shows up- (Cluster 3) and downregulated (Cluster 4) genes in virus-infected dcl2b after excluding the overlapped 916 transcripts. b , d , f , h GO enrichment analysis of Cluster 1-Cluster 4 genes. c , e , g , i Heat map of genes enriched during the mitochondrial RNA metabolic process, hormone signaling, response to stimulus, and lipid and fatty acid metabolism pathways, respectively
Figure Legend Snippet: Gene Ontology enrichment analysis for the differentially expressed genes. a Venn diagrams for numbers of DEGs in the dcl2b mutant. The left panel shows up- (Cluster 1) and downregulated (Cluster 2) genes in virus-free dcl2b . The right panel shows up- (Cluster 3) and downregulated (Cluster 4) genes in virus-infected dcl2b after excluding the overlapped 916 transcripts. b , d , f , h GO enrichment analysis of Cluster 1-Cluster 4 genes. c , e , g , i Heat map of genes enriched during the mitochondrial RNA metabolic process, hormone signaling, response to stimulus, and lipid and fatty acid metabolism pathways, respectively

Techniques Used: Mutagenesis, Infection

The miRNA levels were influenced in dcl2b mutants. a , c Differentially expressed miRNAs in dcl2b mutants. The 22nt miRNAs are colored dark blue. b A small RNA blot analysis of miRNAs. d The alignment of sec-siRNA triggers. Identical nucleotides and conserved nucleotides among sec-siRNA triggers were marked in red and blue, respectively. e Venn diagram for numbers of downregulated precursors of sec-siRNAs in dcl2b mutants
Figure Legend Snippet: The miRNA levels were influenced in dcl2b mutants. a , c Differentially expressed miRNAs in dcl2b mutants. The 22nt miRNAs are colored dark blue. b A small RNA blot analysis of miRNAs. d The alignment of sec-siRNA triggers. Identical nucleotides and conserved nucleotides among sec-siRNA triggers were marked in red and blue, respectively. e Venn diagram for numbers of downregulated precursors of sec-siRNAs in dcl2b mutants

Techniques Used: Northern blot, Size-exclusion Chromatography

2) Product Images from "25-Hydroxycholesterol and 27-hydroxycholesterol inhibit human rotavirus infection by sequestering viral particles into late endosomes"

Article Title: 25-Hydroxycholesterol and 27-hydroxycholesterol inhibit human rotavirus infection by sequestering viral particles into late endosomes

Journal: Redox Biology

doi: 10.1016/j.redox.2018.09.003

Effect of OSBP silencing (panels A, B, and C) or VAP-A silencing (panels D, E, and F) by short interfering RNA (siRNA) transfection on the antiviral activity of 25HC or 27HC. Cells were transfected for 72 h with 20 nM of anti-OSBP siRNA, anti-VAP-A siRNA or control non-interfering siRNA. Cells were then treated for 20 h with oxysterols at sub-optimal concentrations and finally infected with HRV Wa. Viral infections were detected as described in the Material and Methods section. The infectivity titers of virus in the treated samples are expressed as a percentage of the titer obtained in the absence of treatment. Error bars represent the standard error of the mean (SEM) of 3 independent experiments.*p ANOVA
Figure Legend Snippet: Effect of OSBP silencing (panels A, B, and C) or VAP-A silencing (panels D, E, and F) by short interfering RNA (siRNA) transfection on the antiviral activity of 25HC or 27HC. Cells were transfected for 72 h with 20 nM of anti-OSBP siRNA, anti-VAP-A siRNA or control non-interfering siRNA. Cells were then treated for 20 h with oxysterols at sub-optimal concentrations and finally infected with HRV Wa. Viral infections were detected as described in the Material and Methods section. The infectivity titers of virus in the treated samples are expressed as a percentage of the titer obtained in the absence of treatment. Error bars represent the standard error of the mean (SEM) of 3 independent experiments.*p ANOVA

Techniques Used: Small Interfering RNA, Transfection, Activity Assay, Infection

3) Product Images from "KDELR2 Competes with Measles Virus Envelope Proteins for Cellular Chaperones Reducing Their Chaperone-Mediated Cell Surface Transport"

Article Title: KDELR2 Competes with Measles Virus Envelope Proteins for Cellular Chaperones Reducing Their Chaperone-Mediated Cell Surface Transport

Journal: Viruses

doi: 10.3390/v11010027

The A3G upregulated gene KDELR2 reduces MV replication in Vero cells. ( A ) Total RNA from Vero 023 and Vero A3G was isolated and reverse transcribed into cDNA. KDELR2-specific cDNA was then amplified using SYBR-Green Real-Time qPCR ( n = 3). ( B ) The protein expression of KDELR2 was analyzed using Western blot. Equal amounts of cell lysates were separated on 12% SDS-PAGE and transferred on a nitrocellulose (NC) membrane. Target proteins were probed with primary KDELR2 antibody and HRP conjugated secondary antibody then developed using ECL (lane 1: Vero 023, lane 2: Vero A3G, lane 3: Vero KDELR2, lane 4: Vero A3G + KDELR2shRNA). ( C ) Transduced Vero cells were infected with MV eGFP at MOI of 0.1. The titer of newly synthesized virus in these cells was determined 48 h post infection on Vero cells ( n = 3). Significance was calculated using the Student’s t test (** p
Figure Legend Snippet: The A3G upregulated gene KDELR2 reduces MV replication in Vero cells. ( A ) Total RNA from Vero 023 and Vero A3G was isolated and reverse transcribed into cDNA. KDELR2-specific cDNA was then amplified using SYBR-Green Real-Time qPCR ( n = 3). ( B ) The protein expression of KDELR2 was analyzed using Western blot. Equal amounts of cell lysates were separated on 12% SDS-PAGE and transferred on a nitrocellulose (NC) membrane. Target proteins were probed with primary KDELR2 antibody and HRP conjugated secondary antibody then developed using ECL (lane 1: Vero 023, lane 2: Vero A3G, lane 3: Vero KDELR2, lane 4: Vero A3G + KDELR2shRNA). ( C ) Transduced Vero cells were infected with MV eGFP at MOI of 0.1. The titer of newly synthesized virus in these cells was determined 48 h post infection on Vero cells ( n = 3). Significance was calculated using the Student’s t test (** p

Techniques Used: Isolation, Amplification, SYBR Green Assay, Real-time Polymerase Chain Reaction, Expressing, Western Blot, SDS Page, Infection, Synthesized

4) Product Images from "Complement Receptor C5aR1 Inhibition Reduces Pyroptosis in hDPP4-Transgenic Mice Infected with MERS-CoV"

Article Title: Complement Receptor C5aR1 Inhibition Reduces Pyroptosis in hDPP4-Transgenic Mice Infected with MERS-CoV

Journal: Viruses

doi: 10.3390/v11010039

MERS-CoV infection induces pyroptosis in THP-1 macrophages. THP-1 monocytes and macrophages were infected with MERS-CoV for 24 h. Total RNA and protein was then extracted from the cells using TRIzol Reagent. ( A – C ) Total RNA was used for RT-qPCR to detect transcription of pro-caspase-1, pro-IL-1β, and NLRP3. Data are expressed as means ± SEM ( n = 2 per group). ( D ) Samples of total protein were subjected to Western blotting to detect pro-caspase-1, pro-IL-1β, activated IL-1β, and MERS NP.
Figure Legend Snippet: MERS-CoV infection induces pyroptosis in THP-1 macrophages. THP-1 monocytes and macrophages were infected with MERS-CoV for 24 h. Total RNA and protein was then extracted from the cells using TRIzol Reagent. ( A – C ) Total RNA was used for RT-qPCR to detect transcription of pro-caspase-1, pro-IL-1β, and NLRP3. Data are expressed as means ± SEM ( n = 2 per group). ( D ) Samples of total protein were subjected to Western blotting to detect pro-caspase-1, pro-IL-1β, activated IL-1β, and MERS NP.

Techniques Used: Infection, Quantitative RT-PCR, Western Blot

5) Product Images from "Fibroblasts from bank voles inhabiting Chernobyl have increased resistance against oxidative and DNA stresses"

Article Title: Fibroblasts from bank voles inhabiting Chernobyl have increased resistance against oxidative and DNA stresses

Journal: BMC Cell Biology

doi: 10.1186/s12860-018-0169-9

P53-target genes are induced more strongly in control cells than in Chernobyl bank vole fibroblasts. RNA was collected from untreated and etoposide-treated (20 μM) cells 48 and 72 h after the drug treatment. CDNA was prepared and quantitative-PCR was run with gene specific primers, and transcript levels were standardized to beta-actin transcript. Results are shown as a fold increase from untreated samples for a .) p21, b .) Gadd45α, c .) Bax, d .) Puma, and e .) Mdm2. The results are from three separate experiments using the eight Chernobyl ( N = 24) and eight control cell lines ( N = 24), variation is shown by standard deviation, and statistical analysis was done with Student’s t-test (** = p ≤ 0.01, *** = p ≤ 0.001)
Figure Legend Snippet: P53-target genes are induced more strongly in control cells than in Chernobyl bank vole fibroblasts. RNA was collected from untreated and etoposide-treated (20 μM) cells 48 and 72 h after the drug treatment. CDNA was prepared and quantitative-PCR was run with gene specific primers, and transcript levels were standardized to beta-actin transcript. Results are shown as a fold increase from untreated samples for a .) p21, b .) Gadd45α, c .) Bax, d .) Puma, and e .) Mdm2. The results are from three separate experiments using the eight Chernobyl ( N = 24) and eight control cell lines ( N = 24), variation is shown by standard deviation, and statistical analysis was done with Student’s t-test (** = p ≤ 0.01, *** = p ≤ 0.001)

Techniques Used: Real-time Polymerase Chain Reaction, Standard Deviation

6) Product Images from "Elimination of p19ARF‐expressing cells protects against pulmonary emphysema in mice, et al. Elimination of p19ARF‐expressing cells protects against pulmonary emphysema in mice"

Article Title: Elimination of p19ARF‐expressing cells protects against pulmonary emphysema in mice, et al. Elimination of p19ARF‐expressing cells protects against pulmonary emphysema in mice

Journal: Aging Cell

doi: 10.1111/acel.12827

ABT‐263 protected lung tissues from PPE‐induced emphysema. (a) Experimental timeline. Six‐month‐old female ARF‐DTR mice were orally administered ABT‐263 (25 mg kg −1 day −1 ) or vehicle alone. PPE was administered intranasally 4 weeks after the first ABT‐263 treatment. (b) Representative images of the in vivo luciferase analysis. (c) Luminescence in the chest region was quantified. Values were normalized to the average value of the vehicle‐treated group. (d) The total RNA extracted from the lung tissues was analyzed by real‐time PCR for the expression of ARF ( left ) or INK4a ( right ). mRNA levels were normalized to GAPDH in each sample. (e–h) Mice were subjected to pulmonary function tests. Pressure–volume loop (e), Cst (f), Crs (g), and IC (h) are shown. (i) Representative images of vehicle‐ or ABT‐263‐treated mouse lung sections. Sections were stained with hematoxylin and eosin. Bar; 100 μm. (j) Alveolar mean linear intercepts were measured. Bars represent the mean ± SEM . Data were analyzed by an unpaired Student's t test. * p
Figure Legend Snippet: ABT‐263 protected lung tissues from PPE‐induced emphysema. (a) Experimental timeline. Six‐month‐old female ARF‐DTR mice were orally administered ABT‐263 (25 mg kg −1 day −1 ) or vehicle alone. PPE was administered intranasally 4 weeks after the first ABT‐263 treatment. (b) Representative images of the in vivo luciferase analysis. (c) Luminescence in the chest region was quantified. Values were normalized to the average value of the vehicle‐treated group. (d) The total RNA extracted from the lung tissues was analyzed by real‐time PCR for the expression of ARF ( left ) or INK4a ( right ). mRNA levels were normalized to GAPDH in each sample. (e–h) Mice were subjected to pulmonary function tests. Pressure–volume loop (e), Cst (f), Crs (g), and IC (h) are shown. (i) Representative images of vehicle‐ or ABT‐263‐treated mouse lung sections. Sections were stained with hematoxylin and eosin. Bar; 100 μm. (j) Alveolar mean linear intercepts were measured. Bars represent the mean ± SEM . Data were analyzed by an unpaired Student's t test. * p

Techniques Used: Mouse Assay, In Vivo, Luciferase, Real-time Polymerase Chain Reaction, Expressing, Staining

Effects of PPE/DT administration on SASP‐related factors. (a,b) Female ARF‐DTR mice were treated with PPE and/or DT as shown in Figure 1 a. The total RNA was isolated from lung tissues 1 (a) or 3 weeks (b) after the administration of PPE. The expression of the indicated genes was analyzed by real‐time PCR. mRNA levels were normalized to GAPDH in each sample. Bars represent the mean ± SEM . Data were analyzed by a one‐way ANOVA and Steel–Dwass post hoc analysis. * p
Figure Legend Snippet: Effects of PPE/DT administration on SASP‐related factors. (a,b) Female ARF‐DTR mice were treated with PPE and/or DT as shown in Figure 1 a. The total RNA was isolated from lung tissues 1 (a) or 3 weeks (b) after the administration of PPE. The expression of the indicated genes was analyzed by real‐time PCR. mRNA levels were normalized to GAPDH in each sample. Bars represent the mean ± SEM . Data were analyzed by a one‐way ANOVA and Steel–Dwass post hoc analysis. * p

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

DT eliminated p19 ARF ‐expressing cells from the lung tissues of ARF‐DTR mice. (a) Experimental procedure. Five‐month‐old female ARF‐DTR or wild‐type mice pretreated with PBS or DT were administered porcine pancreatic elastase (100 U/kg). DT and PPE were administered intraperitoneally and intranasally, respectively. (b) Representative images of the in vivo imaging of ARF‐DTR mice. Images were taken before (pre) and after (post) the administration of DT and PPE, as shown in panel a. (c) Luciferase activity was measured using in vivo imaging analysis software. Changes in the luciferase activity before and after drug administration in each mouse were plotted. Bars indicate the average values of each group. (d,e) The total RNA extracted from lung tissue was analyzed by real‐time PCR for the expression of ARF (d) or INK4a (e). GAPDH mRNA was used as an internal standard in each sample, and the ∆∆ C t method was used to determine the relative expression level. Bars represent the mean ± SEM . Data were analyzed by a one‐way ANOVA and Steel–Dwass post hoc analysis. * p
Figure Legend Snippet: DT eliminated p19 ARF ‐expressing cells from the lung tissues of ARF‐DTR mice. (a) Experimental procedure. Five‐month‐old female ARF‐DTR or wild‐type mice pretreated with PBS or DT were administered porcine pancreatic elastase (100 U/kg). DT and PPE were administered intraperitoneally and intranasally, respectively. (b) Representative images of the in vivo imaging of ARF‐DTR mice. Images were taken before (pre) and after (post) the administration of DT and PPE, as shown in panel a. (c) Luciferase activity was measured using in vivo imaging analysis software. Changes in the luciferase activity before and after drug administration in each mouse were plotted. Bars indicate the average values of each group. (d,e) The total RNA extracted from lung tissue was analyzed by real‐time PCR for the expression of ARF (d) or INK4a (e). GAPDH mRNA was used as an internal standard in each sample, and the ∆∆ C t method was used to determine the relative expression level. Bars represent the mean ± SEM . Data were analyzed by a one‐way ANOVA and Steel–Dwass post hoc analysis. * p

Techniques Used: Expressing, Mouse Assay, In Vivo Imaging, Luciferase, Activity Assay, Software, Real-time Polymerase Chain Reaction

7) Product Images from "Histone methylation regulates Hif‐1 signaling cascade in activation of hepatic stellate cells"

Article Title: Histone methylation regulates Hif‐1 signaling cascade in activation of hepatic stellate cells

Journal: FEBS Open Bio

doi: 10.1002/2211-5463.12379

Genes related to histone methylation modification were up‐regulated in hypoxia‐induced LX ‐2 cells. Total RNA was extracted and reversely transcribed into cDNA from normal oxygen or CoCl 2 ‐treated hypoxia‐induced human hepatic stellate cell line LX ‐2. (A) Cluster analysis of genomewide expression chips (red: up‐regulated genes, green: down‐regulated genes). (B) The expression of Ogt, a representative up‐regulated gene from genomewide expression chips, was detected at mRNA level by qPCR . Densitometric analysis was performed using pooled data from three such experiments. Data were mean ± SD (* P
Figure Legend Snippet: Genes related to histone methylation modification were up‐regulated in hypoxia‐induced LX ‐2 cells. Total RNA was extracted and reversely transcribed into cDNA from normal oxygen or CoCl 2 ‐treated hypoxia‐induced human hepatic stellate cell line LX ‐2. (A) Cluster analysis of genomewide expression chips (red: up‐regulated genes, green: down‐regulated genes). (B) The expression of Ogt, a representative up‐regulated gene from genomewide expression chips, was detected at mRNA level by qPCR . Densitometric analysis was performed using pooled data from three such experiments. Data were mean ± SD (* P

Techniques Used: Methylation, Modification, Expressing, Real-time Polymerase Chain Reaction

8) Product Images from "A role for long-chain acyl-CoA synthetase-4 (ACSL4) in diet-induced phospholipid remodeling and obesity-associated adipocyte dysfunction"

Article Title: A role for long-chain acyl-CoA synthetase-4 (ACSL4) in diet-induced phospholipid remodeling and obesity-associated adipocyte dysfunction

Journal: Molecular Metabolism

doi: 10.1016/j.molmet.2018.01.012

Ad-KO mice fed HFD have alterations in adipocyte ACSL activity, n-6 PUFA fatty acyl-CoA formation, and FFA and phospholipid composition . (A) ACSL isoform gene expression in isolated gonadal adipocytes of 20-week old male ACSL4 floxed or Ad-KO mice fed LFD or HFD for 12 weeks RNA expression quantified by real-time PCR relative to Cyclophilin A. n = 5–8 mice per group. *p ≤ 0.05 ACSL4 floxed vs. Ad-KO within diet; **p ≤ 0.0125 ACSL4 floxed vs. Ad-KO within diet; #p ≤ 0.05 LFD vs. HFD within genotype; ##p ≤ 0.0125 LFD vs. HFD within genotype; Student's t-test. (B) Total ACSL enzyme activity in isolated gonadal adipocytes of 20 week old males fed LFD or HFD for 12 weeks. n = 4–6 mice/group *p ≤ 0.05 ACSL4 floxed vs. Ad-KO within diet. Student's t-test. (C) Long-chain acyl-CoA species determined in gonadal adipose tissue of male ACSL4 floxed or Ad-KO mice on HFD for 12 weeks as determined by two-way ANOVA. *p ≤ 0.05 ACSL4 floxed vs. Ad-KO within diet; **p ≤ 0.0125 ACSL4 floxed vs. Ad-KO within diet. n = 7–8 mice/group. (D) Isolated adipocyte FFA species (no species less than 20 carbons have significant differences) as determined by two-way ANOVA. *p ≤ 0.05 ACSL4 floxed vs. Ad-KO within diet; **p ≤ 0.0125 ACSL4 floxed vs. Ad-KO within diet. n = 6–9 mice/group. (E) Fold-change of isolated adipocyte arachidonic acid, DHA, and linoleic acid phospholipid species. Significant differences between groups represented in yellow (p ≤ 0.05) and italicized (p ≤ 0.01) as determined by two-way ANOVA (Black boxes = Species not detected). n = 6–9 mice/group. Data represent mean ± SEM.
Figure Legend Snippet: Ad-KO mice fed HFD have alterations in adipocyte ACSL activity, n-6 PUFA fatty acyl-CoA formation, and FFA and phospholipid composition . (A) ACSL isoform gene expression in isolated gonadal adipocytes of 20-week old male ACSL4 floxed or Ad-KO mice fed LFD or HFD for 12 weeks RNA expression quantified by real-time PCR relative to Cyclophilin A. n = 5–8 mice per group. *p ≤ 0.05 ACSL4 floxed vs. Ad-KO within diet; **p ≤ 0.0125 ACSL4 floxed vs. Ad-KO within diet; #p ≤ 0.05 LFD vs. HFD within genotype; ##p ≤ 0.0125 LFD vs. HFD within genotype; Student's t-test. (B) Total ACSL enzyme activity in isolated gonadal adipocytes of 20 week old males fed LFD or HFD for 12 weeks. n = 4–6 mice/group *p ≤ 0.05 ACSL4 floxed vs. Ad-KO within diet. Student's t-test. (C) Long-chain acyl-CoA species determined in gonadal adipose tissue of male ACSL4 floxed or Ad-KO mice on HFD for 12 weeks as determined by two-way ANOVA. *p ≤ 0.05 ACSL4 floxed vs. Ad-KO within diet; **p ≤ 0.0125 ACSL4 floxed vs. Ad-KO within diet. n = 7–8 mice/group. (D) Isolated adipocyte FFA species (no species less than 20 carbons have significant differences) as determined by two-way ANOVA. *p ≤ 0.05 ACSL4 floxed vs. Ad-KO within diet; **p ≤ 0.0125 ACSL4 floxed vs. Ad-KO within diet. n = 6–9 mice/group. (E) Fold-change of isolated adipocyte arachidonic acid, DHA, and linoleic acid phospholipid species. Significant differences between groups represented in yellow (p ≤ 0.05) and italicized (p ≤ 0.01) as determined by two-way ANOVA (Black boxes = Species not detected). n = 6–9 mice/group. Data represent mean ± SEM.

Techniques Used: Mouse Assay, Activity Assay, Expressing, Isolation, RNA Expression, Real-time Polymerase Chain Reaction

Mice that lack ACSL4 specifically in adipocytes are protected against high fat diet-induced obesity . (A) ACSL4 RNA expression quantified by real-time PCR relative to Cyclophilin A in isolated adipocytes and metabolically relevant tissues in 20 week old males fed LFD. n = 3–7 mice per group; *p ≤ 0.05 ACSL4 floxed vs. Ad-KO. Student's t-test. (B) Representative immunoblot against ACSL4 in isolated gonadal adipocytes, whole gonadal adipose tissue, whole subcutaneous adipose tissue, and liver of 20-week-old males. (C) Body weight, (D) fat mass, and (E) lean mass from 8 to 20 weeks of age in male mice fed LFD or HFD; n = 11–12 mice/group LFD, 28–31 mice/group HFD; #p ≤ 0.05 ACSL4 floxed LFD vs. HFD, +p ≤ 0.05 Ad-KO LFD vs. Ad-KO HFD, and *p ≤ 0.05 ACSL4 floxed HFD vs. Ad-KO HFD; Repeated measures ANOVA with Tukey's HSD for multiple comparisons. Data represent ± SEM.
Figure Legend Snippet: Mice that lack ACSL4 specifically in adipocytes are protected against high fat diet-induced obesity . (A) ACSL4 RNA expression quantified by real-time PCR relative to Cyclophilin A in isolated adipocytes and metabolically relevant tissues in 20 week old males fed LFD. n = 3–7 mice per group; *p ≤ 0.05 ACSL4 floxed vs. Ad-KO. Student's t-test. (B) Representative immunoblot against ACSL4 in isolated gonadal adipocytes, whole gonadal adipose tissue, whole subcutaneous adipose tissue, and liver of 20-week-old males. (C) Body weight, (D) fat mass, and (E) lean mass from 8 to 20 weeks of age in male mice fed LFD or HFD; n = 11–12 mice/group LFD, 28–31 mice/group HFD; #p ≤ 0.05 ACSL4 floxed LFD vs. HFD, +p ≤ 0.05 Ad-KO LFD vs. Ad-KO HFD, and *p ≤ 0.05 ACSL4 floxed HFD vs. Ad-KO HFD; Repeated measures ANOVA with Tukey's HSD for multiple comparisons. Data represent ± SEM.

Techniques Used: Mouse Assay, RNA Expression, Real-time Polymerase Chain Reaction, Isolation, Metabolic Labelling

Adipocytes from Ad-KO mice fed HFD are protected against increased 4-HNE production, reductions in glutathione-mediated detoxification gene expression, and 4-HNE mediated p53 activation . (A) Isolated adipocyte 4-HNE content as determined by two-way ANOVA. *p ≤ 0.05 ACSL4 fl oxed vs. Ad-KO within diet. (B) Glutathione-mediated detoxification genes of interest in isolated gonadal adipocytes of 20-week old male ACSL4 floxed or Ad-KO mice fed LFD or HFD for 12 weeks RNA expression quantified by real-time PCR relative to Cyclophilin A. n = 5–8 mice per group. *p ≤ 0.05 ACSL4 floxed vs. Ad-KO within diet; **p ≤ 0.0125 ACSL4 floxed vs. Ad-KO within diet; #p ≤ 0.05 LFD vs. HFD within genotype; ##p ≤ 0.0125 LFD vs. HFD within genotype; Student's t-test with Bonferroni correction for 4 comparisons. (C) p53 downstream target genes in isolated gonadal adipocytes of 20-week old male ACSL4floxed or Ad-KO mice on LFD or HFD for 12 weeks RNA expression quantified by real-time PCR relative to Cyclophilin A. n = 5–7 mice per group. *p
Figure Legend Snippet: Adipocytes from Ad-KO mice fed HFD are protected against increased 4-HNE production, reductions in glutathione-mediated detoxification gene expression, and 4-HNE mediated p53 activation . (A) Isolated adipocyte 4-HNE content as determined by two-way ANOVA. *p ≤ 0.05 ACSL4 fl oxed vs. Ad-KO within diet. (B) Glutathione-mediated detoxification genes of interest in isolated gonadal adipocytes of 20-week old male ACSL4 floxed or Ad-KO mice fed LFD or HFD for 12 weeks RNA expression quantified by real-time PCR relative to Cyclophilin A. n = 5–8 mice per group. *p ≤ 0.05 ACSL4 floxed vs. Ad-KO within diet; **p ≤ 0.0125 ACSL4 floxed vs. Ad-KO within diet; #p ≤ 0.05 LFD vs. HFD within genotype; ##p ≤ 0.0125 LFD vs. HFD within genotype; Student's t-test with Bonferroni correction for 4 comparisons. (C) p53 downstream target genes in isolated gonadal adipocytes of 20-week old male ACSL4floxed or Ad-KO mice on LFD or HFD for 12 weeks RNA expression quantified by real-time PCR relative to Cyclophilin A. n = 5–7 mice per group. *p

Techniques Used: Mouse Assay, Expressing, Activation Assay, Isolation, RNA Expression, Real-time Polymerase Chain Reaction

9) Product Images from "Alternative utrophin mRNAs contribute to phenotypic differences between dystrophin‐deficient mice and Duchenne muscular dystrophy"

Article Title: Alternative utrophin mRNAs contribute to phenotypic differences between dystrophin‐deficient mice and Duchenne muscular dystrophy

Journal: Febs Letters

doi: 10.1002/1873-3468.13099

Identification and characterization of novel 5′ utrophin isoforms. 5′RACE of mouse and human RNA reveals novel utrophin exons within the large genomic interval between utrophin exon 1A and 3. (A) Schematic of (upper) mouse and (lower) human utrophin loci, with exon splicing patterns (grey lines) and type as denoted (untranslated exons: white or light grey; partially or completely translated exons: black or dark grey; lighter coloured exons are novel); distance is not to scale. Arrows signify exons containing transcription start sites with splicing patterns denoted to at least the first common exon (exon 4/3 for mouse/human respectively) represented. Asterisks indicate locations of translation start sites. (B) Embryonic mouse Utrn transcripts display distinctive profiles, indicating regulatory differences. Quantitative RT‐PCR (qRT‐PCR) of Utrn ‐A/A′, Utrn ‐B/B′ and Utrn ‐C,‐D and ‐F isoforms at embryonic stages e7–17 as indicated. (C) Human UTRN mRNAs show isoform variability in their pre and postnatal tissue distribution. Left panel: qRT‐PCR analysis of human isoforms in fetal tissue, right panel: semiquantitative (sq)RT‐PCR of RNA sourced from 20 adult tissues, where values are arbitrary units standardised to 28 s cDNA. The colour scales provided in (B) and (C) represent values obtained for each sample. Human adult tissue sqRT‐PCR products were quantified on agarose gels, with representative bands provided below. Numerical values obtained for all sample sets are provided in Table S1 . sk. musc, skeletal muscle; oesophg, oesophagus; sm. ints, small intestine.
Figure Legend Snippet: Identification and characterization of novel 5′ utrophin isoforms. 5′RACE of mouse and human RNA reveals novel utrophin exons within the large genomic interval between utrophin exon 1A and 3. (A) Schematic of (upper) mouse and (lower) human utrophin loci, with exon splicing patterns (grey lines) and type as denoted (untranslated exons: white or light grey; partially or completely translated exons: black or dark grey; lighter coloured exons are novel); distance is not to scale. Arrows signify exons containing transcription start sites with splicing patterns denoted to at least the first common exon (exon 4/3 for mouse/human respectively) represented. Asterisks indicate locations of translation start sites. (B) Embryonic mouse Utrn transcripts display distinctive profiles, indicating regulatory differences. Quantitative RT‐PCR (qRT‐PCR) of Utrn ‐A/A′, Utrn ‐B/B′ and Utrn ‐C,‐D and ‐F isoforms at embryonic stages e7–17 as indicated. (C) Human UTRN mRNAs show isoform variability in their pre and postnatal tissue distribution. Left panel: qRT‐PCR analysis of human isoforms in fetal tissue, right panel: semiquantitative (sq)RT‐PCR of RNA sourced from 20 adult tissues, where values are arbitrary units standardised to 28 s cDNA. The colour scales provided in (B) and (C) represent values obtained for each sample. Human adult tissue sqRT‐PCR products were quantified on agarose gels, with representative bands provided below. Numerical values obtained for all sample sets are provided in Table S1 . sk. musc, skeletal muscle; oesophg, oesophagus; sm. ints, small intestine.

Techniques Used: Quantitative RT-PCR, Polymerase Chain Reaction

10) Product Images from "Circular RNA CpG island hypermethylation-associated silencing in human cancer"

Article Title: Circular RNA CpG island hypermethylation-associated silencing in human cancer

Journal: Oncotarget

doi: 10.18632/oncotarget.25673

circRNA effects on linear transcripts, miRNAs and tumor growth ( A ) Upon efficient transduction of TUSC3 circ104557 in HCT-116 cells (harboring a methylated CpG island), the TUSC3 linear RNA levels did not change: it was not detected in any of the conditions tested. ( B ) TUSC3 circ104557 transduction in DKO cells (harboring an unmethylated CpG island) did not affect the levels of TUSC3 linear RNA. RNA levels were determined using circular or linear specific qRT-PCR primers. The lentiviral transduction of the empty vector (Mock condition) was used as a control. Experiments were performed in technical triplicates. ( C ) Expression of candidate miRNAs putatively targeted by ATRNL1 circ100686 (miR-378a-3p), SAMD4A circ101356 (miR-660-5p and miR-330-3p) and TUSC3 circ104557 (miR-330-3p) was significantly downregulated in DKO cells, evaluated in three biological replicates by real-time quantitative PCR using TaqMan Advanced MicroRNA Assays. Expression levels were normalized using hsa-miR-345-5p, hsa-miR-191-5p and hsa-miR-423-3p advanced Control miRNA Assays. ( D ) Using the same strategy, expression of miR-330-3p, putatively targeted by TUSC3 circ104557, was also assessed in the gain-of-function cellular model. A significant downregulation of miR-330-3p was detected upon TUSC3 circ104557 transduction in HCT116 cells. ( E ) HCT116-Mock and HCT116-TUSC3 circular cells were injected in the left or right flank of 10 mice, respectively. Tumor volume measured over time (left panel) and tumor weight upon sacrifice (right panel) are shown. Tumor growth was significantly reduced upon TUSC3 circular ectopic expression. ns, nonsignificant; * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001, using Student’s t -test. Error bars show means ± s.d.
Figure Legend Snippet: circRNA effects on linear transcripts, miRNAs and tumor growth ( A ) Upon efficient transduction of TUSC3 circ104557 in HCT-116 cells (harboring a methylated CpG island), the TUSC3 linear RNA levels did not change: it was not detected in any of the conditions tested. ( B ) TUSC3 circ104557 transduction in DKO cells (harboring an unmethylated CpG island) did not affect the levels of TUSC3 linear RNA. RNA levels were determined using circular or linear specific qRT-PCR primers. The lentiviral transduction of the empty vector (Mock condition) was used as a control. Experiments were performed in technical triplicates. ( C ) Expression of candidate miRNAs putatively targeted by ATRNL1 circ100686 (miR-378a-3p), SAMD4A circ101356 (miR-660-5p and miR-330-3p) and TUSC3 circ104557 (miR-330-3p) was significantly downregulated in DKO cells, evaluated in three biological replicates by real-time quantitative PCR using TaqMan Advanced MicroRNA Assays. Expression levels were normalized using hsa-miR-345-5p, hsa-miR-191-5p and hsa-miR-423-3p advanced Control miRNA Assays. ( D ) Using the same strategy, expression of miR-330-3p, putatively targeted by TUSC3 circ104557, was also assessed in the gain-of-function cellular model. A significant downregulation of miR-330-3p was detected upon TUSC3 circ104557 transduction in HCT116 cells. ( E ) HCT116-Mock and HCT116-TUSC3 circular cells were injected in the left or right flank of 10 mice, respectively. Tumor volume measured over time (left panel) and tumor weight upon sacrifice (right panel) are shown. Tumor growth was significantly reduced upon TUSC3 circular ectopic expression. ns, nonsignificant; * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001, using Student’s t -test. Error bars show means ± s.d.

Techniques Used: Transduction, Methylation, Quantitative RT-PCR, Plasmid Preparation, Expressing, Real-time Polymerase Chain Reaction, Injection, Mouse Assay

11) Product Images from "Genome-wide profiling of RNA editing sites in sheep"

Article Title: Genome-wide profiling of RNA editing sites in sheep

Journal: Journal of Animal Science and Biotechnology

doi: 10.1186/s40104-019-0331-z

IGV screenshot for RNA editing sites in BLCAP ( a ) and BEIL1 ( b ). The two IGV screenshots showing the alignments of genomic and RNA reads in BLCAP ( a ) and BEIL1 ( b ). The two editing sites (NC_019470.2:65916359 and NC_019475.2:32339099) were at the center lines. From top to bottom, the tracks are as follows: genomic DNA reads, RNA reads in Kidney, RNA reads in Spleen, reference sequences and transcripts
Figure Legend Snippet: IGV screenshot for RNA editing sites in BLCAP ( a ) and BEIL1 ( b ). The two IGV screenshots showing the alignments of genomic and RNA reads in BLCAP ( a ) and BEIL1 ( b ). The two editing sites (NC_019470.2:65916359 and NC_019475.2:32339099) were at the center lines. From top to bottom, the tracks are as follows: genomic DNA reads, RNA reads in Kidney, RNA reads in Spleen, reference sequences and transcripts

Techniques Used:

12) Product Images from "Hypoxic BMSC-derived exosomal miRNAs promote metastasis of lung cancer cells via STAT3-induced EMT"

Article Title: Hypoxic BMSC-derived exosomal miRNAs promote metastasis of lung cancer cells via STAT3-induced EMT

Journal: Molecular Cancer

doi: 10.1186/s12943-019-0959-5

Exosomal miRNAs from BMSCs promote metastasis by STAT3 driven EMT. C57BL/6 mice were subcutaneously injected with LLC-RFP with or without BMSCs. The red fluorescent protein positive LLC cells were collected from the tumor sites by flow cytometry cell sorting and subjected to RNA sequencing analysis. Timeline of experimental protocol was shown. a The upregulated genes were enriched in the JAK-STAT pathway. Clustering was performed on differentially expressed mRNAs between LLC cells collected from mice that received co-injection of BMSCs and LLC or LLC injection alone. Columns represent individual samples and rows represent each gene. Red and green reflect high and low expression levels, respectively. FPKM for STAT3 transcripts obtained by RNA-Seq was shown in histogram. b Protein expression of STAT3, p-STAT3 were measured by Western blot analysis. Cells were treated with normoxic BMSC-secreted exosomes or hypoxic BMSC-secreted exosomes. β-actin was used as the internal control. c Cell invasion was measured by transwell assay. The increased invasion capability induced by hypoxic BMSC-secreted exosomes was reversed by stat3 inhibitor stattics. Magnification, 100×. d Expression of Snail and Vimentin was measured by quantitative real-time PCR. Cells were treated with hypoxic BMSC-secreted exosomes with or without stat3 inhibitor stattics. Experiments were performed in triplicate.* P
Figure Legend Snippet: Exosomal miRNAs from BMSCs promote metastasis by STAT3 driven EMT. C57BL/6 mice were subcutaneously injected with LLC-RFP with or without BMSCs. The red fluorescent protein positive LLC cells were collected from the tumor sites by flow cytometry cell sorting and subjected to RNA sequencing analysis. Timeline of experimental protocol was shown. a The upregulated genes were enriched in the JAK-STAT pathway. Clustering was performed on differentially expressed mRNAs between LLC cells collected from mice that received co-injection of BMSCs and LLC or LLC injection alone. Columns represent individual samples and rows represent each gene. Red and green reflect high and low expression levels, respectively. FPKM for STAT3 transcripts obtained by RNA-Seq was shown in histogram. b Protein expression of STAT3, p-STAT3 were measured by Western blot analysis. Cells were treated with normoxic BMSC-secreted exosomes or hypoxic BMSC-secreted exosomes. β-actin was used as the internal control. c Cell invasion was measured by transwell assay. The increased invasion capability induced by hypoxic BMSC-secreted exosomes was reversed by stat3 inhibitor stattics. Magnification, 100×. d Expression of Snail and Vimentin was measured by quantitative real-time PCR. Cells were treated with hypoxic BMSC-secreted exosomes with or without stat3 inhibitor stattics. Experiments were performed in triplicate.* P

Techniques Used: Mouse Assay, Injection, Flow Cytometry, Cytometry, FACS, RNA Sequencing Assay, Expressing, Western Blot, Transwell Assay, Real-time Polymerase Chain Reaction

13) Product Images from "Identification and Characterization of Five Cold Stress-Related Rhododendron Dehydrin Genes: Spotlight on a FSK-Type Dehydrin With Multiple F-Segments"

Article Title: Identification and Characterization of Five Cold Stress-Related Rhododendron Dehydrin Genes: Spotlight on a FSK-Type Dehydrin With Multiple F-Segments

Journal: Frontiers in Bioengineering and Biotechnology

doi: 10.3389/fbioe.2019.00030

Regular RT-PCR DNA gel images for monthly expression patterns of dehydrin genes in leaf tissues of field-grown R. catawbiense collected in August, September, October, November, January, and February. Total RNA extracted from leaf tissues were used for cDNA synthesis. Regular RT-PCR was conducted by using each dehydrin's primers, while the Universal 18S Internal Standards primers (Ambion) and gene-specific primers of rhododendron ubiquitin-like ( RcUbql ) gene were used as the mixed primers for the reference genes; the cycle numbers were 32, which was in the exponential phase of the PCR amplification.
Figure Legend Snippet: Regular RT-PCR DNA gel images for monthly expression patterns of dehydrin genes in leaf tissues of field-grown R. catawbiense collected in August, September, October, November, January, and February. Total RNA extracted from leaf tissues were used for cDNA synthesis. Regular RT-PCR was conducted by using each dehydrin's primers, while the Universal 18S Internal Standards primers (Ambion) and gene-specific primers of rhododendron ubiquitin-like ( RcUbql ) gene were used as the mixed primers for the reference genes; the cycle numbers were 32, which was in the exponential phase of the PCR amplification.

Techniques Used: Reverse Transcription Polymerase Chain Reaction, Expressing, Polymerase Chain Reaction, Amplification

Northern blot analysis for the seasonal expression levels of RcDhn 1-5 in NA, CA, and DA leaf tissues. Total RNA (8 μg) was isolated and hybridized with respective cDNA probes. Lower panel shows control hybridization of the filters to rRNA using a blueberry cDNA probe confirming equal loadings among the lanes. Fold change in the expression level during cold acclimation and deacclimation relative to non-acclimated state (defined as ‘1') was estimated by densitometry. The putative genes in red text showed at least 5-fold differences in the intensity of bands of northern blot between NA and CA, thus defined as highly cold-responsive genes. RcDhn 5 's northern blot data were adapted from a previous study (Peng et al., 2008 ). CA, cold-acclimated; DA, de-acclimated; NA, non-acclimated.
Figure Legend Snippet: Northern blot analysis for the seasonal expression levels of RcDhn 1-5 in NA, CA, and DA leaf tissues. Total RNA (8 μg) was isolated and hybridized with respective cDNA probes. Lower panel shows control hybridization of the filters to rRNA using a blueberry cDNA probe confirming equal loadings among the lanes. Fold change in the expression level during cold acclimation and deacclimation relative to non-acclimated state (defined as ‘1') was estimated by densitometry. The putative genes in red text showed at least 5-fold differences in the intensity of bands of northern blot between NA and CA, thus defined as highly cold-responsive genes. RcDhn 5 's northern blot data were adapted from a previous study (Peng et al., 2008 ). CA, cold-acclimated; DA, de-acclimated; NA, non-acclimated.

Techniques Used: Northern Blot, Expressing, Isolation, Hybridization

Real-time RT-PCR analysis for monthly expression patterns of dehydrin genes in leaf tissues of field-grown R. catawbiense collected in August, September, October, November, January, and February (A-D) RcDhn 1-4 . Total RNA extracted from leaf tissues were used for cDNA synthesis. Real-time RT-PCR was conducted by using each dehydrin's primers, while the gene-specific primers of rhododendron ubiquitin-like ( RcUbql ) gene for the reference gene. The monthly expression of each dehydrin gene was presented as the expression levels relative to its expression level in August (which was set at 1). * and ** indicate statistical significance of p
Figure Legend Snippet: Real-time RT-PCR analysis for monthly expression patterns of dehydrin genes in leaf tissues of field-grown R. catawbiense collected in August, September, October, November, January, and February (A-D) RcDhn 1-4 . Total RNA extracted from leaf tissues were used for cDNA synthesis. Real-time RT-PCR was conducted by using each dehydrin's primers, while the gene-specific primers of rhododendron ubiquitin-like ( RcUbql ) gene for the reference gene. The monthly expression of each dehydrin gene was presented as the expression levels relative to its expression level in August (which was set at 1). * and ** indicate statistical significance of p

Techniques Used: Quantitative RT-PCR, Expressing

14) Product Images from "Mechanistic Investigation of the Androgen Receptor DNA-Binding Domain Inhibitor Pyrvinium"

Article Title: Mechanistic Investigation of the Androgen Receptor DNA-Binding Domain Inhibitor Pyrvinium

Journal: ACS Omega

doi: 10.1021/acsomega.8b03205

IP–MS to identify AR-binding proteins affected by pyrvinium. (A) LNCaP cells were treated with 1 nM DHT ± 100 nM PP (indicated by a “–“ or “+” adjacent to “PP”. Cells were lysed, AR was immunoprecipitated and proteins resolved by SDS-PAGE. Sections from a Coomassie-stained gel were isolated and prepared for MS analysis. A western blot was also performed to demonstrate the specificity for AR pull-down and PP activity, as it is known to block the interaction with RNA pol II. The table indicates the arbitrary score from each lane for the detection of AR and DDX proteins, with approximate mass. (B) To confirm the loss of DDX protein binding, LNCaP cells were treated with vehicle, DHT, or DHT + PP for 24 h, at which point the cells were lysed and AR was immunoprecipitated. Western blot for AR and DDX17 demonstrates a loss of coprecipitation of DDX17 with AR in the presence of PP. (C) LNCaP or (D) LAPC4 cells were transfected with a DDX17 expression vector or control vector. The following day, the indicated drugs were added, and 24 h later, RNA was harvested. qPCR demonstrated a decrease in the efficacy of PP to inhibit the transcription of the AR target gene FKBP5 upon DDX17 overexpression. * significantly different compared to DHT alone ( p
Figure Legend Snippet: IP–MS to identify AR-binding proteins affected by pyrvinium. (A) LNCaP cells were treated with 1 nM DHT ± 100 nM PP (indicated by a “–“ or “+” adjacent to “PP”. Cells were lysed, AR was immunoprecipitated and proteins resolved by SDS-PAGE. Sections from a Coomassie-stained gel were isolated and prepared for MS analysis. A western blot was also performed to demonstrate the specificity for AR pull-down and PP activity, as it is known to block the interaction with RNA pol II. The table indicates the arbitrary score from each lane for the detection of AR and DDX proteins, with approximate mass. (B) To confirm the loss of DDX protein binding, LNCaP cells were treated with vehicle, DHT, or DHT + PP for 24 h, at which point the cells were lysed and AR was immunoprecipitated. Western blot for AR and DDX17 demonstrates a loss of coprecipitation of DDX17 with AR in the presence of PP. (C) LNCaP or (D) LAPC4 cells were transfected with a DDX17 expression vector or control vector. The following day, the indicated drugs were added, and 24 h later, RNA was harvested. qPCR demonstrated a decrease in the efficacy of PP to inhibit the transcription of the AR target gene FKBP5 upon DDX17 overexpression. * significantly different compared to DHT alone ( p

Techniques Used: Mass Spectrometry, Binding Assay, Immunoprecipitation, SDS Page, Staining, Isolation, Western Blot, Activity Assay, Blocking Assay, Protein Binding, Transfection, Expressing, Plasmid Preparation, Real-time Polymerase Chain Reaction, Over Expression

15) Product Images from "Identification and characterization of epizootic hemorrhagic disease virus serotype 6 in cattle co-infected with bluetongue virus in Trinidad, West Indies"

Article Title: Identification and characterization of epizootic hemorrhagic disease virus serotype 6 in cattle co-infected with bluetongue virus in Trinidad, West Indies

Journal: Veterinary Microbiology

doi: 10.1016/j.vetmic.2018.12.009

A: Percentage of cattle positive for antibodies (Ab) and RNA for both EHDV and BTV time of arrival in Trinidad (month 0) to the last month of blood collection (month 6).(BTV Ct values from their were taken from Brown-Joseph et al., 2017 ). B: Ct values measured by EHDV group-specific rRT-PCRs on blood samples taken from the first month of virus detection to the last month of blood collection (month 6). The dotted line shows the overall trending increase in Ct values for animal 874 during the five-month period.
Figure Legend Snippet: A: Percentage of cattle positive for antibodies (Ab) and RNA for both EHDV and BTV time of arrival in Trinidad (month 0) to the last month of blood collection (month 6).(BTV Ct values from their were taken from Brown-Joseph et al., 2017 ). B: Ct values measured by EHDV group-specific rRT-PCRs on blood samples taken from the first month of virus detection to the last month of blood collection (month 6). The dotted line shows the overall trending increase in Ct values for animal 874 during the five-month period.

Techniques Used:

16) Product Images from "Follicle-Stimulating Hormone Is an Autocrine Regulator of the Ovarian Cancer Metastatic Niche Through Notch Signaling"

Article Title: Follicle-Stimulating Hormone Is an Autocrine Regulator of the Ovarian Cancer Metastatic Niche Through Notch Signaling

Journal: Journal of the Endocrine Society

doi: 10.1210/js.2018-00272

Expression of FSH and FSHR in primary tumor and spheroids obtained from patients with ovarian cancer. Total RNA was isolated from the primary tumors and spheroids obtained from the ascites of patients with ovarian cancer, and cDNA was prepared from 1 µg total RNA. Cycle threshold values of (A) FSH α subunit, (B) FSH β subunit, and (C) FSHR were examined by RT-PCR, and RNA levels were interpolated from the dilution curves of the respective genes.
Figure Legend Snippet: Expression of FSH and FSHR in primary tumor and spheroids obtained from patients with ovarian cancer. Total RNA was isolated from the primary tumors and spheroids obtained from the ascites of patients with ovarian cancer, and cDNA was prepared from 1 µg total RNA. Cycle threshold values of (A) FSH α subunit, (B) FSH β subunit, and (C) FSHR were examined by RT-PCR, and RNA levels were interpolated from the dilution curves of the respective genes.

Techniques Used: Expressing, Isolation, Reverse Transcription Polymerase Chain Reaction

17) Product Images from "Bioinformatic analysis of long-lasting transcriptional and translational changes in the basolateral amygdala following acute stress"

Article Title: Bioinformatic analysis of long-lasting transcriptional and translational changes in the basolateral amygdala following acute stress

Journal: PLoS ONE

doi: 10.1371/journal.pone.0209846

Acute restraint stress induces long lasting RNA changes in the BLC. (A) A summary of the global transcriptome profile of the BLC in mice that underwent a single acute restraint session 30 days prior. (B) A volcano plot of the RNA-seq data with significantly changed genes colored in red and unchanged genes colored in gray. (C) qPCR validation of a subset of genes significantly upregulated in the RNA-seq. *p
Figure Legend Snippet: Acute restraint stress induces long lasting RNA changes in the BLC. (A) A summary of the global transcriptome profile of the BLC in mice that underwent a single acute restraint session 30 days prior. (B) A volcano plot of the RNA-seq data with significantly changed genes colored in red and unchanged genes colored in gray. (C) qPCR validation of a subset of genes significantly upregulated in the RNA-seq. *p

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

18) Product Images from "RNA–Protein Interactions Prevent Long RNA Duplex Formation: Implications for the Design of RNA-Based Therapeutics"

Article Title: RNA–Protein Interactions Prevent Long RNA Duplex Formation: Implications for the Design of RNA-Based Therapeutics

Journal: Molecules

doi: 10.3390/molecules23123329

Digestion of cell lysate and reannealed RNA with RNAse A/T1. ( A ) HEK293T cell lysate spiked with different kinds of RNA duplex oligos were digested with 1:200 dilution of RNAse A/T1 at 37 °C for 30 min. RNA was extracted after digestion and separated on 15% acrylamide TBE native gels and stained with SYBR gold. Lanes with different amounts of spike-in RNA were labeled with different shapes (Δ, 0.1 μg; ◊, 0.5 μg; ☐, 5 μg). The arrow indicates the band that was subject to more detailed investigation as described in Section 2.3 . ( B ) Cell lysate and reannealed RNA with or without spiked-in were digested with 1:200 dilution of RNAse A/T1. * indicates where the bands were cut for cloning and sequencing.
Figure Legend Snippet: Digestion of cell lysate and reannealed RNA with RNAse A/T1. ( A ) HEK293T cell lysate spiked with different kinds of RNA duplex oligos were digested with 1:200 dilution of RNAse A/T1 at 37 °C for 30 min. RNA was extracted after digestion and separated on 15% acrylamide TBE native gels and stained with SYBR gold. Lanes with different amounts of spike-in RNA were labeled with different shapes (Δ, 0.1 μg; ◊, 0.5 μg; ☐, 5 μg). The arrow indicates the band that was subject to more detailed investigation as described in Section 2.3 . ( B ) Cell lysate and reannealed RNA with or without spiked-in were digested with 1:200 dilution of RNAse A/T1. * indicates where the bands were cut for cloning and sequencing.

Techniques Used: Staining, Labeling, Clone Assay, Sequencing

19) Product Images from "Contribution of the Alkylquinolone Quorum-Sensing System to the Interaction of Pseudomonas aeruginosa With Bronchial Epithelial Cells"

Article Title: Contribution of the Alkylquinolone Quorum-Sensing System to the Interaction of Pseudomonas aeruginosa With Bronchial Epithelial Cells

Journal: Frontiers in Microbiology

doi: 10.3389/fmicb.2018.03018

Analysis of pqsA , pqsE , mexG, and lecA transcription in WT and Δ pqsA Calu-3-ALI-infected cultures in the presence and absence of exogenous PQS. Expression of pqsA , pqsE , mexG, and lecA during infection of Calu-3-ALI cultures with PAO1 and Δ pqsA PAO1 at 3 hpi was assessed by RT-PCR. Calu-3-ALI cultures exposed to PQS (40 μM) or DMSO were infected at MOI 50 and total RNA prepared at 3 hpi. A 250-bp DNA region within the pqsA gene and a 200-bp DNA region within pqsE, oprL , mexG, and lecA genes were amplified from PAO1 genomic DNA (positive control); 1, cDNA; 2, corresponding RNA (negative control). L, 50 bp DNA ladder. Data are representative from 2 independent experiments.
Figure Legend Snippet: Analysis of pqsA , pqsE , mexG, and lecA transcription in WT and Δ pqsA Calu-3-ALI-infected cultures in the presence and absence of exogenous PQS. Expression of pqsA , pqsE , mexG, and lecA during infection of Calu-3-ALI cultures with PAO1 and Δ pqsA PAO1 at 3 hpi was assessed by RT-PCR. Calu-3-ALI cultures exposed to PQS (40 μM) or DMSO were infected at MOI 50 and total RNA prepared at 3 hpi. A 250-bp DNA region within the pqsA gene and a 200-bp DNA region within pqsE, oprL , mexG, and lecA genes were amplified from PAO1 genomic DNA (positive control); 1, cDNA; 2, corresponding RNA (negative control). L, 50 bp DNA ladder. Data are representative from 2 independent experiments.

Techniques Used: Infection, Expressing, Reverse Transcription Polymerase Chain Reaction, Amplification, Positive Control, Negative Control

20) Product Images from "Tetramethylpyrazine-Inducible Promoter Region from Rhodococcus jostii TMP1"

Article Title: Tetramethylpyrazine-Inducible Promoter Region from Rhodococcus jostii TMP1

Journal: Molecules : A Journal of Synthetic Chemistry and Natural Product Chemistry

doi: 10.3390/molecules23071530

Determination of the transcription start site by primer extension analysis using the total RNA from R. jostii TMP1 harbouring the pART3-5′UTR-gfp plasmid. The extended product was analysed alongside a DNA sequencing reaction, using the same primer. The transcription initiation sites are indicated by arrows, and the corresponding nucleotides in the DNA sequence are marked by asterisks.
Figure Legend Snippet: Determination of the transcription start site by primer extension analysis using the total RNA from R. jostii TMP1 harbouring the pART3-5′UTR-gfp plasmid. The extended product was analysed alongside a DNA sequencing reaction, using the same primer. The transcription initiation sites are indicated by arrows, and the corresponding nucleotides in the DNA sequence are marked by asterisks.

Techniques Used: Plasmid Preparation, DNA Sequencing, Sequencing

21) Product Images from "MiR‐130b promotes the progression of oesophageal squamous cell carcinoma by targeting SASH1, et al. MiR‐130b promotes the progression of oesophageal squamous cell carcinoma by targeting SASH1"

Article Title: MiR‐130b promotes the progression of oesophageal squamous cell carcinoma by targeting SASH1, et al. MiR‐130b promotes the progression of oesophageal squamous cell carcinoma by targeting SASH1

Journal: Journal of Cellular and Molecular Medicine

doi: 10.1111/jcmm.13887

MiR‐130b is highly expressed in ESCC tissues and cell lines. (A) Data from GSE 55857: the differentially expressed mi RNA s are illustrated in a heatmap. MiR‐130b was part of the overexpression category. (B) Data from GSE 97051: the differentially expressed mi RNA s are illustrated in a heatmap. MiR‐130b was part of the overexpression category. (C) The qRT ‐ PCR results showed the overexpression of miR‐130b in the tumour tissues. ** P
Figure Legend Snippet: MiR‐130b is highly expressed in ESCC tissues and cell lines. (A) Data from GSE 55857: the differentially expressed mi RNA s are illustrated in a heatmap. MiR‐130b was part of the overexpression category. (B) Data from GSE 97051: the differentially expressed mi RNA s are illustrated in a heatmap. MiR‐130b was part of the overexpression category. (C) The qRT ‐ PCR results showed the overexpression of miR‐130b in the tumour tissues. ** P

Techniques Used: Over Expression, Quantitative RT-PCR

22) Product Images from "The human adenovirus type 5 E1B 55kDa protein interacts with RNA promoting timely DNA replication and viral late mRNA metabolism"

Article Title: The human adenovirus type 5 E1B 55kDa protein interacts with RNA promoting timely DNA replication and viral late mRNA metabolism

Journal: PLoS ONE

doi: 10.1371/journal.pone.0214882

Substitutions in E1B 55K RNP affect viral late mRNA biogenesis. HFF cells infected with Ad5 WT or E1B 55K mutants were harvested at 36 hpi and total RNA was isolated. Viral late pre-mRNA levels were determined for L5 RNA by RT qPCR against an (A) intron-exon (L5NP) or (B) Exon-exon (L5P) junction, for the unspliced and spliced L5 mRNA species, respectively. (C) To compare the splicing efficiency the L5P:L5NP ratios were calculated. β actin mRNA was used as endogenous control. Data from two independent experiments performed in triplicate are shown. ** P
Figure Legend Snippet: Substitutions in E1B 55K RNP affect viral late mRNA biogenesis. HFF cells infected with Ad5 WT or E1B 55K mutants were harvested at 36 hpi and total RNA was isolated. Viral late pre-mRNA levels were determined for L5 RNA by RT qPCR against an (A) intron-exon (L5NP) or (B) Exon-exon (L5P) junction, for the unspliced and spliced L5 mRNA species, respectively. (C) To compare the splicing efficiency the L5P:L5NP ratios were calculated. β actin mRNA was used as endogenous control. Data from two independent experiments performed in triplicate are shown. ** P

Techniques Used: Infection, Isolation, Quantitative RT-PCR

E1B 55K interacts with RNA in Ad5 WT-infected cells and RNP substitutions affect RNA binding. HFF cells infected with the indicated viruses were harvested at 36hpi. E1B 55K was immunoprecipitated with the 2A6 MAb, RNA was isolated and RT qPCR were performed to detect a sequence corresponding to intron 2 in the TPL. (A) Western blot of immunoprecipitated samples with the anti-E1B 55K 2A6 MAb. (B) RT qPCR of immunoprecipitated viral RNA. Immunoprecipitation data was normalized as described in Materials and Methods and it is represented as the percentage of the input RNA. Standard deviations from three independent experiments performed in triplicate are shown. *** P
Figure Legend Snippet: E1B 55K interacts with RNA in Ad5 WT-infected cells and RNP substitutions affect RNA binding. HFF cells infected with the indicated viruses were harvested at 36hpi. E1B 55K was immunoprecipitated with the 2A6 MAb, RNA was isolated and RT qPCR were performed to detect a sequence corresponding to intron 2 in the TPL. (A) Western blot of immunoprecipitated samples with the anti-E1B 55K 2A6 MAb. (B) RT qPCR of immunoprecipitated viral RNA. Immunoprecipitation data was normalized as described in Materials and Methods and it is represented as the percentage of the input RNA. Standard deviations from three independent experiments performed in triplicate are shown. *** P

Techniques Used: Infection, RNA Binding Assay, Immunoprecipitation, Isolation, Quantitative RT-PCR, Sequencing, Western Blot

23) Product Images from "Gene-Specific Intron Retention Serves as Molecular Signature that Distinguishes Melanoma from Non-Melanoma Cancer Cells in Greek Patients"

Article Title: Gene-Specific Intron Retention Serves as Molecular Signature that Distinguishes Melanoma from Non-Melanoma Cancer Cells in Greek Patients

Journal: International Journal of Molecular Sciences

doi: 10.3390/ijms20040937

Intron retention of Sestrin-1 gene molecularly differentiates BCC and SCC from melanoma cells. Expression profiles of Sestrin-1 gene, via employment of RT-sqPCR protocols and utilization of total RNA preparations purified from BCC and SCC ( A ) or melanoma ( B ) biopsy collections. GAPDH served as gene of reference (also, see Figure 6 F and Table S1 ). ( C ) Representative DNA chromatogram derived from cycle sequencing of the melanoma-specific 1253 bp long PCR band. A characteristic Sestrin-1 intron 9/10 (912 bp) sequenced area is indicated. Besides intron 9/10 retention (1253 bp), note the absence of Sestrin-1 normal transcriptional activity (lack of 341 bp) in all melanoma cDNA preparations (B). p: patient, c: cancer tissue (biopsy), h: healthy tissue (biopsy), m: melanoma (biopsy), bp: base pair, F: forward (primer) and R: reverse (primer).
Figure Legend Snippet: Intron retention of Sestrin-1 gene molecularly differentiates BCC and SCC from melanoma cells. Expression profiles of Sestrin-1 gene, via employment of RT-sqPCR protocols and utilization of total RNA preparations purified from BCC and SCC ( A ) or melanoma ( B ) biopsy collections. GAPDH served as gene of reference (also, see Figure 6 F and Table S1 ). ( C ) Representative DNA chromatogram derived from cycle sequencing of the melanoma-specific 1253 bp long PCR band. A characteristic Sestrin-1 intron 9/10 (912 bp) sequenced area is indicated. Besides intron 9/10 retention (1253 bp), note the absence of Sestrin-1 normal transcriptional activity (lack of 341 bp) in all melanoma cDNA preparations (B). p: patient, c: cancer tissue (biopsy), h: healthy tissue (biopsy), m: melanoma (biopsy), bp: base pair, F: forward (primer) and R: reverse (primer).

Techniques Used: Expressing, Purification, Derivative Assay, Sequencing, Polymerase Chain Reaction, Activity Assay

MCT4 intron retention is activated both in non-melanoma (BCC and SCC) and melanoma biopsy collections. Patterns of MCT4 transcriptional activity, through engagement of RT-sqPCR platforms based on total RNA extraction from BCC and SCC ( A ), or melanoma ( B ) biopsy samples. GAPDH was used as control gene (also, see Figure 6 F and Table S1 ). ( C ) Representative DNA sequence chromatogram of the melanoma-specific 443 bp long PCR fragment. A characteristic MCT4 intron 2/3 (265 bp) sequenced area is indicated. Besides the retention of MCT4 intron 2/3 (443 bp), note the absence of gene’s normal transcriptional activity (lack of 178 bp) in all melanoma cDNA samples (B). p: patient, c: cancer tissue (biopsy), h: healthy tissue (biopsy), m: melanoma (biopsy), bp: base pair, F: forward (primer), R: reverse (primer) and asterisk (*): aberrant splicing-derived PCR band.
Figure Legend Snippet: MCT4 intron retention is activated both in non-melanoma (BCC and SCC) and melanoma biopsy collections. Patterns of MCT4 transcriptional activity, through engagement of RT-sqPCR platforms based on total RNA extraction from BCC and SCC ( A ), or melanoma ( B ) biopsy samples. GAPDH was used as control gene (also, see Figure 6 F and Table S1 ). ( C ) Representative DNA sequence chromatogram of the melanoma-specific 443 bp long PCR fragment. A characteristic MCT4 intron 2/3 (265 bp) sequenced area is indicated. Besides the retention of MCT4 intron 2/3 (443 bp), note the absence of gene’s normal transcriptional activity (lack of 178 bp) in all melanoma cDNA samples (B). p: patient, c: cancer tissue (biopsy), h: healthy tissue (biopsy), m: melanoma (biopsy), bp: base pair, F: forward (primer), R: reverse (primer) and asterisk (*): aberrant splicing-derived PCR band.

Techniques Used: Activity Assay, RNA Extraction, Sequencing, Polymerase Chain Reaction, Derivative Assay

The c-MYC gene is subjected to an intron retention mechanism in human melanoma. Gene expression profiles of c-MYC , via employment of RT-sqPCR protocols, using total RNA preparations derived from BCC and SCC ( A ) or melanoma ( B ) biopsy specimens. GAPDH served as gene of reference (also, see Figure 6 F and Table S1 ). ( C ) Representative DNA chromatogram derived from cycle sequencing of the melanoma-specific 1564 bp long PCR product. Characteristic c-MYC intron 2/3 (1376 bp) sequenced areas are indicated. Besides intron 2/3 retention (1564 bp), note the absence of c-MYC normal transcriptional activity (lack of 188 bp) in all melanoma cDNA preparations ( B ). p: patient, c: cancer tissue (biopsy), h: healthy tissue (biopsy), m: melanoma (biopsy), bp: base pair, F: forward (primer) and R: reverse (primer).
Figure Legend Snippet: The c-MYC gene is subjected to an intron retention mechanism in human melanoma. Gene expression profiles of c-MYC , via employment of RT-sqPCR protocols, using total RNA preparations derived from BCC and SCC ( A ) or melanoma ( B ) biopsy specimens. GAPDH served as gene of reference (also, see Figure 6 F and Table S1 ). ( C ) Representative DNA chromatogram derived from cycle sequencing of the melanoma-specific 1564 bp long PCR product. Characteristic c-MYC intron 2/3 (1376 bp) sequenced areas are indicated. Besides intron 2/3 retention (1564 bp), note the absence of c-MYC normal transcriptional activity (lack of 188 bp) in all melanoma cDNA preparations ( B ). p: patient, c: cancer tissue (biopsy), h: healthy tissue (biopsy), m: melanoma (biopsy), bp: base pair, F: forward (primer) and R: reverse (primer).

Techniques Used: Expressing, Derivative Assay, Sequencing, Polymerase Chain Reaction, Activity Assay

Lack of regular transcriptional activity is not a common mechanism in human melanoma. Expression profiles of a number of genes critically implicated in cell metabolism ( A , B , F ), cell signaling ( B ) and cell survival or death ( C – E ), via engagement of RT-sqPCR platforms based on total RNA extraction from melanoma biopsy specimens. ( A ) MCT1 gene. (B) Sestrin-2 gene. (C) Survivin gene. ( D ) XIAP gene. ( E ) NOXA gene. ( F ) GAPDH gene. Note the readily detectable ( MCT1 ), or strongly activated ( Sestrin-2 , Survivin , XIAP , NOXA , and GAPDH ) transcriptional expression of the herein examined genes. GAPDH was used as -housekeeping- gene of reference (also, see Figure 1 , Figure 2 and Figure 3 and Figure 5 , Table S1 , and Figures S1 and S2 ). m: melanoma (biopsy) and bp: base pair.
Figure Legend Snippet: Lack of regular transcriptional activity is not a common mechanism in human melanoma. Expression profiles of a number of genes critically implicated in cell metabolism ( A , B , F ), cell signaling ( B ) and cell survival or death ( C – E ), via engagement of RT-sqPCR platforms based on total RNA extraction from melanoma biopsy specimens. ( A ) MCT1 gene. (B) Sestrin-2 gene. (C) Survivin gene. ( D ) XIAP gene. ( E ) NOXA gene. ( F ) GAPDH gene. Note the readily detectable ( MCT1 ), or strongly activated ( Sestrin-2 , Survivin , XIAP , NOXA , and GAPDH ) transcriptional expression of the herein examined genes. GAPDH was used as -housekeeping- gene of reference (also, see Figure 1 , Figure 2 and Figure 3 and Figure 5 , Table S1 , and Figures S1 and S2 ). m: melanoma (biopsy) and bp: base pair.

Techniques Used: Activity Assay, Expressing, RNA Extraction

SRPX2 gene is subjected to a strong intron retention mechanism in melanoma, but not in non-melanoma cells. Gene expression patterns of SRPX2 , through engagement of RT-sqPCR protocols and utilization of total RNA extracts prepared from BCC and SCC ( A ) or melanoma ( B ) cDNA collections. GAPDH served as control gene (also, see Figure 6 F and Table S1 ). ( C ) Representative DNA sequence chromatogram derived from the melanoma-specific 1274 bp long PCR product. A characteristic SRPX2 intron 7/8 (1096 bp) sequenced area is indicated. Besides intron 7/8 retention (1274 bp), note the absence of SRPX2 normal transcriptional activity (lack of 178 bp) in all melanoma biopsy samples ( B ). p: patient, c: cancer tissue (biopsy), h: healthy tissue (biopsy), m: melanoma (biopsy), bp: base pair, F: forward (primer) and R: reverse (primer).
Figure Legend Snippet: SRPX2 gene is subjected to a strong intron retention mechanism in melanoma, but not in non-melanoma cells. Gene expression patterns of SRPX2 , through engagement of RT-sqPCR protocols and utilization of total RNA extracts prepared from BCC and SCC ( A ) or melanoma ( B ) cDNA collections. GAPDH served as control gene (also, see Figure 6 F and Table S1 ). ( C ) Representative DNA sequence chromatogram derived from the melanoma-specific 1274 bp long PCR product. A characteristic SRPX2 intron 7/8 (1096 bp) sequenced area is indicated. Besides intron 7/8 retention (1274 bp), note the absence of SRPX2 normal transcriptional activity (lack of 178 bp) in all melanoma biopsy samples ( B ). p: patient, c: cancer tissue (biopsy), h: healthy tissue (biopsy), m: melanoma (biopsy), bp: base pair, F: forward (primer) and R: reverse (primer).

Techniques Used: Expressing, Sequencing, Derivative Assay, Polymerase Chain Reaction, Activity Assay

24) Product Images from "A determining factor for insect feeding preference in the silkworm, Bombyx mori"

Article Title: A determining factor for insect feeding preference in the silkworm, Bombyx mori

Journal: PLoS Biology

doi: 10.1371/journal.pbio.3000162

GR66 expression in tissues and cellular localization of GR66. (A) Scanning electron micrographs of first-instar heads. The area inside the white box is enlarged on the right. (B) Relative mRNA levels of BmGR66 in Bombyx mori tissues as determined by qRT-PCR. Total RNA was isolated from the antennae, labra, mandibles, maxillae, labia, thoracic legs, and midguts. The RNA was converted to cDNA, which was used as a template to quantify BmGR66 mRNA levels using Bmrp49 as a reference gene. The data shown was the mean ± SEM ( n = 3). Underlying data can be found in S1 Data . (C) Photographs of HEK293T cells expressing the EGFP-GR66 fusion protein and stained with DAPI. n = 32. Scale bars: 10 μm. cDNA, complementary DNA; EGFP, enhanced green fluorescent protein; FITC, fluorescein isothiocyanate; GR66, gustatory receptor 66; HEK293T, human embryonic kidney 239T; Ls, lateral sensilla; Mp, maxillary palp; Ms, medial sensilla; qRT-PCR, quantitative real-time PCR; SEM, standard error of the mean.
Figure Legend Snippet: GR66 expression in tissues and cellular localization of GR66. (A) Scanning electron micrographs of first-instar heads. The area inside the white box is enlarged on the right. (B) Relative mRNA levels of BmGR66 in Bombyx mori tissues as determined by qRT-PCR. Total RNA was isolated from the antennae, labra, mandibles, maxillae, labia, thoracic legs, and midguts. The RNA was converted to cDNA, which was used as a template to quantify BmGR66 mRNA levels using Bmrp49 as a reference gene. The data shown was the mean ± SEM ( n = 3). Underlying data can be found in S1 Data . (C) Photographs of HEK293T cells expressing the EGFP-GR66 fusion protein and stained with DAPI. n = 32. Scale bars: 10 μm. cDNA, complementary DNA; EGFP, enhanced green fluorescent protein; FITC, fluorescein isothiocyanate; GR66, gustatory receptor 66; HEK293T, human embryonic kidney 239T; Ls, lateral sensilla; Mp, maxillary palp; Ms, medial sensilla; qRT-PCR, quantitative real-time PCR; SEM, standard error of the mean.

Techniques Used: Expressing, Quantitative RT-PCR, Isolation, Staining, Mass Spectrometry, Real-time Polymerase Chain Reaction

25) Product Images from "RNA Catabolites Contribute to the Nitrogen Pool and Support Growth Recovery of Wheat"

Article Title: RNA Catabolites Contribute to the Nitrogen Pool and Support Growth Recovery of Wheat

Journal: Frontiers in Plant Science

doi: 10.3389/fpls.2018.01539

Diagram of the purine pathway characterized in Arabidopsis adapted from Bernard et al. (2011) . The diagram shows the cellular localization of RNA degradation, purine salvage and catabolism. Proteins (enzymes and transporters) that were examined here at the gene transcriptional level are shown in red; RNS2, ribonuclease type II; ENT1, equilibrative nucleoside transporter 1; ENT3, equlibrative nucleoside transporter 3; XDH, xanthine dehydrogenase; ADK, adenosine kinase; ALN, allantoinase and AAH, allantoate amidohydrolase. Additional enzymes shown include NSH, nucleoside hydrolase and APT, adenine phosphoribosyltransferase. Cellular localization of these proteins is not known in wheat.
Figure Legend Snippet: Diagram of the purine pathway characterized in Arabidopsis adapted from Bernard et al. (2011) . The diagram shows the cellular localization of RNA degradation, purine salvage and catabolism. Proteins (enzymes and transporters) that were examined here at the gene transcriptional level are shown in red; RNS2, ribonuclease type II; ENT1, equilibrative nucleoside transporter 1; ENT3, equlibrative nucleoside transporter 3; XDH, xanthine dehydrogenase; ADK, adenosine kinase; ALN, allantoinase and AAH, allantoate amidohydrolase. Additional enzymes shown include NSH, nucleoside hydrolase and APT, adenine phosphoribosyltransferase. Cellular localization of these proteins is not known in wheat.

Techniques Used:

Transcriptional response to N starvation of genes with putative functions in RNA degradation, nucleoside transport and salvage. Plants of the genotypes Mace and RAC875 were grown for 14 days with full nutrition before applying the treatments +N (1 mM N) or –N (0 mM N) for 1 day, 3 days or 5 days. n = 6 with bulking of 3 plants per n. Transcriptional profile of the putative ribonuclease type II gene ( TaRNS2 ), the putative equilibrative nucleoside transporter 1 gene ( TaENT1 ), the putative equilibrative nucleoside transporter 3 gene ( TaENT3 ) and the putative adenosine kinase gene ( TaADK ) from Mace and RAC875 youngest fully expanded leaf (YFEL, A ), oldest leaf (B) and roots (C) expressed as calibrated normalized relative quantities (CNRQ). Statistical significance between treatments within a single time point and a single genotype at ∗∗∗ p ≤ 0.001, ∗∗ p ≤ 0.02, ∗ p ≤ 0.05 as determined by T -test analysis and post hoc analysis using the Holm-Sidak method.
Figure Legend Snippet: Transcriptional response to N starvation of genes with putative functions in RNA degradation, nucleoside transport and salvage. Plants of the genotypes Mace and RAC875 were grown for 14 days with full nutrition before applying the treatments +N (1 mM N) or –N (0 mM N) for 1 day, 3 days or 5 days. n = 6 with bulking of 3 plants per n. Transcriptional profile of the putative ribonuclease type II gene ( TaRNS2 ), the putative equilibrative nucleoside transporter 1 gene ( TaENT1 ), the putative equilibrative nucleoside transporter 3 gene ( TaENT3 ) and the putative adenosine kinase gene ( TaADK ) from Mace and RAC875 youngest fully expanded leaf (YFEL, A ), oldest leaf (B) and roots (C) expressed as calibrated normalized relative quantities (CNRQ). Statistical significance between treatments within a single time point and a single genotype at ∗∗∗ p ≤ 0.001, ∗∗ p ≤ 0.02, ∗ p ≤ 0.05 as determined by T -test analysis and post hoc analysis using the Holm-Sidak method.

Techniques Used:

26) Product Images from "Properties of LINE-1 proteins and repeat element expression in the context of amyotrophic lateral sclerosis"

Article Title: Properties of LINE-1 proteins and repeat element expression in the context of amyotrophic lateral sclerosis

Journal: Mobile DNA

doi: 10.1186/s13100-018-0138-z

Nuclear localization of L1 ORF1 protein. a Endogenous ORF1p detected in 2102Ep cells by the α-4H1-ORF1 antibody. ORF1p is mostly cytoplasmic where it concentrates in granules and occasionally at the nuclear membrane. It is faintly seen in the nucleoplasm and concentrates in nucleoli of a subset of cells. b , c Exogenously expressed EGFP-tagged ORF1p strongly concentrates at the nuclear membrane and in perinucleolar foci of 5% or fewer human ( b ) U2OS or ( c ) HEK 293T cells with attendant reduction in size and number of cytoplasmic granules. Cotransfected mCherry-PSP1 marks nuclei and is excluded from nucleoli. d Endogenous ORF1p detected by α-4H1-ORF1 also forms discrete nuclear foci in a minor percentage of 2102Ep cells. Selected foci are enlarged in panels to the right. e Alu RNA, tagged with six MS2 coat protein recognition stem loops and expressed from construct pBS 7SL Alu-MS2 (Ya5), was detected by FISH using a Cy3-tagged DNA probe to the MS2 stem loops. Alu RNA colocalizes with nuclear foci marked by EGFP-tagged ORF1p in HEK 293T cells. f Nuclear foci of MS2 stem loop-tagged full-length SVA RNA detected by the Cy3-MS2 DNA probe do not colocalize with foci marked by ORF1p-EGFP. g RNA having 31 tandem G4C2 repeats detected by FISH using a Cy3-conjugated (C4G2) 4 DNA probe induces intense intranuclear or cytoplasmic RNA aggregates that colocalize with ORF1p-EGFP in a minor percentage of HEK 293T cells (nuclear granules are marked by small arrows and cytoplasmic granules by large arrows). Size bars are 10 μm
Figure Legend Snippet: Nuclear localization of L1 ORF1 protein. a Endogenous ORF1p detected in 2102Ep cells by the α-4H1-ORF1 antibody. ORF1p is mostly cytoplasmic where it concentrates in granules and occasionally at the nuclear membrane. It is faintly seen in the nucleoplasm and concentrates in nucleoli of a subset of cells. b , c Exogenously expressed EGFP-tagged ORF1p strongly concentrates at the nuclear membrane and in perinucleolar foci of 5% or fewer human ( b ) U2OS or ( c ) HEK 293T cells with attendant reduction in size and number of cytoplasmic granules. Cotransfected mCherry-PSP1 marks nuclei and is excluded from nucleoli. d Endogenous ORF1p detected by α-4H1-ORF1 also forms discrete nuclear foci in a minor percentage of 2102Ep cells. Selected foci are enlarged in panels to the right. e Alu RNA, tagged with six MS2 coat protein recognition stem loops and expressed from construct pBS 7SL Alu-MS2 (Ya5), was detected by FISH using a Cy3-tagged DNA probe to the MS2 stem loops. Alu RNA colocalizes with nuclear foci marked by EGFP-tagged ORF1p in HEK 293T cells. f Nuclear foci of MS2 stem loop-tagged full-length SVA RNA detected by the Cy3-MS2 DNA probe do not colocalize with foci marked by ORF1p-EGFP. g RNA having 31 tandem G4C2 repeats detected by FISH using a Cy3-conjugated (C4G2) 4 DNA probe induces intense intranuclear or cytoplasmic RNA aggregates that colocalize with ORF1p-EGFP in a minor percentage of HEK 293T cells (nuclear granules are marked by small arrows and cytoplasmic granules by large arrows). Size bars are 10 μm

Techniques Used: Construct, Fluorescence In Situ Hybridization

27) Product Images from "Comprehensive analysis of the BC200 ribonucleoprotein reveals a reciprocal regulatory function with CSDE1/UNR"

Article Title: Comprehensive analysis of the BC200 ribonucleoprotein reveals a reciprocal regulatory function with CSDE1/UNR

Journal: Nucleic Acids Research

doi: 10.1093/nar/gky860

CSDE1 can bind directly to BC200 whereas STRAP interactions are CSDE1 dependent. ( A ) Immunoprecipitation experiments performed as described for Figure 3A under conditions of CSDE1 and STRAP knock-down by siRNA (48 h post transfection, CSDE1 siRNA_2, STRAP siRNA_2). Co-immunoprecipitating BC200 RNA was detected by RT-qPCR and compared to total RNA extracted from 10% of input. Data represents the mean of three independent replicates ± standard deviation. ( B ) Immunoprecipitation efficiency was monitored as in Figure 3C . ( C ) Panel 1: Coomassie stain gel of purified CSDE1 and STRAP separated by SDS/PAGE. Panel 2: Western blot with antibodies against CSDE1 of 2 ng purified CSDE1 and STRAP protein separated by SDS/PAGE. Panel 3: As in Panel 1, with antibodies to STRAP. Panel 4: As in Panel 1, with antibodies to FLAG peptide. ( D ) Electrophoretic mobility shift assays of binding reactions prepared with 50 nM BC200, BCSCR or BC119 and a concentration gradient of the indicated proteins. Serial dilutions of protein were used from 1000 to 7.8 nM. Gels were stained with SYBR Gold nucleic acid stain.
Figure Legend Snippet: CSDE1 can bind directly to BC200 whereas STRAP interactions are CSDE1 dependent. ( A ) Immunoprecipitation experiments performed as described for Figure 3A under conditions of CSDE1 and STRAP knock-down by siRNA (48 h post transfection, CSDE1 siRNA_2, STRAP siRNA_2). Co-immunoprecipitating BC200 RNA was detected by RT-qPCR and compared to total RNA extracted from 10% of input. Data represents the mean of three independent replicates ± standard deviation. ( B ) Immunoprecipitation efficiency was monitored as in Figure 3C . ( C ) Panel 1: Coomassie stain gel of purified CSDE1 and STRAP separated by SDS/PAGE. Panel 2: Western blot with antibodies against CSDE1 of 2 ng purified CSDE1 and STRAP protein separated by SDS/PAGE. Panel 3: As in Panel 1, with antibodies to STRAP. Panel 4: As in Panel 1, with antibodies to FLAG peptide. ( D ) Electrophoretic mobility shift assays of binding reactions prepared with 50 nM BC200, BCSCR or BC119 and a concentration gradient of the indicated proteins. Serial dilutions of protein were used from 1000 to 7.8 nM. Gels were stained with SYBR Gold nucleic acid stain.

Techniques Used: Immunoprecipitation, Transfection, Quantitative RT-PCR, Standard Deviation, Staining, Purification, SDS Page, Western Blot, Electrophoretic Mobility Shift Assay, Binding Assay, Concentration Assay

RNA Immunoprecipitation experiments of confirmed BC200 targets. ( A ) RT-qPCR analysis of BC200, GAPDH and 7SL enrichment by immunoprecipitation of the indicated proteins. RNA extracted from 10% of the input sample was used as a reference to calculate percent of input for each RNA that was bound to the immunoprecipitated protein. Data represents the mean of three independent replicates ± standard deviation. Dashed line represents the threshold value of 5% input. ( B ) Percent input values of BC200 and 7SL were compared to GAPDH to demonstrate the degree of specificity of the interactions analyzed in (A). Dashed line represents the threshold value of 2-fold enrichment. ( C ) Immunoprecipitation efficiency was monitored by performing western blot on 50 μg of PRE and POST IP samples as well as 2% of the IP.
Figure Legend Snippet: RNA Immunoprecipitation experiments of confirmed BC200 targets. ( A ) RT-qPCR analysis of BC200, GAPDH and 7SL enrichment by immunoprecipitation of the indicated proteins. RNA extracted from 10% of the input sample was used as a reference to calculate percent of input for each RNA that was bound to the immunoprecipitated protein. Data represents the mean of three independent replicates ± standard deviation. Dashed line represents the threshold value of 5% input. ( B ) Percent input values of BC200 and 7SL were compared to GAPDH to demonstrate the degree of specificity of the interactions analyzed in (A). Dashed line represents the threshold value of 2-fold enrichment. ( C ) Immunoprecipitation efficiency was monitored by performing western blot on 50 μg of PRE and POST IP samples as well as 2% of the IP.

Techniques Used: Immunoprecipitation, Quantitative RT-PCR, Standard Deviation, Western Blot

CSDE1 knock-down decreases stability of the BC200 RNA. ( A ) RT-qPCR analysis of BC200 expression following 72-hour CSDE1 or control knock-down and Actinomycin D treatment at T = 0. Indicated half-lives were calculated by fitting the data to a one-phase decay equation with GraphPad Prism software. Data represents the mean of three independent biological replicates measured in duplicate. ( B ) Relative total RNA quantities purified at the indicated time points from an equal number of cells to monitor total RNA decay. Data represents the mean of three independent replicates ± standard deviation. ( C ) MCF-7 cells were reverse transfected with control or CSDE1 siRNA and following 24 h were forward transfected with the plasmids containing BC200 under control of either the U6 snRNA promoter or endogenous BC200 promoter. Absolute expression levels of RNA from plasmid was calculated by RT-qPCR in parallel to a standard curve generated with purified BC200 RNA. Data represents the mean of three independent replicates ± standard deviation. ( D ) Relative CSDE1 mRNA expression was monitored by RT-qPCR analysis of the same RNA samples as used in (C).
Figure Legend Snippet: CSDE1 knock-down decreases stability of the BC200 RNA. ( A ) RT-qPCR analysis of BC200 expression following 72-hour CSDE1 or control knock-down and Actinomycin D treatment at T = 0. Indicated half-lives were calculated by fitting the data to a one-phase decay equation with GraphPad Prism software. Data represents the mean of three independent biological replicates measured in duplicate. ( B ) Relative total RNA quantities purified at the indicated time points from an equal number of cells to monitor total RNA decay. Data represents the mean of three independent replicates ± standard deviation. ( C ) MCF-7 cells were reverse transfected with control or CSDE1 siRNA and following 24 h were forward transfected with the plasmids containing BC200 under control of either the U6 snRNA promoter or endogenous BC200 promoter. Absolute expression levels of RNA from plasmid was calculated by RT-qPCR in parallel to a standard curve generated with purified BC200 RNA. Data represents the mean of three independent replicates ± standard deviation. ( D ) Relative CSDE1 mRNA expression was monitored by RT-qPCR analysis of the same RNA samples as used in (C).

Techniques Used: Quantitative RT-PCR, Expressing, Software, Purification, Standard Deviation, Transfection, Plasmid Preparation, Generated

BC200 and CSDE1 expression are mutually codependent. ( A ) RT-qPCR analysis of BC200 RNA expression following transfection with the indicated RNA interference oligonucleotides. Data represents the mean of three independent replicates ± standard deviation. ( B ) RT-qPCR analysis of CSDE1 mRNA expression following transfection with the indicated RNA interference oligonucleotides. Data represents the mean of three independent replicates ± standard deviation. ( C ) Western blot analysis of protein samples from a 72-hour knock-down time-course with the indicated RNA interference oligonucleotides and antibodies. Data is representative of three independent replicates. ( D ) Densitometry measurements of CSDE1 protein expression from (C) as well as replicate experiments. Data represents the mean of three independent replicates ± standard deviation
Figure Legend Snippet: BC200 and CSDE1 expression are mutually codependent. ( A ) RT-qPCR analysis of BC200 RNA expression following transfection with the indicated RNA interference oligonucleotides. Data represents the mean of three independent replicates ± standard deviation. ( B ) RT-qPCR analysis of CSDE1 mRNA expression following transfection with the indicated RNA interference oligonucleotides. Data represents the mean of three independent replicates ± standard deviation. ( C ) Western blot analysis of protein samples from a 72-hour knock-down time-course with the indicated RNA interference oligonucleotides and antibodies. Data is representative of three independent replicates. ( D ) Densitometry measurements of CSDE1 protein expression from (C) as well as replicate experiments. Data represents the mean of three independent replicates ± standard deviation

Techniques Used: Expressing, Quantitative RT-PCR, RNA Expression, Transfection, Standard Deviation, Western Blot

28) Product Images from "Epigenetic modifications acetylation and deacetylation play important roles in juvenile hormone action"

Article Title: Epigenetic modifications acetylation and deacetylation play important roles in juvenile hormone action

Journal: BMC Genomics

doi: 10.1186/s12864-018-5323-4

TcA cells respond to both juvenile hormone and TSA after dsmalE and dsCBP treatment. Total RNA was isolated from 100,000 cells that were cultured in the medium containing either dsmalE or dsCBP for 72 h. The cells were then exposed to DMSO (
Figure Legend Snippet: TcA cells respond to both juvenile hormone and TSA after dsmalE and dsCBP treatment. Total RNA was isolated from 100,000 cells that were cultured in the medium containing either dsmalE or dsCBP for 72 h. The cells were then exposed to DMSO (

Techniques Used: Isolation, Cell Culture

29) Product Images from "Cryptococcus neoformans and Cryptococcus gattii clinical isolates from Thailand display diverse phenotypic interactions with macrophages"

Article Title: Cryptococcus neoformans and Cryptococcus gattii clinical isolates from Thailand display diverse phenotypic interactions with macrophages

Journal: Virulence

doi: 10.1080/21505594.2018.1556150

Infection with high-uptake C. neoformans strains induced alveolar macrophages with higher expression of genes related to M2 macrophage. BALB/c mice received PBS or were infected with the top 4 high-and the top 4 low-uptake C. neoformans isolates. (a) Alveolar macrophages were isolated from the bronchoalveolar lavage of each mouse at 14 d postinfection and total RNA was isolated and subjected to cDNA synthesis, followed by real-time PCR analysis of gene for M1 and M2 including Arg1, Fizz1, Ym1, Il13, Ccl17, Nos2, Ifng, Il6, Tnfa, Mcp1, Csf2 and Ip10 . Data were expressed as fold induction over actin ( Actb ) expression, with the mRNA levels in the PBS control set as 1. (b) Bronchoalveolar lavages of PBS-treated mice and high-uptake and low-uptake C. neoformans -infected mice were collected and the arginase enzyme activity was determined by photometric measurement of produced urea concentration. Graphs depict mean ± SD and are representative of 3 experiments with 3 to 5 mice per group. Significance was determined by one-way ANOVA with Tukey’s post hoc analysis * p
Figure Legend Snippet: Infection with high-uptake C. neoformans strains induced alveolar macrophages with higher expression of genes related to M2 macrophage. BALB/c mice received PBS or were infected with the top 4 high-and the top 4 low-uptake C. neoformans isolates. (a) Alveolar macrophages were isolated from the bronchoalveolar lavage of each mouse at 14 d postinfection and total RNA was isolated and subjected to cDNA synthesis, followed by real-time PCR analysis of gene for M1 and M2 including Arg1, Fizz1, Ym1, Il13, Ccl17, Nos2, Ifng, Il6, Tnfa, Mcp1, Csf2 and Ip10 . Data were expressed as fold induction over actin ( Actb ) expression, with the mRNA levels in the PBS control set as 1. (b) Bronchoalveolar lavages of PBS-treated mice and high-uptake and low-uptake C. neoformans -infected mice were collected and the arginase enzyme activity was determined by photometric measurement of produced urea concentration. Graphs depict mean ± SD and are representative of 3 experiments with 3 to 5 mice per group. Significance was determined by one-way ANOVA with Tukey’s post hoc analysis * p

Techniques Used: Infection, Expressing, Mouse Assay, Isolation, Real-time Polymerase Chain Reaction, Activity Assay, Produced, Concentration Assay

30) Product Images from "Exosomal Transmission of MicroRNA from HCV Replicating Cells Stimulates Transdifferentiation in Hepatic Stellate Cells"

Article Title: Exosomal Transmission of MicroRNA from HCV Replicating Cells Stimulates Transdifferentiation in Hepatic Stellate Cells

Journal: Molecular Therapy. Nucleic Acids

doi: 10.1016/j.omtn.2019.01.006

Effects of Supernatant from HCV-Replicating Cells on miR-192 Expression in HSCs (A) Experimental scheme (left). LX-2 cells were treated with supernatant from naive Huh-7 or JFH-1 stable cells for 3 days. The RNA levels of COL1A1, α-SMA, and TGF-β1 and miR-192 levels in LX-2 cells treated with JFH-1 supernatant were quantified relative to those in cells treated with naive Huh-7 supernatant (right). (B) Effects of TGF-β1 on miR-192 expression in HSCs are shown. LX-2 cells were cultured with serum-free medium for 16 h and then treated with different concentrations of recombinant TGF-β1 protein for 72 h. The mRNA levels of COL1A1, α-SMA, and TGF-β1 and miR-192 levels in LX-2 cells were assessed relative to those in control PBS-treated cells. (C) Effects of miR-192 introduction or depletion on Huh-7 cells are shown. Experimental scheme is shown (left). Huh-7 cells were transfected with miR-192 mimic RNA or anti-miR-192 for 48 h and then the supernatant was collected and used to treat LX-2 cells for 72 h. The miR-192 copy number in each supernatant was quantified (middle). miR-192 levels in LX-2 cells treated with each type of supernatant were assessed relative to those in cells treated with supernatant from negative-control miRNA (siNTC)- or scramble RNA (scr)-treated Huh-7 cells (right). All data are presented as the means of at least three independent experiments, each performed in triplicate. Error bars represent the SEM. p values were determined via a one-tailed unpaired Student’s t test. * p
Figure Legend Snippet: Effects of Supernatant from HCV-Replicating Cells on miR-192 Expression in HSCs (A) Experimental scheme (left). LX-2 cells were treated with supernatant from naive Huh-7 or JFH-1 stable cells for 3 days. The RNA levels of COL1A1, α-SMA, and TGF-β1 and miR-192 levels in LX-2 cells treated with JFH-1 supernatant were quantified relative to those in cells treated with naive Huh-7 supernatant (right). (B) Effects of TGF-β1 on miR-192 expression in HSCs are shown. LX-2 cells were cultured with serum-free medium for 16 h and then treated with different concentrations of recombinant TGF-β1 protein for 72 h. The mRNA levels of COL1A1, α-SMA, and TGF-β1 and miR-192 levels in LX-2 cells were assessed relative to those in control PBS-treated cells. (C) Effects of miR-192 introduction or depletion on Huh-7 cells are shown. Experimental scheme is shown (left). Huh-7 cells were transfected with miR-192 mimic RNA or anti-miR-192 for 48 h and then the supernatant was collected and used to treat LX-2 cells for 72 h. The miR-192 copy number in each supernatant was quantified (middle). miR-192 levels in LX-2 cells treated with each type of supernatant were assessed relative to those in cells treated with supernatant from negative-control miRNA (siNTC)- or scramble RNA (scr)-treated Huh-7 cells (right). All data are presented as the means of at least three independent experiments, each performed in triplicate. Error bars represent the SEM. p values were determined via a one-tailed unpaired Student’s t test. * p

Techniques Used: Expressing, Cell Culture, Recombinant, Transfection, Negative Control, One-tailed Test

Effects of Exosomes from miR-192-Transfected Hepatocytes or Exosomes Packaged with miR-192 (A) Experimental scheme: negative-control miRNA (siNTC) or miR-192 mimic RNA was transfected into Huh-7 cells, and exosomes were purified from the transfected cells (siNTC and miR-192 exo, respectively) and used to treat LX-2 cells. (B) Comparisons of protein levels of the exosome markers LAMP2 and CD63 using western blotting (left) and exosome total protein levels using a BCA assay (right) within exosomes from each transfected cell type are shown. (C) Quantification of miR-192 (left: 3.98 ± 1.71 × 10 6 and 2.08 ± 2.09 × 10 8 copies per 20 μL of control siNTC exo and miR-192 exo, respectively) and miR-122 levels (right: 2.04 ± 1.48 × 10 6 and 1.88 ± 1.27 × 10 6 copies per 20 μL of siNTC exo and miR-192 exo, respectively) in exosomes from each transfected cell type is shown. (D) Immunoblot analysis of the fibrosis markers COL1A1 and α-SMA, as well as TGF-β1, in LX-2 cells treated with each exosome type is shown. (E) LX-2 cells were treated with each type of exosomes for 72 h, fixed, stained, and visualized via fluorescence microscopy. Representative images of α-SMA and DAPI staining in each cell type (left). Scale bars represent 50 μm. The intensity of α-SMA staining was quantified using more than five fields from at least three independent experiments (right). Data are also shown in Figure S8 A. (F) Intracellular miR-192 levels in LX-2 cells treated with each type of exosomes were quantified. (G) Experimental scheme: exosomes were first purified from naive Huh-7 cells and then packaged with siNTC or miR-192 (siNTC- and miR-192-packaged exosomes, respectively). (H) Immunoblot analysis of COL1A1, α-SMA, and TGF-β1 expression in LX-2 cells treated with each packaged exosome type is shown (left). Protein levels were quantified relative to those of tubulin (right). (I) LX-2 cells were treated with each packaged exosome type for 72 h, fixed, stained, and visualized using fluorescence microscopy. Representative images of α-SMA and DAPI staining in each cell are shown (left). Scale bars represent 50 μm. Intensity of α-SMA staining was quantified using more than five fields from at least three independent experiments is shown (right). Data are also shown in Figure S8 B. (J) Intracellular miR-192 levels in LX-2 cells treated with each packaged exosome type were quantified. All data are presented as the means of at least three independent experiments, each performed in triplicate. Error bars represent SEM. p values were determined using a one-tailed unpaired Student’s t test. * p
Figure Legend Snippet: Effects of Exosomes from miR-192-Transfected Hepatocytes or Exosomes Packaged with miR-192 (A) Experimental scheme: negative-control miRNA (siNTC) or miR-192 mimic RNA was transfected into Huh-7 cells, and exosomes were purified from the transfected cells (siNTC and miR-192 exo, respectively) and used to treat LX-2 cells. (B) Comparisons of protein levels of the exosome markers LAMP2 and CD63 using western blotting (left) and exosome total protein levels using a BCA assay (right) within exosomes from each transfected cell type are shown. (C) Quantification of miR-192 (left: 3.98 ± 1.71 × 10 6 and 2.08 ± 2.09 × 10 8 copies per 20 μL of control siNTC exo and miR-192 exo, respectively) and miR-122 levels (right: 2.04 ± 1.48 × 10 6 and 1.88 ± 1.27 × 10 6 copies per 20 μL of siNTC exo and miR-192 exo, respectively) in exosomes from each transfected cell type is shown. (D) Immunoblot analysis of the fibrosis markers COL1A1 and α-SMA, as well as TGF-β1, in LX-2 cells treated with each exosome type is shown. (E) LX-2 cells were treated with each type of exosomes for 72 h, fixed, stained, and visualized via fluorescence microscopy. Representative images of α-SMA and DAPI staining in each cell type (left). Scale bars represent 50 μm. The intensity of α-SMA staining was quantified using more than five fields from at least three independent experiments (right). Data are also shown in Figure S8 A. (F) Intracellular miR-192 levels in LX-2 cells treated with each type of exosomes were quantified. (G) Experimental scheme: exosomes were first purified from naive Huh-7 cells and then packaged with siNTC or miR-192 (siNTC- and miR-192-packaged exosomes, respectively). (H) Immunoblot analysis of COL1A1, α-SMA, and TGF-β1 expression in LX-2 cells treated with each packaged exosome type is shown (left). Protein levels were quantified relative to those of tubulin (right). (I) LX-2 cells were treated with each packaged exosome type for 72 h, fixed, stained, and visualized using fluorescence microscopy. Representative images of α-SMA and DAPI staining in each cell are shown (left). Scale bars represent 50 μm. Intensity of α-SMA staining was quantified using more than five fields from at least three independent experiments is shown (right). Data are also shown in Figure S8 B. (J) Intracellular miR-192 levels in LX-2 cells treated with each packaged exosome type were quantified. All data are presented as the means of at least three independent experiments, each performed in triplicate. Error bars represent SEM. p values were determined using a one-tailed unpaired Student’s t test. * p

Techniques Used: Transfection, Negative Control, Purification, Western Blot, BIA-KA, Staining, Fluorescence, Microscopy, Expressing, One-tailed Test

Transmission of Exosomal miR-192 Released from Hepatocytes into HSCs and Effects of miR-192 Downregulation on Activated HSCs (A) Experimental scheme: Huh-7 cells were transfected with Cy3-labeled miR-192, and exosomes were purified and used to treat LX-2 cells. (B) Confocal microscopy image of Cy3-labeled miR-192 (red), α-SMA, and DAPI staining in exosome-treated LX-2 cells is shown. An image of two representative fields is shown, and the scale bars represent 50 μm. (C) LX-2 cells were exposed to PBS or JFH-1 exosomes and then treated with scrambled RNA (scr) or anti-miR-192. Oil Red O staining image of LX-2 cells using a light microscope (left) and quantification of Oil-Red-O-stained cells using a microplate reader after destaining are shown (right). The results are representative of at least three independent experiments. (D) Immunoblot analysis of COL1A1, α-SMA, and TGF-β1 expression in exosome-exposed LX-2 cells treated with each RNA is shown. (E) Intracellular miR-192 levels in exosome-exposed LX-2 cells treated with each RNA were quantified and presented as the means of at least three independent experiments, each performed in triplicate. Error bars represent the SEM. p values were determined using a one-tailed unpaired Student’s t test. * p
Figure Legend Snippet: Transmission of Exosomal miR-192 Released from Hepatocytes into HSCs and Effects of miR-192 Downregulation on Activated HSCs (A) Experimental scheme: Huh-7 cells were transfected with Cy3-labeled miR-192, and exosomes were purified and used to treat LX-2 cells. (B) Confocal microscopy image of Cy3-labeled miR-192 (red), α-SMA, and DAPI staining in exosome-treated LX-2 cells is shown. An image of two representative fields is shown, and the scale bars represent 50 μm. (C) LX-2 cells were exposed to PBS or JFH-1 exosomes and then treated with scrambled RNA (scr) or anti-miR-192. Oil Red O staining image of LX-2 cells using a light microscope (left) and quantification of Oil-Red-O-stained cells using a microplate reader after destaining are shown (right). The results are representative of at least three independent experiments. (D) Immunoblot analysis of COL1A1, α-SMA, and TGF-β1 expression in exosome-exposed LX-2 cells treated with each RNA is shown. (E) Intracellular miR-192 levels in exosome-exposed LX-2 cells treated with each RNA were quantified and presented as the means of at least three independent experiments, each performed in triplicate. Error bars represent the SEM. p values were determined using a one-tailed unpaired Student’s t test. * p

Techniques Used: Transmission Assay, Transfection, Labeling, Purification, Confocal Microscopy, Staining, Light Microscopy, Expressing, One-tailed Test

Effects of HCV Replication on Exosome Release, Exosomal RNA Level, and HSC Activation (A) Comparison of exosome release from naive Huh-7 (Huh-7 exo) and JFH-1 stable cells (JFH-1 exo). Immunoblot analysis of exosome marker proteins (CD63, LAMP2, HSP70, and CD81) in increasing volume of exosomes purified from each cell type is shown (left). The level of each exosome marker protein was quantified and presented as a percentage of that in Huh-7 exo (middle). Exosome total proteins were quantified using a bicinchoninic acid assay (BCA) (right). (B) Immunoblot analysis of total cell lysates (lysate) and isolated exosomes (exo) of naive Huh-7 or JFH-1 stable cells is shown. (C and D) Quantification of miRNAs and HCV RNA within exosomes from naive Huh-7 or JFH-1 cells is shown. After isolating exosomes, we re-suspended the exosomes in 100 μL PBS buffer and used 2 μL or 20 μL of the solution for the analysis of (C) the RNA levels of miR-192 (left: Huh-7 exo, 3.03 ± 2.69 × 10 5 copies per 2 μL and 3.09 ± 2.26 × 10 6 copies per 20 μL; JFH-1 exo, 7.20 ± 4.74 × 10 5 copies per 2 μL and 8.80 ± 7.89 × 10 6 copies per 20 μL), miR-122 (right: Huh-7 exo, 2.25 ± 1.80 × 10 5 copies per 2 μL and 1.60 ± 1.43 × 10 6 copies per 20 μL; JFH-1 exo, 4.43 ± 2.29 × 10 5 copies per 2 μL and 5.06 ± 4.90 × 10 6 copies per 20 μL), and (D) HCV RNA (JFH-1 exo, 2.42 ± 3.07 × 10 5 copies per 2 μL and 2.15 ± 2.79 × 10 6 copies per 20 μL). (E and F) Quantification of fibrotic marker RNAs and proteins in LX-2 cells treated with each type of exosomes is shown. (E) Relative intracellular mRNA levels of fibrosis markers (left) and copy numbers of miR-192 (right) in LX-2 cells treated with each type of exosomes are shown. Copy numbers of miR-192 were 3.30 ± 1.67 × 10 5 and 6.18 ± 2.78 × 10 5 per 100 ng of total RNA in Huh-7 exo-treated and JFH-1 exo-treated LX-2 cells, respectively. (F) Immunoblot analysis of the fibrosis markers COL1A1 and α-SMA, as well as TGF-β1, in LX-2 cells treated with each type of exosome (left) and quantification of their levels relative to those of tubulin (right) are shown. (G) Effects of exosomes on HSC activation are shown. LX-2 cells were treated with each exosome type for 72 h, fixed, stained, and visualized using fluorescence microscopy. Representative images (left) of α-SMA and DAPI staining in LX-2 cells are shown. Scale bars represent 50 μm. α-SMA protein levels (right) in LX-2 cells treated with each type of exosomes were quantified from more than five fields of three independent experiments and shown relative to the levels in cells treated with Huh-7 exo. Data are also shown in Figure S4 . All data are presented as the means of at least three independent experiments, each performed in triplicate. Error bars represent the SEM. p values were determined using a one-tailed unpaired Student’s t test. * p
Figure Legend Snippet: Effects of HCV Replication on Exosome Release, Exosomal RNA Level, and HSC Activation (A) Comparison of exosome release from naive Huh-7 (Huh-7 exo) and JFH-1 stable cells (JFH-1 exo). Immunoblot analysis of exosome marker proteins (CD63, LAMP2, HSP70, and CD81) in increasing volume of exosomes purified from each cell type is shown (left). The level of each exosome marker protein was quantified and presented as a percentage of that in Huh-7 exo (middle). Exosome total proteins were quantified using a bicinchoninic acid assay (BCA) (right). (B) Immunoblot analysis of total cell lysates (lysate) and isolated exosomes (exo) of naive Huh-7 or JFH-1 stable cells is shown. (C and D) Quantification of miRNAs and HCV RNA within exosomes from naive Huh-7 or JFH-1 cells is shown. After isolating exosomes, we re-suspended the exosomes in 100 μL PBS buffer and used 2 μL or 20 μL of the solution for the analysis of (C) the RNA levels of miR-192 (left: Huh-7 exo, 3.03 ± 2.69 × 10 5 copies per 2 μL and 3.09 ± 2.26 × 10 6 copies per 20 μL; JFH-1 exo, 7.20 ± 4.74 × 10 5 copies per 2 μL and 8.80 ± 7.89 × 10 6 copies per 20 μL), miR-122 (right: Huh-7 exo, 2.25 ± 1.80 × 10 5 copies per 2 μL and 1.60 ± 1.43 × 10 6 copies per 20 μL; JFH-1 exo, 4.43 ± 2.29 × 10 5 copies per 2 μL and 5.06 ± 4.90 × 10 6 copies per 20 μL), and (D) HCV RNA (JFH-1 exo, 2.42 ± 3.07 × 10 5 copies per 2 μL and 2.15 ± 2.79 × 10 6 copies per 20 μL). (E and F) Quantification of fibrotic marker RNAs and proteins in LX-2 cells treated with each type of exosomes is shown. (E) Relative intracellular mRNA levels of fibrosis markers (left) and copy numbers of miR-192 (right) in LX-2 cells treated with each type of exosomes are shown. Copy numbers of miR-192 were 3.30 ± 1.67 × 10 5 and 6.18 ± 2.78 × 10 5 per 100 ng of total RNA in Huh-7 exo-treated and JFH-1 exo-treated LX-2 cells, respectively. (F) Immunoblot analysis of the fibrosis markers COL1A1 and α-SMA, as well as TGF-β1, in LX-2 cells treated with each type of exosome (left) and quantification of their levels relative to those of tubulin (right) are shown. (G) Effects of exosomes on HSC activation are shown. LX-2 cells were treated with each exosome type for 72 h, fixed, stained, and visualized using fluorescence microscopy. Representative images (left) of α-SMA and DAPI staining in LX-2 cells are shown. Scale bars represent 50 μm. α-SMA protein levels (right) in LX-2 cells treated with each type of exosomes were quantified from more than five fields of three independent experiments and shown relative to the levels in cells treated with Huh-7 exo. Data are also shown in Figure S4 . All data are presented as the means of at least three independent experiments, each performed in triplicate. Error bars represent the SEM. p values were determined using a one-tailed unpaired Student’s t test. * p

Techniques Used: Activation Assay, Marker, Purification, Acid Assay, BIA-KA, Isolation, Staining, Fluorescence, Microscopy, One-tailed Test

Effects of HCV Replication and HCV-Induced miR-192 on HSC Activation (A) Scheme of co-culture of LX-2 cells with naive Huh-7 or HCV (JFH-1) stable cells. (B) RNA levels of two activation markers (COL1A1 and α-SMA) and TGF-β1, as well as miR-192 levels, in co-cultured LX-2 cells are shown. (C) Effects of miR-192 on HSC activation are shown. LX-2 cells were transfected with miR-192 mimic RNA or negative-control miRNA (siNTC). RNA level of COL1A1, α-SMA, and TGF-β1 was quantified relative to those in siNTC-transfected LX-2 cells. (D) Effects of miR-192 depletion on HCV stable cells are shown. Experimental scheme is shown (left). JFH-1 stable cells were transfected with anti-miR-192 RNA or scramble siRNA (scr) and co-cultured with LX-2 cells starting 6 h after transfection. RNA levels of COL1A1, α-SMA, TGF-β1, and miR-192 in co-cultured LX-2 cells were quantified relative to those in cells co-cultured with scr-transfected JFH-1 stable cells (right). All data are the means of at least three independent experiments, each performed in triplicate. Error bars represent the SEM. p values were determined via a one-tailed unpaired Student’s t test. * p
Figure Legend Snippet: Effects of HCV Replication and HCV-Induced miR-192 on HSC Activation (A) Scheme of co-culture of LX-2 cells with naive Huh-7 or HCV (JFH-1) stable cells. (B) RNA levels of two activation markers (COL1A1 and α-SMA) and TGF-β1, as well as miR-192 levels, in co-cultured LX-2 cells are shown. (C) Effects of miR-192 on HSC activation are shown. LX-2 cells were transfected with miR-192 mimic RNA or negative-control miRNA (siNTC). RNA level of COL1A1, α-SMA, and TGF-β1 was quantified relative to those in siNTC-transfected LX-2 cells. (D) Effects of miR-192 depletion on HCV stable cells are shown. Experimental scheme is shown (left). JFH-1 stable cells were transfected with anti-miR-192 RNA or scramble siRNA (scr) and co-cultured with LX-2 cells starting 6 h after transfection. RNA levels of COL1A1, α-SMA, TGF-β1, and miR-192 in co-cultured LX-2 cells were quantified relative to those in cells co-cultured with scr-transfected JFH-1 stable cells (right). All data are the means of at least three independent experiments, each performed in triplicate. Error bars represent the SEM. p values were determined via a one-tailed unpaired Student’s t test. * p

Techniques Used: Activation Assay, Co-Culture Assay, Cell Culture, Transfection, Negative Control, One-tailed Test

31) Product Images from "TyrR is involved in the transcriptional regulation of biofilm formation and D-alanine catabolism in Azospirillum brasilense Sp7."

Article Title: TyrR is involved in the transcriptional regulation of biofilm formation and D-alanine catabolism in Azospirillum brasilense Sp7.

Journal: PLoS ONE

doi: 10.1371/journal.pone.0211904

RT-PCR analysis of dadA , tyrR and gyrA expression from the A . brasilense Sp7, 2116, and 2117 strains. RT-PCR analysis of tyrR , dadA and gyrA expression from the A . brasilense Sp7, 2116 and 2117 strains. Total RNA (1 μg) from each strain was subjected to reverse transcription, and the obtained cDNA was amplified using pairs of primers specific for tyrR . ( A ) dadA , ( B ), and gyrA ( C ) A . brasilense strains are indicated at the top of each lane by their conventional denomination. The expected PCR products of 196 bp ( tyrR ), 145 bp ( dadA ) and 143 bp ( gyrA ) were visualized by ethidium bromide staining on a 2.5% agarose gel. A positive control (+), represented by genomic DNA. A negative reaction control is represented by (-), without reverse transcriptase, both were included in each experiment. The image is representative of three independents experiments.
Figure Legend Snippet: RT-PCR analysis of dadA , tyrR and gyrA expression from the A . brasilense Sp7, 2116, and 2117 strains. RT-PCR analysis of tyrR , dadA and gyrA expression from the A . brasilense Sp7, 2116 and 2117 strains. Total RNA (1 μg) from each strain was subjected to reverse transcription, and the obtained cDNA was amplified using pairs of primers specific for tyrR . ( A ) dadA , ( B ), and gyrA ( C ) A . brasilense strains are indicated at the top of each lane by their conventional denomination. The expected PCR products of 196 bp ( tyrR ), 145 bp ( dadA ) and 143 bp ( gyrA ) were visualized by ethidium bromide staining on a 2.5% agarose gel. A positive control (+), represented by genomic DNA. A negative reaction control is represented by (-), without reverse transcriptase, both were included in each experiment. The image is representative of three independents experiments.

Techniques Used: Reverse Transcription Polymerase Chain Reaction, Expressing, Amplification, Polymerase Chain Reaction, Staining, Agarose Gel Electrophoresis, Positive Control

32) Product Images from "Piscine orthoreovirus demonstrates high infectivity but low virulence in Atlantic salmon of Pacific Canada"

Article Title: Piscine orthoreovirus demonstrates high infectivity but low virulence in Atlantic salmon of Pacific Canada

Journal: Scientific Reports

doi: 10.1038/s41598-019-40025-7

The relative proportion of PRV genomic and messenger RNA in blood changes over the time course of infection. ( A) RNA from PRV 16-005ND infected fish at 10 wpc was used to validate differential detection of PRV L1 ssRNA (mRNA) and dsRNA (gRNA) transcripts by qPCR. Total RNA exposed to either no enzyme, RNAse A, or RNAse A in 0.5 M sodium chloride (which selectively protects dsRNA but not ssRNA from RNAse A degradation 60 ) was heated at 55 °C for 5 min, 80 °C for 1 min, or 95 °C for 5 min prior to reverse transcription and 30-cycles of qPCR. PRV dsRNA template was only present following 95 °C denaturation as seen in cropped gel images relative to host β-actin ssRNA (for Ct values see Supplement 1 ). (B) The relative quantities (scaled to the minimum value per time point) of PRV L1 at 2, 4, and 10 wpc for both PRV 16-005ND and 16-011D are compared between pre-amplification denaturation temperatures. The proportion of dsRNA to ssRNA significantly increased at 10 wpc in both PRV challenged groups (*p
Figure Legend Snippet: The relative proportion of PRV genomic and messenger RNA in blood changes over the time course of infection. ( A) RNA from PRV 16-005ND infected fish at 10 wpc was used to validate differential detection of PRV L1 ssRNA (mRNA) and dsRNA (gRNA) transcripts by qPCR. Total RNA exposed to either no enzyme, RNAse A, or RNAse A in 0.5 M sodium chloride (which selectively protects dsRNA but not ssRNA from RNAse A degradation 60 ) was heated at 55 °C for 5 min, 80 °C for 1 min, or 95 °C for 5 min prior to reverse transcription and 30-cycles of qPCR. PRV dsRNA template was only present following 95 °C denaturation as seen in cropped gel images relative to host β-actin ssRNA (for Ct values see Supplement 1 ). (B) The relative quantities (scaled to the minimum value per time point) of PRV L1 at 2, 4, and 10 wpc for both PRV 16-005ND and 16-011D are compared between pre-amplification denaturation temperatures. The proportion of dsRNA to ssRNA significantly increased at 10 wpc in both PRV challenged groups (*p

Techniques Used: Infection, Fluorescence In Situ Hybridization, Real-time Polymerase Chain Reaction, Amplification

PRV sourced from cohorts with and without HSMI-like lesions have high sequence similarity. ( A) RNA-seq read libraries (n = 4) were pooled specific to PRV source material (n = 2) and assembled de novo using Trinity. (B) Concatenated PRV segments are phylogenetically compared to two previously published genomes of PRV (Pacific Canada isolate B5690 8 and Norway isolate V3621 1 ). Bootstrap probabilities (percentages) are provided at branch nodes if less than 100%; scale bar indicates nucleotide substitutions per site. (C) Predicted amino acid sequences are compared in the same manner as nucleotide sequences. (D) The comparative identity between nucleotide (nt) and predicted amino acid (aa) sequences as well as the number of substitutions () per alignment are provided in relation to 16-005ND. Substitutions unique to either 16-005ND (no HSMI) or V3621 (only Norwegian isolate; HSMI) are also indicated.
Figure Legend Snippet: PRV sourced from cohorts with and without HSMI-like lesions have high sequence similarity. ( A) RNA-seq read libraries (n = 4) were pooled specific to PRV source material (n = 2) and assembled de novo using Trinity. (B) Concatenated PRV segments are phylogenetically compared to two previously published genomes of PRV (Pacific Canada isolate B5690 8 and Norway isolate V3621 1 ). Bootstrap probabilities (percentages) are provided at branch nodes if less than 100%; scale bar indicates nucleotide substitutions per site. (C) Predicted amino acid sequences are compared in the same manner as nucleotide sequences. (D) The comparative identity between nucleotide (nt) and predicted amino acid (aa) sequences as well as the number of substitutions () per alignment are provided in relation to 16-005ND. Substitutions unique to either 16-005ND (no HSMI) or V3621 (only Norwegian isolate; HSMI) are also indicated.

Techniques Used: Sequencing, RNA Sequencing Assay

Contrast summary for trends in PRV phenotypic infection dynamics between Norway and Canada laboratory challenge of Atlantic salmon. In comparing the present challenge trials conducted in ( A ) Pacific Canada with results from similar challenge trials conducted in ( B ) Norway 1 , 20 , 21 , 40 , 45 , the kinetics of viral RNA, protein (indirectly measured in Canada by mRNA or directly measured in Norway by florescent antibody staining) and erythrocytic inclusion body formation follow a similar pattern. However, the kinetics regarding the plasma load of PRV, transcriptional induction of genes involved in innate host recognition of virus by Atlantic salmon, as well as the severity and timing of PRV associated heart inflammation appear notably discrete between the two countries. For these comparisons, timing is presented relative to first signs of infection at 10-12 °C and not necessarily the initiation of a challenge since detectable infections take longer to develop following cohabitation than by i.p. injection. It should also be noted that no data are yet available with regard to the transcriptional host innate immune responses or plasma PRV loads beyond 8 weeks post infection in Norwegian based studies and thus their late stage kinetics are unknown and not presented. Lastly, Y-axis scale is not intended to be interpreted as absolute.
Figure Legend Snippet: Contrast summary for trends in PRV phenotypic infection dynamics between Norway and Canada laboratory challenge of Atlantic salmon. In comparing the present challenge trials conducted in ( A ) Pacific Canada with results from similar challenge trials conducted in ( B ) Norway 1 , 20 , 21 , 40 , 45 , the kinetics of viral RNA, protein (indirectly measured in Canada by mRNA or directly measured in Norway by florescent antibody staining) and erythrocytic inclusion body formation follow a similar pattern. However, the kinetics regarding the plasma load of PRV, transcriptional induction of genes involved in innate host recognition of virus by Atlantic salmon, as well as the severity and timing of PRV associated heart inflammation appear notably discrete between the two countries. For these comparisons, timing is presented relative to first signs of infection at 10-12 °C and not necessarily the initiation of a challenge since detectable infections take longer to develop following cohabitation than by i.p. injection. It should also be noted that no data are yet available with regard to the transcriptional host innate immune responses or plasma PRV loads beyond 8 weeks post infection in Norwegian based studies and thus their late stage kinetics are unknown and not presented. Lastly, Y-axis scale is not intended to be interpreted as absolute.

Techniques Used: Infection, Staining, Injection

Expression of PRV segments in host blood is temporally similar but with slight proportional variation. ( A) The relative quantity (scaled to the minimum value) of each PRV RNA segment in the blood of 16-005ND challenged fish were statistically similar at 2, 4 or 10 wpc. (B) The single-stranded mRNA contribution to total PRV load was also similar between segments at each time point. However, (C) the cumulative proportional contribution of L2, L3, and M1 was significantly less, whereas S2 and S4 was significantly more, relative to all other segments independent of time (letters indicate significant groupings at p
Figure Legend Snippet: Expression of PRV segments in host blood is temporally similar but with slight proportional variation. ( A) The relative quantity (scaled to the minimum value) of each PRV RNA segment in the blood of 16-005ND challenged fish were statistically similar at 2, 4 or 10 wpc. (B) The single-stranded mRNA contribution to total PRV load was also similar between segments at each time point. However, (C) the cumulative proportional contribution of L2, L3, and M1 was significantly less, whereas S2 and S4 was significantly more, relative to all other segments independent of time (letters indicate significant groupings at p

Techniques Used: Expressing, Fluorescence In Situ Hybridization

PRV from Atlantic salmon with and without HSMI-like lesions generate extensive and persistent infections in naïve recipients. ( A , B ) PRV was obtained from the blood of donor fish without heart lesions (16-005ND) or ( C , D ) with HSMI-like lesions (16-011D). Lesions in diseased fish included epicarditis (*), endocardial cell hypertrophy (arrows) and small foci of myocardial necrosis (open arrowheads). Black boxes within left images outline the area shown at higher magnification in right images. ( E ) The quantity of L1 transcripts of PRV 16-005ND and 16-011D inoculated into each naïve recipient was estimated by qPCR, and ( F ) the systemic blood load of PRV L1 transcripts was assessed every 7 days in recipient fish (n = 6 per treatment where available). The unique relative qPCR threshold cycle (Ct) associated with each sample (dots) as well as the mean estimated quantity of L1 copies per µg total RNA at each time point (lines) are shown.
Figure Legend Snippet: PRV from Atlantic salmon with and without HSMI-like lesions generate extensive and persistent infections in naïve recipients. ( A , B ) PRV was obtained from the blood of donor fish without heart lesions (16-005ND) or ( C , D ) with HSMI-like lesions (16-011D). Lesions in diseased fish included epicarditis (*), endocardial cell hypertrophy (arrows) and small foci of myocardial necrosis (open arrowheads). Black boxes within left images outline the area shown at higher magnification in right images. ( E ) The quantity of L1 transcripts of PRV 16-005ND and 16-011D inoculated into each naïve recipient was estimated by qPCR, and ( F ) the systemic blood load of PRV L1 transcripts was assessed every 7 days in recipient fish (n = 6 per treatment where available). The unique relative qPCR threshold cycle (Ct) associated with each sample (dots) as well as the mean estimated quantity of L1 copies per µg total RNA at each time point (lines) are shown.

Techniques Used: Fluorescence In Situ Hybridization, Real-time Polymerase Chain Reaction

33) Product Images from "LINC01554-Mediated Glucose Metabolism Reprogramming Suppresses Tumorigenicity in Hepatocellular Carcinoma via Downregulating PKM2 Expression and Inhibiting Akt/mTOR Signaling Pathway"

Article Title: LINC01554-Mediated Glucose Metabolism Reprogramming Suppresses Tumorigenicity in Hepatocellular Carcinoma via Downregulating PKM2 Expression and Inhibiting Akt/mTOR Signaling Pathway

Journal: Theranostics

doi: 10.7150/thno.28992

LINC01554 is frequently downregulated in HCCs. (A) Relative expression level of LINC01554 detected by qRT-PCR in 167 HCC tissues and adjacent non-tumor tissues. Expression of β-actin was used as an internal control. (B) LINC01554 expression fractions in 167 HCC tissues and adjacent non-tumor tissues. (C) Relative expression level of LINC01554 detected by qRT-PCR in an immortalized liver cell line (MIHA) and HCC cell lines. Expression of β-actin was used as internal control. (D) RNA sequencing expression of LINC01554 was analyzed in different cohorts, including Lee's cohort, Llovet's cohort, Wurmbach's cohort in oncomine ( https://www.oncomine.org ), and TCGA database. (E) Nuclear and cytoplasmic fractions of LINC01554 in MIHA cells and SMMC7721 cells determined by qRT-PCR with U6 or β-actin as a nuclear or cytoplasmic internal control. (F) Representative images of subcellular localization of LINC01554 (red) in MIHA cells, HCC tissues and adjacent non-tumor tissues detected by RNA FISH. Nuclei were stained with DAPI (blue). Scale bar, 20 μm in image of MIHA cells and 50 μm in images of adjacent non-tumor and HCC tissues. (G) Kaplan-Meier analysis revealed that low expression of LINC01554 was related to poorer overall survival of HCC patients in both SYSUCC cohort (n = 167, P = 0.035) and TCGA database (n = 364, P
Figure Legend Snippet: LINC01554 is frequently downregulated in HCCs. (A) Relative expression level of LINC01554 detected by qRT-PCR in 167 HCC tissues and adjacent non-tumor tissues. Expression of β-actin was used as an internal control. (B) LINC01554 expression fractions in 167 HCC tissues and adjacent non-tumor tissues. (C) Relative expression level of LINC01554 detected by qRT-PCR in an immortalized liver cell line (MIHA) and HCC cell lines. Expression of β-actin was used as internal control. (D) RNA sequencing expression of LINC01554 was analyzed in different cohorts, including Lee's cohort, Llovet's cohort, Wurmbach's cohort in oncomine ( https://www.oncomine.org ), and TCGA database. (E) Nuclear and cytoplasmic fractions of LINC01554 in MIHA cells and SMMC7721 cells determined by qRT-PCR with U6 or β-actin as a nuclear or cytoplasmic internal control. (F) Representative images of subcellular localization of LINC01554 (red) in MIHA cells, HCC tissues and adjacent non-tumor tissues detected by RNA FISH. Nuclei were stained with DAPI (blue). Scale bar, 20 μm in image of MIHA cells and 50 μm in images of adjacent non-tumor and HCC tissues. (G) Kaplan-Meier analysis revealed that low expression of LINC01554 was related to poorer overall survival of HCC patients in both SYSUCC cohort (n = 167, P = 0.035) and TCGA database (n = 364, P

Techniques Used: Expressing, Quantitative RT-PCR, RNA Sequencing Assay, Fluorescence In Situ Hybridization, Staining

34) Product Images from "More than just inflammation: Ureaplasma species induce apoptosis in human brain microvascular endothelial cells"

Article Title: More than just inflammation: Ureaplasma species induce apoptosis in human brain microvascular endothelial cells

Journal: Journal of Neuroinflammation

doi: 10.1186/s12974-019-1413-8

Ureaplasma -driven pyroptosis in HBMEC. Key genes in pyroptosis (Fig. 1 ) were assessed for mRNA responses upon stimulation of HBMEC for 4 h and 30 h. Caspase 1 mRNA expression was determined via RNA sequencing ( a ) and qRT-PCR ( b ). Similarly, RNA sequencing ( c ) and qRT-PCR ( d ) were used to assess caspase 4 mRNA levels. RNA sequencing furthermore determined mRNA expression of NLRP3 ( e ) and gasdermin D ( f ). Data are presented as means ± SD (* p
Figure Legend Snippet: Ureaplasma -driven pyroptosis in HBMEC. Key genes in pyroptosis (Fig. 1 ) were assessed for mRNA responses upon stimulation of HBMEC for 4 h and 30 h. Caspase 1 mRNA expression was determined via RNA sequencing ( a ) and qRT-PCR ( b ). Similarly, RNA sequencing ( c ) and qRT-PCR ( d ) were used to assess caspase 4 mRNA levels. RNA sequencing furthermore determined mRNA expression of NLRP3 ( e ) and gasdermin D ( f ). Data are presented as means ± SD (* p

Techniques Used: Expressing, RNA Sequencing Assay, Quantitative RT-PCR

Ureaplasma -driven apoptosis in HBMEC. Enzymes and other proteins involved in the apoptotic cascade (Fig. 1 ) were analyzed upon stimulation of HBMEC for 4 h, 24 h, or 30 h. For caspase 3, mRNA expression was determined via RNA sequencing ( a ) and qRT-PCR ( b ) and enzyme activity (cleaved caspase 3) was assessed via flow cytometry ( c ). For caspase 8, RNA sequencing ( d ) and qRT-PCR ( e ) were used to evaluate mRNA levels and flow cytometry ( f ) was employed to determine protein expression. Caspase 9 mRNA levels were also assessed via RNA sequencing ( g ) and qRT-PCR ( h ), and levels of active caspase 9 were determined using flow cytometry ( i ). RNA sequencing was used to assess mRNA expression of caspase 7 ( j ), BAK ( k ), BAX ( l ), p53 ( m ), FOS ( n ), and APAF1 ( o ). Data are shown as means ± SD (* p
Figure Legend Snippet: Ureaplasma -driven apoptosis in HBMEC. Enzymes and other proteins involved in the apoptotic cascade (Fig. 1 ) were analyzed upon stimulation of HBMEC for 4 h, 24 h, or 30 h. For caspase 3, mRNA expression was determined via RNA sequencing ( a ) and qRT-PCR ( b ) and enzyme activity (cleaved caspase 3) was assessed via flow cytometry ( c ). For caspase 8, RNA sequencing ( d ) and qRT-PCR ( e ) were used to evaluate mRNA levels and flow cytometry ( f ) was employed to determine protein expression. Caspase 9 mRNA levels were also assessed via RNA sequencing ( g ) and qRT-PCR ( h ), and levels of active caspase 9 were determined using flow cytometry ( i ). RNA sequencing was used to assess mRNA expression of caspase 7 ( j ), BAK ( k ), BAX ( l ), p53 ( m ), FOS ( n ), and APAF1 ( o ). Data are shown as means ± SD (* p

Techniques Used: Expressing, RNA Sequencing Assay, Quantitative RT-PCR, Activity Assay, Flow Cytometry, Cytometry

35) Product Images from "A genetic variant associated with multiple sclerosis inversely affects the expression of CD58 and microRNA-548ac from the same gene"

Article Title: A genetic variant associated with multiple sclerosis inversely affects the expression of CD58 and microRNA-548ac from the same gene

Journal: PLoS Genetics

doi: 10.1371/journal.pgen.1007961

eQTL analysis of CD58 and microRNA-548ac based on three different data sets. Expression values of CD58 mRNA (labeled in green) and hsa-miR-548ac molecules (labeled in red) measured using microarrays ( A ), RNA-sequencing ( B ), and quantitative real-time PCR ( C ) were plotted for each genotype group. Genotypes 0, 1, and 2 denote the number of MS risk alleles carried, defined either by SNP rs1335532 ( A ) or SNP rs1414273 ( B and C ). The average expression level per group is indicated by a red line. Welch t -test p -values are shown above the brackets for all pairwise genotype comparisons. ( A ) HapMap cohort data (in log2 scale) demonstrated a significant relationship between the MS-associated SNP and CD58 transcript levels in independent populations (n = 82 JPT and n = 82 GIH displayed). ( B ) This could be confirmed by Geuvadis cohort data, presented here for LCLs collected from 282 individuals living in Europe. Interestingly, the eQTL effect is in the opposite direction for hsa-miR-548ac: Significantly higher levels of this miRNA were seen in individuals with increased genetic risk of MS. Numbers below the data points specify the proportion of samples (of n = 276 analyzed LCLs) with zero miRNA read counts. ( C ) Data of the regional MS cohort (n = 32) further substantiated the differences in miRNA levels among the genotypes. A non-significant positive correlation of CD58 mRNA and hsa-miR-548ac expression was found in both the RNA-sequencing data ( B ) and the real-time PCR data ( C ). ( D ) The table gives the F -test p -values calculated for the complete data of the HapMap and Geuvadis cohorts, establishing the cis -mRNA-/miR-eQTL when accounting for population structure in the analysis of covariance (ANCOVA). ANOVA = analysis of variance, eQTL = expression quantitative trait locus, FIN = Finnish in Finland, GBR = British in England and Scotland, GIH = Gujarati Indians in Houston, JPT = Japanese in Tokyo, LCL = lymphoblastoid cell line, MS = multiple sclerosis, PBMC = peripheral blood mononuclear cells, PCR = polymerase chain reaction, SLR = simple linear regression, SNP = single-nucleotide polymorphism, TSI = Toscani in Italia.
Figure Legend Snippet: eQTL analysis of CD58 and microRNA-548ac based on three different data sets. Expression values of CD58 mRNA (labeled in green) and hsa-miR-548ac molecules (labeled in red) measured using microarrays ( A ), RNA-sequencing ( B ), and quantitative real-time PCR ( C ) were plotted for each genotype group. Genotypes 0, 1, and 2 denote the number of MS risk alleles carried, defined either by SNP rs1335532 ( A ) or SNP rs1414273 ( B and C ). The average expression level per group is indicated by a red line. Welch t -test p -values are shown above the brackets for all pairwise genotype comparisons. ( A ) HapMap cohort data (in log2 scale) demonstrated a significant relationship between the MS-associated SNP and CD58 transcript levels in independent populations (n = 82 JPT and n = 82 GIH displayed). ( B ) This could be confirmed by Geuvadis cohort data, presented here for LCLs collected from 282 individuals living in Europe. Interestingly, the eQTL effect is in the opposite direction for hsa-miR-548ac: Significantly higher levels of this miRNA were seen in individuals with increased genetic risk of MS. Numbers below the data points specify the proportion of samples (of n = 276 analyzed LCLs) with zero miRNA read counts. ( C ) Data of the regional MS cohort (n = 32) further substantiated the differences in miRNA levels among the genotypes. A non-significant positive correlation of CD58 mRNA and hsa-miR-548ac expression was found in both the RNA-sequencing data ( B ) and the real-time PCR data ( C ). ( D ) The table gives the F -test p -values calculated for the complete data of the HapMap and Geuvadis cohorts, establishing the cis -mRNA-/miR-eQTL when accounting for population structure in the analysis of covariance (ANCOVA). ANOVA = analysis of variance, eQTL = expression quantitative trait locus, FIN = Finnish in Finland, GBR = British in England and Scotland, GIH = Gujarati Indians in Houston, JPT = Japanese in Tokyo, LCL = lymphoblastoid cell line, MS = multiple sclerosis, PBMC = peripheral blood mononuclear cells, PCR = polymerase chain reaction, SLR = simple linear regression, SNP = single-nucleotide polymorphism, TSI = Toscani in Italia.

Techniques Used: Expressing, Labeling, RNA Sequencing Assay, Real-time Polymerase Chain Reaction, Mass Spectrometry, Polymerase Chain Reaction

Biogenesis of microRNA-548ac and genetic variants in the CD58 gene locus. ( A ) Diagram of the processing of an mRNA and an intronic miRNA from the same transcript (adapted from [ 49 ]). Both RNA splicing by the spliceosome and miRNA stem-loop cropping by the Drosha-DGCR8 complex occur cotranscriptionally. Drosha may cleave the miRNA-harboring intron before splicing commitment of the flanking exons. The resulting precursor miRNA is subsequently processed into a mature miRNA, which is loaded into the RNA-induced silencing complex (RISC). ( B ) Annotated secondary structure of hsa-mir-548ac. Highlighted in gray is the 22 nt long sequence of the mature miRNA isoform as assigned by Jima et al . (miRBase accession MIMAT0018938) [ 43 ]. The red circle marks the only common single-nucleotide polymorphism (SNP) within the stem-loop region. The G allele is overrepresented in MS patients. ( C ) Genetic variants in pairwise linkage disequilibrium (LD) with SNP rs1414273. This plot was generated using the LDproxy module of the web-based analysis tool LDlink [ 97 ]. Shown are r 2 LD values of proximal SNPs based on all subpopulations of the 1000 Genomes project (orange dots), recombination rate as estimated from HapMap data (gray line), and the position and exon-intron structure of nearby genes on chromosome 1 (chr1, GRCh37 assembly). The MS-associated SNP rs1335532 is in strong LD with SNP rs1414273 (blue) (correlated forward strand alleles: A = C, G = T). The entire block of LD spans about 50 kb but does not include the promoter region of CD58, which is encoded on the minus strand in the reference genome. ( D ) Worldwide distribution of SNP rs1335532 alleles. Global allele frequencies were visualized as two-color pie charts with the HGDP Selection Browser [ 98 ]. The disease susceptibility variant (A, blue) is the major allele in European populations and the minor allele in East Asian and Southern African populations. cM/Mb = centimorgan per megabase, HGDP = Human Genome Diversity Panel, MS = multiple sclerosis.
Figure Legend Snippet: Biogenesis of microRNA-548ac and genetic variants in the CD58 gene locus. ( A ) Diagram of the processing of an mRNA and an intronic miRNA from the same transcript (adapted from [ 49 ]). Both RNA splicing by the spliceosome and miRNA stem-loop cropping by the Drosha-DGCR8 complex occur cotranscriptionally. Drosha may cleave the miRNA-harboring intron before splicing commitment of the flanking exons. The resulting precursor miRNA is subsequently processed into a mature miRNA, which is loaded into the RNA-induced silencing complex (RISC). ( B ) Annotated secondary structure of hsa-mir-548ac. Highlighted in gray is the 22 nt long sequence of the mature miRNA isoform as assigned by Jima et al . (miRBase accession MIMAT0018938) [ 43 ]. The red circle marks the only common single-nucleotide polymorphism (SNP) within the stem-loop region. The G allele is overrepresented in MS patients. ( C ) Genetic variants in pairwise linkage disequilibrium (LD) with SNP rs1414273. This plot was generated using the LDproxy module of the web-based analysis tool LDlink [ 97 ]. Shown are r 2 LD values of proximal SNPs based on all subpopulations of the 1000 Genomes project (orange dots), recombination rate as estimated from HapMap data (gray line), and the position and exon-intron structure of nearby genes on chromosome 1 (chr1, GRCh37 assembly). The MS-associated SNP rs1335532 is in strong LD with SNP rs1414273 (blue) (correlated forward strand alleles: A = C, G = T). The entire block of LD spans about 50 kb but does not include the promoter region of CD58, which is encoded on the minus strand in the reference genome. ( D ) Worldwide distribution of SNP rs1335532 alleles. Global allele frequencies were visualized as two-color pie charts with the HGDP Selection Browser [ 98 ]. The disease susceptibility variant (A, blue) is the major allele in European populations and the minor allele in East Asian and Southern African populations. cM/Mb = centimorgan per megabase, HGDP = Human Genome Diversity Panel, MS = multiple sclerosis.

Techniques Used: Sequencing, Mass Spectrometry, Generated, Blocking Assay, Selection, Variant Assay

36) Product Images from "A genetic variant associated with multiple sclerosis inversely affects the expression of CD58 and microRNA-548ac from the same gene"

Article Title: A genetic variant associated with multiple sclerosis inversely affects the expression of CD58 and microRNA-548ac from the same gene

Journal: PLoS Genetics

doi: 10.1371/journal.pgen.1007961

eQTL analysis of CD58 and microRNA-548ac based on three different data sets. Expression values of CD58 mRNA (labeled in green) and hsa-miR-548ac molecules (labeled in red) measured using microarrays ( A ), RNA-sequencing ( B ), and quantitative real-time PCR ( C ) were plotted for each genotype group. Genotypes 0, 1, and 2 denote the number of MS risk alleles carried, defined either by SNP rs1335532 ( A ) or SNP rs1414273 ( B and C ). The average expression level per group is indicated by a red line. Welch t -test p -values are shown above the brackets for all pairwise genotype comparisons. ( A ) HapMap cohort data (in log2 scale) demonstrated a significant relationship between the MS-associated SNP and CD58 transcript levels in independent populations (n = 82 JPT and n = 82 GIH displayed). ( B ) This could be confirmed by Geuvadis cohort data, presented here for LCLs collected from 282 individuals living in Europe. Interestingly, the eQTL effect is in the opposite direction for hsa-miR-548ac: Significantly higher levels of this miRNA were seen in individuals with increased genetic risk of MS. Numbers below the data points specify the proportion of samples (of n = 276 analyzed LCLs) with zero miRNA read counts. ( C ) Data of the regional MS cohort (n = 32) further substantiated the differences in miRNA levels among the genotypes. A non-significant positive correlation of CD58 mRNA and hsa-miR-548ac expression was found in both the RNA-sequencing data ( B ) and the real-time PCR data ( C ). ( D ) The table gives the F -test p -values calculated for the complete data of the HapMap and Geuvadis cohorts, establishing the cis -mRNA-/miR-eQTL when accounting for population structure in the analysis of covariance (ANCOVA). ANOVA = analysis of variance, eQTL = expression quantitative trait locus, FIN = Finnish in Finland, GBR = British in England and Scotland, GIH = Gujarati Indians in Houston, JPT = Japanese in Tokyo, LCL = lymphoblastoid cell line, MS = multiple sclerosis, PBMC = peripheral blood mononuclear cells, PCR = polymerase chain reaction, SLR = simple linear regression, SNP = single-nucleotide polymorphism, TSI = Toscani in Italia.
Figure Legend Snippet: eQTL analysis of CD58 and microRNA-548ac based on three different data sets. Expression values of CD58 mRNA (labeled in green) and hsa-miR-548ac molecules (labeled in red) measured using microarrays ( A ), RNA-sequencing ( B ), and quantitative real-time PCR ( C ) were plotted for each genotype group. Genotypes 0, 1, and 2 denote the number of MS risk alleles carried, defined either by SNP rs1335532 ( A ) or SNP rs1414273 ( B and C ). The average expression level per group is indicated by a red line. Welch t -test p -values are shown above the brackets for all pairwise genotype comparisons. ( A ) HapMap cohort data (in log2 scale) demonstrated a significant relationship between the MS-associated SNP and CD58 transcript levels in independent populations (n = 82 JPT and n = 82 GIH displayed). ( B ) This could be confirmed by Geuvadis cohort data, presented here for LCLs collected from 282 individuals living in Europe. Interestingly, the eQTL effect is in the opposite direction for hsa-miR-548ac: Significantly higher levels of this miRNA were seen in individuals with increased genetic risk of MS. Numbers below the data points specify the proportion of samples (of n = 276 analyzed LCLs) with zero miRNA read counts. ( C ) Data of the regional MS cohort (n = 32) further substantiated the differences in miRNA levels among the genotypes. A non-significant positive correlation of CD58 mRNA and hsa-miR-548ac expression was found in both the RNA-sequencing data ( B ) and the real-time PCR data ( C ). ( D ) The table gives the F -test p -values calculated for the complete data of the HapMap and Geuvadis cohorts, establishing the cis -mRNA-/miR-eQTL when accounting for population structure in the analysis of covariance (ANCOVA). ANOVA = analysis of variance, eQTL = expression quantitative trait locus, FIN = Finnish in Finland, GBR = British in England and Scotland, GIH = Gujarati Indians in Houston, JPT = Japanese in Tokyo, LCL = lymphoblastoid cell line, MS = multiple sclerosis, PBMC = peripheral blood mononuclear cells, PCR = polymerase chain reaction, SLR = simple linear regression, SNP = single-nucleotide polymorphism, TSI = Toscani in Italia.

Techniques Used: Expressing, Labeling, RNA Sequencing Assay, Real-time Polymerase Chain Reaction, Mass Spectrometry, Polymerase Chain Reaction

Biogenesis of microRNA-548ac and genetic variants in the CD58 gene locus. ( A ) Diagram of the processing of an mRNA and an intronic miRNA from the same transcript (adapted from [ 49 ]). Both RNA splicing by the spliceosome and miRNA stem-loop cropping by the Drosha-DGCR8 complex occur cotranscriptionally. Drosha may cleave the miRNA-harboring intron before splicing commitment of the flanking exons. The resulting precursor miRNA is subsequently processed into a mature miRNA, which is loaded into the RNA-induced silencing complex (RISC). ( B ) Annotated secondary structure of hsa-mir-548ac. Highlighted in gray is the 22 nt long sequence of the mature miRNA isoform as assigned by Jima et al . (miRBase accession MIMAT0018938) [ 43 ]. The red circle marks the only common single-nucleotide polymorphism (SNP) within the stem-loop region. The G allele is overrepresented in MS patients. ( C ) Genetic variants in pairwise linkage disequilibrium (LD) with SNP rs1414273. This plot was generated using the LDproxy module of the web-based analysis tool LDlink [ 97 ]. Shown are r 2 LD values of proximal SNPs based on all subpopulations of the 1000 Genomes project (orange dots), recombination rate as estimated from HapMap data (gray line), and the position and exon-intron structure of nearby genes on chromosome 1 (chr1, GRCh37 assembly). The MS-associated SNP rs1335532 is in strong LD with SNP rs1414273 (blue) (correlated forward strand alleles: A = C, G = T). The entire block of LD spans about 50 kb but does not include the promoter region of CD58, which is encoded on the minus strand in the reference genome. ( D ) Worldwide distribution of SNP rs1335532 alleles. Global allele frequencies were visualized as two-color pie charts with the HGDP Selection Browser [ 98 ]. The disease susceptibility variant (A, blue) is the major allele in European populations and the minor allele in East Asian and Southern African populations. cM/Mb = centimorgan per megabase, HGDP = Human Genome Diversity Panel, MS = multiple sclerosis.
Figure Legend Snippet: Biogenesis of microRNA-548ac and genetic variants in the CD58 gene locus. ( A ) Diagram of the processing of an mRNA and an intronic miRNA from the same transcript (adapted from [ 49 ]). Both RNA splicing by the spliceosome and miRNA stem-loop cropping by the Drosha-DGCR8 complex occur cotranscriptionally. Drosha may cleave the miRNA-harboring intron before splicing commitment of the flanking exons. The resulting precursor miRNA is subsequently processed into a mature miRNA, which is loaded into the RNA-induced silencing complex (RISC). ( B ) Annotated secondary structure of hsa-mir-548ac. Highlighted in gray is the 22 nt long sequence of the mature miRNA isoform as assigned by Jima et al . (miRBase accession MIMAT0018938) [ 43 ]. The red circle marks the only common single-nucleotide polymorphism (SNP) within the stem-loop region. The G allele is overrepresented in MS patients. ( C ) Genetic variants in pairwise linkage disequilibrium (LD) with SNP rs1414273. This plot was generated using the LDproxy module of the web-based analysis tool LDlink [ 97 ]. Shown are r 2 LD values of proximal SNPs based on all subpopulations of the 1000 Genomes project (orange dots), recombination rate as estimated from HapMap data (gray line), and the position and exon-intron structure of nearby genes on chromosome 1 (chr1, GRCh37 assembly). The MS-associated SNP rs1335532 is in strong LD with SNP rs1414273 (blue) (correlated forward strand alleles: A = C, G = T). The entire block of LD spans about 50 kb but does not include the promoter region of CD58, which is encoded on the minus strand in the reference genome. ( D ) Worldwide distribution of SNP rs1335532 alleles. Global allele frequencies were visualized as two-color pie charts with the HGDP Selection Browser [ 98 ]. The disease susceptibility variant (A, blue) is the major allele in European populations and the minor allele in East Asian and Southern African populations. cM/Mb = centimorgan per megabase, HGDP = Human Genome Diversity Panel, MS = multiple sclerosis.

Techniques Used: Sequencing, Mass Spectrometry, Generated, Blocking Assay, Selection, Variant Assay

37) Product Images from "Conserved motifs in the hypervariable domain of chikungunya virus nsP3 required for transmission by Aedes aegypti mosquitoes"

Article Title: Conserved motifs in the hypervariable domain of chikungunya virus nsP3 required for transmission by Aedes aegypti mosquitoes

Journal: PLoS Neglected Tropical Diseases

doi: 10.1371/journal.pntd.0006958

At least one FGDF motif is required for the interaction of nsP3 with G3BP and mosquito Rin. (A) Schematic overview of the used plasmids expressing Rasputin (Rin) fused to EGFP Rin-EGFP or mCherry Rin-mC and CHIKV nsP3 EGFP fusion proteins of wild type and FGDF single- and double-mutant nsP3. CMV cytomegalovirus promoter; PUB Aedes aegypti poly-ubiquitin promoter; NTF2 nuclear transport factor 2-like domain; RRM RNA recognition motif; RGG arginine glycine rich region; HVD hypervariable domain. (B) Vero cells were infected with CHIK IC nsP3mC, CHIK IC nsP3mC-FG N or CHIK IC nsP3mC-FG C . At 24 hours post infection (hpi) cells were fixed, permeabilized, stained with α-G3BP and visualized by fluorescent microscopy. (C) Aag2 cells were transfected with pPUB-Rin-EGFP and at 24 hours post transfection (hpt) cells were infected with CHIK IC nsP3mC, CHIK IC nsP3mC-FG N or CHIK IC nsP3mC-FG C . At 24 hpi cells were fixed and visualized by fluorescence microscopy. (D) Vero cells were transfected with CMV driven plasmids expressing EGFP, nsP3EGFP, nsP3EGFP-FG C , nsP3EGFP-FG N or nsP3EGFP-FG NC . At 24 hpt cells were lysed and lysates were subjected to co-immunoprecipitation with α-GFP beads. Lysates and co-precipitates were subjected to western blot with α-G3BP and α-GFP antibodies. (E) Aag2 cells were transfected with PUB driven plasmids expressing EGFP, nsP3EGFP, nsP3EGFP-FG N , nsP3EGFP-FG C or nsP3EGFP-FG NC and co-transfected with pPUB-Rin-mC. At 24 hpt cells were lysed and lysates were subjected to co-immunoprecipitation with α-GFP beads. Lysates and co-precipitates were subjected to western blot with α-GFP and α-mCherry antibodies.
Figure Legend Snippet: At least one FGDF motif is required for the interaction of nsP3 with G3BP and mosquito Rin. (A) Schematic overview of the used plasmids expressing Rasputin (Rin) fused to EGFP Rin-EGFP or mCherry Rin-mC and CHIKV nsP3 EGFP fusion proteins of wild type and FGDF single- and double-mutant nsP3. CMV cytomegalovirus promoter; PUB Aedes aegypti poly-ubiquitin promoter; NTF2 nuclear transport factor 2-like domain; RRM RNA recognition motif; RGG arginine glycine rich region; HVD hypervariable domain. (B) Vero cells were infected with CHIK IC nsP3mC, CHIK IC nsP3mC-FG N or CHIK IC nsP3mC-FG C . At 24 hours post infection (hpi) cells were fixed, permeabilized, stained with α-G3BP and visualized by fluorescent microscopy. (C) Aag2 cells were transfected with pPUB-Rin-EGFP and at 24 hours post transfection (hpt) cells were infected with CHIK IC nsP3mC, CHIK IC nsP3mC-FG N or CHIK IC nsP3mC-FG C . At 24 hpi cells were fixed and visualized by fluorescence microscopy. (D) Vero cells were transfected with CMV driven plasmids expressing EGFP, nsP3EGFP, nsP3EGFP-FG C , nsP3EGFP-FG N or nsP3EGFP-FG NC . At 24 hpt cells were lysed and lysates were subjected to co-immunoprecipitation with α-GFP beads. Lysates and co-precipitates were subjected to western blot with α-G3BP and α-GFP antibodies. (E) Aag2 cells were transfected with PUB driven plasmids expressing EGFP, nsP3EGFP, nsP3EGFP-FG N , nsP3EGFP-FG C or nsP3EGFP-FG NC and co-transfected with pPUB-Rin-mC. At 24 hpt cells were lysed and lysates were subjected to co-immunoprecipitation with α-GFP beads. Lysates and co-precipitates were subjected to western blot with α-GFP and α-mCherry antibodies.

Techniques Used: Expressing, Mutagenesis, Infection, Staining, Microscopy, Transfection, Fluorescence, Immunoprecipitation, Western Blot

At least one FGDF motif is required for chikungunya virus replication in mammalian and mosquito cells. (A) Vero and C6/36 cells were transfected with in vitro transcribed RNA of CHIK IC nsP3mC, CHIK IC nsP3mC-FG N , CHIK IC nsP3mC-FG C , or CHIK IC nsP3mC-FG NC . Cells were fixed at 36 hours post transfection, stained with Hoechst, and fluorescence was observed by fluorescence microscopy. (B) Vero cells were transfected with in vitro transcribed RNA of CHIK IC nsP3mC or CHIK IC nsP3-FG NC either individually or co-transfected with in vitro transcribed RNA of CHIK IC . Cells were fixed at 36 hpt, stained with Hoechst and fluorescence was observed by fluorescence microscopy. (C) Vero and C6/36 cells were transfected with in vitro transcribed RNA of CHIK rep or CHIK rep -FG NC and the relative luciferase expression was quantified at 24 hpt. Bars indicate the mean relative light units (RLU) ±SEM, normalized to the wild type replicon from at least three independent experiments. (D) Growth curves of CHIKV IC , CHIKV IC -FG N and CHIKV IC -FG C on Vero cells infected in duplicate with an MOI of 0.01 based on end-point dilution assay (EPDA) on Vero cells. At the indicated time-points the TCID 50 /ml was determined by EPDA on Vero cells. (E) Growth curves of CHIKV IC , CHIKV IC -FG N and CHIKV IC -FG C on Aag2 cells infected in duplicate with an MOI of 0.01 based on infectivity on Aag2 cells. At the indicated time-points the TCID 50 /ml was determined by EPDA on Aag2 cells. Statistics were performed by one-way ANOVA with Tukey’s post-hoc test on Log 10 transformed data at each time-point (α = 0.05). Asterisks indicate significance compared to the wild type virus. The dotted line in panels D-E indicates the EPDA detection limit.
Figure Legend Snippet: At least one FGDF motif is required for chikungunya virus replication in mammalian and mosquito cells. (A) Vero and C6/36 cells were transfected with in vitro transcribed RNA of CHIK IC nsP3mC, CHIK IC nsP3mC-FG N , CHIK IC nsP3mC-FG C , or CHIK IC nsP3mC-FG NC . Cells were fixed at 36 hours post transfection, stained with Hoechst, and fluorescence was observed by fluorescence microscopy. (B) Vero cells were transfected with in vitro transcribed RNA of CHIK IC nsP3mC or CHIK IC nsP3-FG NC either individually or co-transfected with in vitro transcribed RNA of CHIK IC . Cells were fixed at 36 hpt, stained with Hoechst and fluorescence was observed by fluorescence microscopy. (C) Vero and C6/36 cells were transfected with in vitro transcribed RNA of CHIK rep or CHIK rep -FG NC and the relative luciferase expression was quantified at 24 hpt. Bars indicate the mean relative light units (RLU) ±SEM, normalized to the wild type replicon from at least three independent experiments. (D) Growth curves of CHIKV IC , CHIKV IC -FG N and CHIKV IC -FG C on Vero cells infected in duplicate with an MOI of 0.01 based on end-point dilution assay (EPDA) on Vero cells. At the indicated time-points the TCID 50 /ml was determined by EPDA on Vero cells. (E) Growth curves of CHIKV IC , CHIKV IC -FG N and CHIKV IC -FG C on Aag2 cells infected in duplicate with an MOI of 0.01 based on infectivity on Aag2 cells. At the indicated time-points the TCID 50 /ml was determined by EPDA on Aag2 cells. Statistics were performed by one-way ANOVA with Tukey’s post-hoc test on Log 10 transformed data at each time-point (α = 0.05). Asterisks indicate significance compared to the wild type virus. The dotted line in panels D-E indicates the EPDA detection limit.

Techniques Used: Transfection, In Vitro, Staining, Fluorescence, Microscopy, Luciferase, Expressing, Infection, End-point Dilution Assay, Transformation Assay

The P-rich motif is important but not essential for chikungunya virus replication in mammalian and mosquito cells. (A) Vero and Aag2 cells were transfected with in vitro transcribed RNA of CHIK rep or CHIK rep ΔPVA and the relative luciferase expression was quantified at 24 hpt. Bars indicate the mean relative light units (RLU) ±standard error of the mean from three independent experiments. (B/C) Vero (B) and Aag2 (C) cells were infected in duplicate with CHIK IC , CHIK IC P398A or CHIK IC PPR401AAA at a multiplicity of infection (MOI) of 0.01. TCID 50 /ml was determined by EPDA on Vero cells at the indicated time-points. (D) Aag2 cells were infected in duplicate with CHIK IC , CHIK IC P398A or CHIK IC PPR401AAA at an MOI of 5 and the TCID 50 /ml was determined by EPDA on Vero cells at the indicated time-points. Asterisks indicate significant differences compared to the wild type virus by one-way ANOVA with Tukey’s post-hoc test on Log 10 transformed data at each time-point (α = 0.05). The dotted line in panels B-D indicates the EPDA detection limit.
Figure Legend Snippet: The P-rich motif is important but not essential for chikungunya virus replication in mammalian and mosquito cells. (A) Vero and Aag2 cells were transfected with in vitro transcribed RNA of CHIK rep or CHIK rep ΔPVA and the relative luciferase expression was quantified at 24 hpt. Bars indicate the mean relative light units (RLU) ±standard error of the mean from three independent experiments. (B/C) Vero (B) and Aag2 (C) cells were infected in duplicate with CHIK IC , CHIK IC P398A or CHIK IC PPR401AAA at a multiplicity of infection (MOI) of 0.01. TCID 50 /ml was determined by EPDA on Vero cells at the indicated time-points. (D) Aag2 cells were infected in duplicate with CHIK IC , CHIK IC P398A or CHIK IC PPR401AAA at an MOI of 5 and the TCID 50 /ml was determined by EPDA on Vero cells at the indicated time-points. Asterisks indicate significant differences compared to the wild type virus by one-way ANOVA with Tukey’s post-hoc test on Log 10 transformed data at each time-point (α = 0.05). The dotted line in panels B-D indicates the EPDA detection limit.

Techniques Used: Transfection, In Vitro, Luciferase, Expressing, Infection, End-point Dilution Assay, Transformation Assay

38) Product Images from "Small RNA sequences derived from pre-microRNAs in the supraspliceosome"

Article Title: Small RNA sequences derived from pre-microRNAs in the supraspliceosome

Journal: Nucleic Acids Research

doi: 10.1093/nar/gky791

Regulation of gene expression by nuclear miR-7704. ( A ) Genome browser view for miR-7704 and the overlap with HOXD1 and HAGLR, a long non-coding RNA gene. ( B ) Average expression levels of miR-7704 from the SF based on HeLa (six samples, blue) and MCF-10A cells (three samples, orange). The expression values are based on normalizing each sample to miRNA read counts (100k per sample). The statistical bars are the calculated standard errors. ( C ) Comparison of the results of RT-PCR assays of HOXD1 and HAGLR expression in HeLa and mammary epithelial MCF-10A cells. Results represent RT-PCR from three biological repeats. The identity of the extracted bands was confirmed by DNA sequencing. (D–F) Effect of down-regulation of miR-7704 on HAGLR expression in HeLa cells. Transfection with Anti-miR-7704 inhibitor resulted in down-regulation of miR-7704 ( D ), increase in the expression level of HAGLR mRNA ( E ), and mild elevation of HOXD1 ( F ). (G–I) Effect of down-regulation of miR-7704 on HAGLR expression in MCF-10A cells. Transfection with Anti-miR-7704 inhibitor resulted in down-regulation of miR-7704 ( G ), increase in the expression level of HAGLR mRNA ( H ) and mild elevation of HOXD1 ( I ). D–I, each, show results of qPCR of three independent biological preparations. The expression levels of HAGLR and HOXD1 were normalized to β-actin expression from the same preparation.
Figure Legend Snippet: Regulation of gene expression by nuclear miR-7704. ( A ) Genome browser view for miR-7704 and the overlap with HOXD1 and HAGLR, a long non-coding RNA gene. ( B ) Average expression levels of miR-7704 from the SF based on HeLa (six samples, blue) and MCF-10A cells (three samples, orange). The expression values are based on normalizing each sample to miRNA read counts (100k per sample). The statistical bars are the calculated standard errors. ( C ) Comparison of the results of RT-PCR assays of HOXD1 and HAGLR expression in HeLa and mammary epithelial MCF-10A cells. Results represent RT-PCR from three biological repeats. The identity of the extracted bands was confirmed by DNA sequencing. (D–F) Effect of down-regulation of miR-7704 on HAGLR expression in HeLa cells. Transfection with Anti-miR-7704 inhibitor resulted in down-regulation of miR-7704 ( D ), increase in the expression level of HAGLR mRNA ( E ), and mild elevation of HOXD1 ( F ). (G–I) Effect of down-regulation of miR-7704 on HAGLR expression in MCF-10A cells. Transfection with Anti-miR-7704 inhibitor resulted in down-regulation of miR-7704 ( G ), increase in the expression level of HAGLR mRNA ( H ) and mild elevation of HOXD1 ( I ). D–I, each, show results of qPCR of three independent biological preparations. The expression levels of HAGLR and HOXD1 were normalized to β-actin expression from the same preparation.

Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction, DNA Sequencing, Transfection, Real-time Polymerase Chain Reaction

39) Product Images from "USP14 as a novel prognostic marker promotes cisplatin resistance via Akt/ ERK signaling pathways in gastric cancer, et al. USP14 as a novel prognostic marker promotes cisplatin resistance via Akt/ERK signaling pathways in gastric cancer"

Article Title: USP14 as a novel prognostic marker promotes cisplatin resistance via Akt/ ERK signaling pathways in gastric cancer, et al. USP14 as a novel prognostic marker promotes cisplatin resistance via Akt/ERK signaling pathways in gastric cancer

Journal: Cancer Medicine

doi: 10.1002/cam4.1770

Suppressing USP 14 expression sensitized GC cells to cisplatin in vitro. A, B, USP 14 has high expression at both mRNA and protein level in the indicated six GC cell lines. C, D, A pool of three si RNA s (100 nmol/L in total) were introduced into MKN 45 (C) and KATO III (D) cells, respectively. The USP 14 expressions in these cells were measured via RT ‐ qPCR and western blotting. Data, ** P
Figure Legend Snippet: Suppressing USP 14 expression sensitized GC cells to cisplatin in vitro. A, B, USP 14 has high expression at both mRNA and protein level in the indicated six GC cell lines. C, D, A pool of three si RNA s (100 nmol/L in total) were introduced into MKN 45 (C) and KATO III (D) cells, respectively. The USP 14 expressions in these cells were measured via RT ‐ qPCR and western blotting. Data, ** P

Techniques Used: Expressing, In Vitro, Quantitative RT-PCR, Western Blot

40) Product Images from "Novel Class of Viral Ankyrin Proteins Targeting the Host E3 Ubiquitin Ligase Cullin-2"

Article Title: Novel Class of Viral Ankyrin Proteins Targeting the Host E3 Ubiquitin Ligase Cullin-2

Journal: Journal of Virology

doi: 10.1128/JVI.01374-18

ANK/BC proteins suppress the production of CXCL10, CCL5, and IFN. (A) HEK293T-REx cells were induced with 2 μg/ml of doxycycline (Dox) for 16 h and subjected to immunoblotting against the indicated proteins. (B to D) The cells were induced with Dox as described for panel A and subsequently stimulated with SeV for a further 24 h. RNA expression levels for (B) IFN-β, (C) CXCL10, and (D) CCL5 were measured by quantitative PCR (qPCR). Data were normalized to 18S expression levels; data represent fold increase over the levels seen under nonstimulated conditions. (E and F) The cells were treated as described for panel B, and the resulting cell culture medium was subjected to ELISA to detect soluble levels of (E) CXCL10 and (F) CCL5. In all assays, data are presented as means ± SD and represent results from one experiment that is representative of at least three, each performed in triplicate. **, P
Figure Legend Snippet: ANK/BC proteins suppress the production of CXCL10, CCL5, and IFN. (A) HEK293T-REx cells were induced with 2 μg/ml of doxycycline (Dox) for 16 h and subjected to immunoblotting against the indicated proteins. (B to D) The cells were induced with Dox as described for panel A and subsequently stimulated with SeV for a further 24 h. RNA expression levels for (B) IFN-β, (C) CXCL10, and (D) CCL5 were measured by quantitative PCR (qPCR). Data were normalized to 18S expression levels; data represent fold increase over the levels seen under nonstimulated conditions. (E and F) The cells were treated as described for panel B, and the resulting cell culture medium was subjected to ELISA to detect soluble levels of (E) CXCL10 and (F) CCL5. In all assays, data are presented as means ± SD and represent results from one experiment that is representative of at least three, each performed in triplicate. **, P

Techniques Used: RNA Expression, Real-time Polymerase Chain Reaction, Expressing, Cell Culture, Enzyme-linked Immunosorbent Assay

Related Articles

Real-time Polymerase Chain Reaction:

Article Title: Elimination of p19ARF‐expressing cells protects against pulmonary emphysema in mice, et al. Elimination of p19ARF‐expressing cells protects against pulmonary emphysema in mice
Article Snippet: .. 4.6 Real‐time PCR analysis The total RNA was isolated from lung tissues using the PureLink® RNA Mini kit (Thermo Fischer Scientific) and reverse‐transcribed using the PrimeScript RT reagent kit with a gDNA eraser (TAKARA BIO) according to the manufacturer's instructions. .. PCR was performed on a Chromo4 PCR system (Bio‐Rad) using the KOD SYBR qPCR mix (TOYOBO).

Article Title: A role for long-chain acyl-CoA synthetase-4 (ACSL4) in diet-induced phospholipid remodeling and obesity-associated adipocyte dysfunction
Article Snippet: .. RNA was quantified and checked for purity using the Nanodrop spectrophotometer (Nanodrop 1000, Wilmington, DE). cDNA was generated from equal amounts of RNA, and real-time quantitative PCR was performed using SYBR Green (Applied Biosystems 7300, Carlsbad, CA). ..

Transfection:

Article Title: 25-Hydroxycholesterol and 27-hydroxycholesterol inhibit human rotavirus infection by sequestering viral particles into late endosomes
Article Snippet: .. Briefly, extraction of total RNA from transfected or untransfected Caco2 cells was performed 72 h after transfection using TRIzol Reagent (Applied Biosystems, Monza, Italy). .. Concentration and purity of the extracted RNA were assessed by spectrophotometry (A260/A280).

Mutagenesis:

Article Title: Tomato DCL2b is required for the biosynthesis of 22-nt small RNAs, the resulting secondary siRNAs, and the host defense against ToMV
Article Snippet: .. High-throughput sequencing of RNAs and sRNAs The total RNA samples were prepared from WT and DCL2b mutant adult leaves using TRIzol reagent (Invitrogen, USA). .. Paired-end mRNA libraries were generated using NEBNext® UltraTM RNA Library Prep Kit for Illumina® (NEB, USA) according to the manufacturer’s recommendations and were sequenced on an Illumina HiSeq 4000 platform; 150 bp reads were generated.

Isolation:

Article Title: KDELR2 Competes with Measles Virus Envelope Proteins for Cellular Chaperones Reducing Their Chaperone-Mediated Cell Surface Transport
Article Snippet: .. Isolated RNA was reverse transcribed in cDNA using the RevertAid first strand cDNA synthesis kit (Fermentas). ..

Article Title: Complement Receptor C5aR1 Inhibition Reduces Pyroptosis in hDPP4-Transgenic Mice Infected with MERS-CoV
Article Snippet: .. Isolation of RNA and Proteins THP-1 differentiated macrophages were lysed in TRIzol™ Reagent (Life Technologies, Carlsbad, CA, USA) at 24 h post-infection with MERS-CoV. .. Total RNA and proteins were isolated according to the reagent user guide.

Article Title: Elimination of p19ARF‐expressing cells protects against pulmonary emphysema in mice, et al. Elimination of p19ARF‐expressing cells protects against pulmonary emphysema in mice
Article Snippet: .. 4.6 Real‐time PCR analysis The total RNA was isolated from lung tissues using the PureLink® RNA Mini kit (Thermo Fischer Scientific) and reverse‐transcribed using the PrimeScript RT reagent kit with a gDNA eraser (TAKARA BIO) according to the manufacturer's instructions. .. PCR was performed on a Chromo4 PCR system (Bio‐Rad) using the KOD SYBR qPCR mix (TOYOBO).

Spectrophotometry:

Article Title: A role for long-chain acyl-CoA synthetase-4 (ACSL4) in diet-induced phospholipid remodeling and obesity-associated adipocyte dysfunction
Article Snippet: .. RNA was quantified and checked for purity using the Nanodrop spectrophotometer (Nanodrop 1000, Wilmington, DE). cDNA was generated from equal amounts of RNA, and real-time quantitative PCR was performed using SYBR Green (Applied Biosystems 7300, Carlsbad, CA). ..

SYBR Green Assay:

Article Title: A role for long-chain acyl-CoA synthetase-4 (ACSL4) in diet-induced phospholipid remodeling and obesity-associated adipocyte dysfunction
Article Snippet: .. RNA was quantified and checked for purity using the Nanodrop spectrophotometer (Nanodrop 1000, Wilmington, DE). cDNA was generated from equal amounts of RNA, and real-time quantitative PCR was performed using SYBR Green (Applied Biosystems 7300, Carlsbad, CA). ..

Sequencing:

Article Title: Tomato DCL2b is required for the biosynthesis of 22-nt small RNAs, the resulting secondary siRNAs, and the host defense against ToMV
Article Snippet: .. High-throughput sequencing of RNAs and sRNAs The total RNA samples were prepared from WT and DCL2b mutant adult leaves using TRIzol reagent (Invitrogen, USA). .. Paired-end mRNA libraries were generated using NEBNext® UltraTM RNA Library Prep Kit for Illumina® (NEB, USA) according to the manufacturer’s recommendations and were sequenced on an Illumina HiSeq 4000 platform; 150 bp reads were generated.

Generated:

Article Title: A role for long-chain acyl-CoA synthetase-4 (ACSL4) in diet-induced phospholipid remodeling and obesity-associated adipocyte dysfunction
Article Snippet: .. RNA was quantified and checked for purity using the Nanodrop spectrophotometer (Nanodrop 1000, Wilmington, DE). cDNA was generated from equal amounts of RNA, and real-time quantitative PCR was performed using SYBR Green (Applied Biosystems 7300, Carlsbad, CA). ..

High Throughput Screening Assay:

Article Title: Tomato DCL2b is required for the biosynthesis of 22-nt small RNAs, the resulting secondary siRNAs, and the host defense against ToMV
Article Snippet: .. High-throughput sequencing of RNAs and sRNAs The total RNA samples were prepared from WT and DCL2b mutant adult leaves using TRIzol reagent (Invitrogen, USA). .. Paired-end mRNA libraries were generated using NEBNext® UltraTM RNA Library Prep Kit for Illumina® (NEB, USA) according to the manufacturer’s recommendations and were sequenced on an Illumina HiSeq 4000 platform; 150 bp reads were generated.

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  • 94
    Thermo Fisher qrt pcr rna
    MARF1 binds the CCR4-NOT deadenylase complex and cyclin A mRNA. a Co-immunoprecipitation using anti-HA antibody-conjugated beads followed by Western blotting. Ovary lysates expressing 3xHA-MARF1 (MAT67Tub-Gal4 → UASP-3xHA-MARF1) and those from w 1118 negative control were tested. b Co-immunoprecipitation using anti-HA antibody-conjugated beads followed by Western blotting. Ovary lysates expressing 3xHA-tagged MARF1 (MAT67Tub-Gal4 → UASP-3xHA-MARF1 full-length or fragments) and those from w 1118 negative control were tested. Black triangles indicate the detected 3xHA-tagged MARF1 proteins. c Co-immunoprecipitation using ovary lysates from w 1118 and anti-Not1 antibody or negative control IgG-bound protein G beads followed by Western blotting. d Fold enrichment of mRNAs relative to a control gapdh mRNA that were co-immunoprecipitated with MARF1 by anti-HA antibody-conjugated beads and were eluted from the beads normalized by w 1118 negative control, determined by <t>RNA-immunoprecipitation</t> followed by <t>qRT-PCR.</t> Mean ± SD ( n = 3). P -value
    Qrt Pcr Rna, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 94/100, based on 106 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/qrt pcr rna/product/Thermo Fisher
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    88
    Thermo Fisher genomic hcv rna
    Disruption in the 5′UTR nt95–110:NS5B nt8528–8543 duplex reduces intracellular <t>HCV</t> <t>RNA</t> levels. ( A ) Huh-7.5 cells transfected with HCV genomic RNA (WT) or HCV genomic RNA containing mutations at 5′UTR nt 95–110 and NS5B nt 8528–8543 express NS5B antigen 24 h post-transfection. Scale bar = 50 µm. ( B ) Plots represent the average number of HCV RNA genome copies ± SE in 1 μg of cellular RNA at 48 h post-transfection. p -values ≤ 0.05 (*) or ≤ 0.01 (**) were determined by the Student’s t -test and represent four or more independent experiments.
    Genomic Hcv Rna, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 88/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    Thermo Fisher rna
    P53-target genes are induced more strongly in control cells than in Chernobyl bank vole fibroblasts. <t>RNA</t> was collected from untreated and etoposide-treated (20 μM) cells 48 and 72 h after the drug treatment. <t>CDNA</t> was prepared and quantitative-PCR was run with gene specific primers, and transcript levels were standardized to beta-actin transcript. Results are shown as a fold increase from untreated samples for a .) p21, b .) Gadd45α, c .) Bax, d .) Puma, and e .) Mdm2. The results are from three separate experiments using the eight Chernobyl ( N = 24) and eight control cell lines ( N = 24), variation is shown by standard deviation, and statistical analysis was done with Student’s t-test (** = p ≤ 0.01, *** = p ≤ 0.001)
    Rna, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 95/100, based on 3653 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    MARF1 binds the CCR4-NOT deadenylase complex and cyclin A mRNA. a Co-immunoprecipitation using anti-HA antibody-conjugated beads followed by Western blotting. Ovary lysates expressing 3xHA-MARF1 (MAT67Tub-Gal4 → UASP-3xHA-MARF1) and those from w 1118 negative control were tested. b Co-immunoprecipitation using anti-HA antibody-conjugated beads followed by Western blotting. Ovary lysates expressing 3xHA-tagged MARF1 (MAT67Tub-Gal4 → UASP-3xHA-MARF1 full-length or fragments) and those from w 1118 negative control were tested. Black triangles indicate the detected 3xHA-tagged MARF1 proteins. c Co-immunoprecipitation using ovary lysates from w 1118 and anti-Not1 antibody or negative control IgG-bound protein G beads followed by Western blotting. d Fold enrichment of mRNAs relative to a control gapdh mRNA that were co-immunoprecipitated with MARF1 by anti-HA antibody-conjugated beads and were eluted from the beads normalized by w 1118 negative control, determined by RNA-immunoprecipitation followed by qRT-PCR. Mean ± SD ( n = 3). P -value

    Journal: Nature Communications

    Article Title: LOTUS domain protein MARF1 binds CCR4-NOT deadenylase complex to post-transcriptionally regulate gene expression in oocytes

    doi: 10.1038/s41467-018-06404-w

    Figure Lengend Snippet: MARF1 binds the CCR4-NOT deadenylase complex and cyclin A mRNA. a Co-immunoprecipitation using anti-HA antibody-conjugated beads followed by Western blotting. Ovary lysates expressing 3xHA-MARF1 (MAT67Tub-Gal4 → UASP-3xHA-MARF1) and those from w 1118 negative control were tested. b Co-immunoprecipitation using anti-HA antibody-conjugated beads followed by Western blotting. Ovary lysates expressing 3xHA-tagged MARF1 (MAT67Tub-Gal4 → UASP-3xHA-MARF1 full-length or fragments) and those from w 1118 negative control were tested. Black triangles indicate the detected 3xHA-tagged MARF1 proteins. c Co-immunoprecipitation using ovary lysates from w 1118 and anti-Not1 antibody or negative control IgG-bound protein G beads followed by Western blotting. d Fold enrichment of mRNAs relative to a control gapdh mRNA that were co-immunoprecipitated with MARF1 by anti-HA antibody-conjugated beads and were eluted from the beads normalized by w 1118 negative control, determined by RNA-immunoprecipitation followed by qRT-PCR. Mean ± SD ( n = 3). P -value

    Article Snippet: qRT-PCR RNA from oocytes was prepared using miRVana (Thermo Fisher Scientific).

    Techniques: Immunoprecipitation, Western Blot, Expressing, Negative Control, Quantitative RT-PCR

    Tethered MARF1 shortens reporter mRNA poly-A tail and reduces reporter protein level. a GFP-5x BoxB reporter structure, harboring a ubiquitous tubulin promoter, GFP-coding sequence, and a 3′ UTR containing five BoxB hairpins. LambdaN-HA-fused control peptide, MARF1, GW182, and Piwi under a UASP promoter were expressed in germline cells using maternal-alpha-tubulin-Gal4 driver. b Confocal images of GFP signal in stage 14 oocytes. Scale bar is 100 μm. c Western blots using stage 14 oocyte lysates. Black triangles indicate the transgenic lambda-HA-fused proteins. d Quantification of band intensities in c . e ePAT assay measuring GFP reporter mRNA poly-A tail length in stage 14 oocytes. The amplified DNA sizes were analyzed on an agarose gel. f ePAT assay measuring poly-A tail lengths of GFP reporter mRNA and a negative control cdk1 mRNA in stage 14 oocytes. Amplified DNA sizes were analyzed by capillary fragment analysis. g Relative abundance of GFP-5xBoxB mRNA normalized by gapdh mRNA determined by qRT-PCR. Mean ± SD ( n = 6 for the control and n = 4 for all the others)

    Journal: Nature Communications

    Article Title: LOTUS domain protein MARF1 binds CCR4-NOT deadenylase complex to post-transcriptionally regulate gene expression in oocytes

    doi: 10.1038/s41467-018-06404-w

    Figure Lengend Snippet: Tethered MARF1 shortens reporter mRNA poly-A tail and reduces reporter protein level. a GFP-5x BoxB reporter structure, harboring a ubiquitous tubulin promoter, GFP-coding sequence, and a 3′ UTR containing five BoxB hairpins. LambdaN-HA-fused control peptide, MARF1, GW182, and Piwi under a UASP promoter were expressed in germline cells using maternal-alpha-tubulin-Gal4 driver. b Confocal images of GFP signal in stage 14 oocytes. Scale bar is 100 μm. c Western blots using stage 14 oocyte lysates. Black triangles indicate the transgenic lambda-HA-fused proteins. d Quantification of band intensities in c . e ePAT assay measuring GFP reporter mRNA poly-A tail length in stage 14 oocytes. The amplified DNA sizes were analyzed on an agarose gel. f ePAT assay measuring poly-A tail lengths of GFP reporter mRNA and a negative control cdk1 mRNA in stage 14 oocytes. Amplified DNA sizes were analyzed by capillary fragment analysis. g Relative abundance of GFP-5xBoxB mRNA normalized by gapdh mRNA determined by qRT-PCR. Mean ± SD ( n = 6 for the control and n = 4 for all the others)

    Article Snippet: qRT-PCR RNA from oocytes was prepared using miRVana (Thermo Fisher Scientific).

    Techniques: Sequencing, Western Blot, Transgenic Assay, Amplification, Agarose Gel Electrophoresis, Negative Control, Quantitative RT-PCR

    Disruption in the 5′UTR nt95–110:NS5B nt8528–8543 duplex reduces intracellular HCV RNA levels. ( A ) Huh-7.5 cells transfected with HCV genomic RNA (WT) or HCV genomic RNA containing mutations at 5′UTR nt 95–110 and NS5B nt 8528–8543 express NS5B antigen 24 h post-transfection. Scale bar = 50 µm. ( B ) Plots represent the average number of HCV RNA genome copies ± SE in 1 μg of cellular RNA at 48 h post-transfection. p -values ≤ 0.05 (*) or ≤ 0.01 (**) were determined by the Student’s t -test and represent four or more independent experiments.

    Journal: Viruses

    Article Title: Genomic-Scale Interaction Involving Complementary Sequences in the Hepatitis C Virus 5′UTR Domain IIa and the RNA-Dependent RNA Polymerase Coding Region Promotes Efficient Virus Replication

    doi: 10.3390/v11010017

    Figure Lengend Snippet: Disruption in the 5′UTR nt95–110:NS5B nt8528–8543 duplex reduces intracellular HCV RNA levels. ( A ) Huh-7.5 cells transfected with HCV genomic RNA (WT) or HCV genomic RNA containing mutations at 5′UTR nt 95–110 and NS5B nt 8528–8543 express NS5B antigen 24 h post-transfection. Scale bar = 50 µm. ( B ) Plots represent the average number of HCV RNA genome copies ± SE in 1 μg of cellular RNA at 48 h post-transfection. p -values ≤ 0.05 (*) or ≤ 0.01 (**) were determined by the Student’s t -test and represent four or more independent experiments.

    Article Snippet: Genomic HCV RNA was synthesized using the MEGAscript T7 kit (Thermo Fisher Scientific) with 1 µg of Xba I-linearized, mung bean nuclease-treated pER-1b or pER-1b containing 5′UTR or NS5B mutations (Thermo Fisher Scientific).

    Techniques: Transfection

    Disruption in the 5′UTR nt 95–110:NS5B nt 8528–8543 duplex reduced progeny virus titers. Huh-7.5 cells were infected with HCV parental virus (WT) or HCV containing mutations at 5′UTR nt 95–110 and NS5B nt 8528–8543. Plots represent the average number of infectious virus titers as focus-forming units (FFU)/mL or HCV RNA genome copies ± SE in 1 μg of cellular RNA taken at 48 h post-infection. p -values ≤ 0.05 (*) or ≤ 0.01 (**) were determined by the Student’s t -test and represent six independent experiments.

    Journal: Viruses

    Article Title: Genomic-Scale Interaction Involving Complementary Sequences in the Hepatitis C Virus 5′UTR Domain IIa and the RNA-Dependent RNA Polymerase Coding Region Promotes Efficient Virus Replication

    doi: 10.3390/v11010017

    Figure Lengend Snippet: Disruption in the 5′UTR nt 95–110:NS5B nt 8528–8543 duplex reduced progeny virus titers. Huh-7.5 cells were infected with HCV parental virus (WT) or HCV containing mutations at 5′UTR nt 95–110 and NS5B nt 8528–8543. Plots represent the average number of infectious virus titers as focus-forming units (FFU)/mL or HCV RNA genome copies ± SE in 1 μg of cellular RNA taken at 48 h post-infection. p -values ≤ 0.05 (*) or ≤ 0.01 (**) were determined by the Student’s t -test and represent six independent experiments.

    Article Snippet: Genomic HCV RNA was synthesized using the MEGAscript T7 kit (Thermo Fisher Scientific) with 1 µg of Xba I-linearized, mung bean nuclease-treated pER-1b or pER-1b containing 5′UTR or NS5B mutations (Thermo Fisher Scientific).

    Techniques: Infection

    P53-target genes are induced more strongly in control cells than in Chernobyl bank vole fibroblasts. RNA was collected from untreated and etoposide-treated (20 μM) cells 48 and 72 h after the drug treatment. CDNA was prepared and quantitative-PCR was run with gene specific primers, and transcript levels were standardized to beta-actin transcript. Results are shown as a fold increase from untreated samples for a .) p21, b .) Gadd45α, c .) Bax, d .) Puma, and e .) Mdm2. The results are from three separate experiments using the eight Chernobyl ( N = 24) and eight control cell lines ( N = 24), variation is shown by standard deviation, and statistical analysis was done with Student’s t-test (** = p ≤ 0.01, *** = p ≤ 0.001)

    Journal: BMC Cell Biology

    Article Title: Fibroblasts from bank voles inhabiting Chernobyl have increased resistance against oxidative and DNA stresses

    doi: 10.1186/s12860-018-0169-9

    Figure Lengend Snippet: P53-target genes are induced more strongly in control cells than in Chernobyl bank vole fibroblasts. RNA was collected from untreated and etoposide-treated (20 μM) cells 48 and 72 h after the drug treatment. CDNA was prepared and quantitative-PCR was run with gene specific primers, and transcript levels were standardized to beta-actin transcript. Results are shown as a fold increase from untreated samples for a .) p21, b .) Gadd45α, c .) Bax, d .) Puma, and e .) Mdm2. The results are from three separate experiments using the eight Chernobyl ( N = 24) and eight control cell lines ( N = 24), variation is shown by standard deviation, and statistical analysis was done with Student’s t-test (** = p ≤ 0.01, *** = p ≤ 0.001)

    Article Snippet: CDNA was prepared from 0.5 μg of RNA using RevertAid H Minus First Strand cDNA Synthesis Kit (Thermofisher) with random primers as suggested by the manufacturer.

    Techniques: Real-time Polymerase Chain Reaction, Standard Deviation

    ABT‐263 protected lung tissues from PPE‐induced emphysema. (a) Experimental timeline. Six‐month‐old female ARF‐DTR mice were orally administered ABT‐263 (25 mg kg −1 day −1 ) or vehicle alone. PPE was administered intranasally 4 weeks after the first ABT‐263 treatment. (b) Representative images of the in vivo luciferase analysis. (c) Luminescence in the chest region was quantified. Values were normalized to the average value of the vehicle‐treated group. (d) The total RNA extracted from the lung tissues was analyzed by real‐time PCR for the expression of ARF ( left ) or INK4a ( right ). mRNA levels were normalized to GAPDH in each sample. (e–h) Mice were subjected to pulmonary function tests. Pressure–volume loop (e), Cst (f), Crs (g), and IC (h) are shown. (i) Representative images of vehicle‐ or ABT‐263‐treated mouse lung sections. Sections were stained with hematoxylin and eosin. Bar; 100 μm. (j) Alveolar mean linear intercepts were measured. Bars represent the mean ± SEM . Data were analyzed by an unpaired Student's t test. * p

    Journal: Aging Cell

    Article Title: Elimination of p19ARF‐expressing cells protects against pulmonary emphysema in mice, et al. Elimination of p19ARF‐expressing cells protects against pulmonary emphysema in mice

    doi: 10.1111/acel.12827

    Figure Lengend Snippet: ABT‐263 protected lung tissues from PPE‐induced emphysema. (a) Experimental timeline. Six‐month‐old female ARF‐DTR mice were orally administered ABT‐263 (25 mg kg −1 day −1 ) or vehicle alone. PPE was administered intranasally 4 weeks after the first ABT‐263 treatment. (b) Representative images of the in vivo luciferase analysis. (c) Luminescence in the chest region was quantified. Values were normalized to the average value of the vehicle‐treated group. (d) The total RNA extracted from the lung tissues was analyzed by real‐time PCR for the expression of ARF ( left ) or INK4a ( right ). mRNA levels were normalized to GAPDH in each sample. (e–h) Mice were subjected to pulmonary function tests. Pressure–volume loop (e), Cst (f), Crs (g), and IC (h) are shown. (i) Representative images of vehicle‐ or ABT‐263‐treated mouse lung sections. Sections were stained with hematoxylin and eosin. Bar; 100 μm. (j) Alveolar mean linear intercepts were measured. Bars represent the mean ± SEM . Data were analyzed by an unpaired Student's t test. * p

    Article Snippet: 4.6 Real‐time PCR analysis The total RNA was isolated from lung tissues using the PureLink® RNA Mini kit (Thermo Fischer Scientific) and reverse‐transcribed using the PrimeScript RT reagent kit with a gDNA eraser (TAKARA BIO) according to the manufacturer's instructions.

    Techniques: Mouse Assay, In Vivo, Luciferase, Real-time Polymerase Chain Reaction, Expressing, Staining

    Effects of PPE/DT administration on SASP‐related factors. (a,b) Female ARF‐DTR mice were treated with PPE and/or DT as shown in Figure 1 a. The total RNA was isolated from lung tissues 1 (a) or 3 weeks (b) after the administration of PPE. The expression of the indicated genes was analyzed by real‐time PCR. mRNA levels were normalized to GAPDH in each sample. Bars represent the mean ± SEM . Data were analyzed by a one‐way ANOVA and Steel–Dwass post hoc analysis. * p

    Journal: Aging Cell

    Article Title: Elimination of p19ARF‐expressing cells protects against pulmonary emphysema in mice, et al. Elimination of p19ARF‐expressing cells protects against pulmonary emphysema in mice

    doi: 10.1111/acel.12827

    Figure Lengend Snippet: Effects of PPE/DT administration on SASP‐related factors. (a,b) Female ARF‐DTR mice were treated with PPE and/or DT as shown in Figure 1 a. The total RNA was isolated from lung tissues 1 (a) or 3 weeks (b) after the administration of PPE. The expression of the indicated genes was analyzed by real‐time PCR. mRNA levels were normalized to GAPDH in each sample. Bars represent the mean ± SEM . Data were analyzed by a one‐way ANOVA and Steel–Dwass post hoc analysis. * p

    Article Snippet: 4.6 Real‐time PCR analysis The total RNA was isolated from lung tissues using the PureLink® RNA Mini kit (Thermo Fischer Scientific) and reverse‐transcribed using the PrimeScript RT reagent kit with a gDNA eraser (TAKARA BIO) according to the manufacturer's instructions.

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

    DT eliminated p19 ARF ‐expressing cells from the lung tissues of ARF‐DTR mice. (a) Experimental procedure. Five‐month‐old female ARF‐DTR or wild‐type mice pretreated with PBS or DT were administered porcine pancreatic elastase (100 U/kg). DT and PPE were administered intraperitoneally and intranasally, respectively. (b) Representative images of the in vivo imaging of ARF‐DTR mice. Images were taken before (pre) and after (post) the administration of DT and PPE, as shown in panel a. (c) Luciferase activity was measured using in vivo imaging analysis software. Changes in the luciferase activity before and after drug administration in each mouse were plotted. Bars indicate the average values of each group. (d,e) The total RNA extracted from lung tissue was analyzed by real‐time PCR for the expression of ARF (d) or INK4a (e). GAPDH mRNA was used as an internal standard in each sample, and the ∆∆ C t method was used to determine the relative expression level. Bars represent the mean ± SEM . Data were analyzed by a one‐way ANOVA and Steel–Dwass post hoc analysis. * p

    Journal: Aging Cell

    Article Title: Elimination of p19ARF‐expressing cells protects against pulmonary emphysema in mice, et al. Elimination of p19ARF‐expressing cells protects against pulmonary emphysema in mice

    doi: 10.1111/acel.12827

    Figure Lengend Snippet: DT eliminated p19 ARF ‐expressing cells from the lung tissues of ARF‐DTR mice. (a) Experimental procedure. Five‐month‐old female ARF‐DTR or wild‐type mice pretreated with PBS or DT were administered porcine pancreatic elastase (100 U/kg). DT and PPE were administered intraperitoneally and intranasally, respectively. (b) Representative images of the in vivo imaging of ARF‐DTR mice. Images were taken before (pre) and after (post) the administration of DT and PPE, as shown in panel a. (c) Luciferase activity was measured using in vivo imaging analysis software. Changes in the luciferase activity before and after drug administration in each mouse were plotted. Bars indicate the average values of each group. (d,e) The total RNA extracted from lung tissue was analyzed by real‐time PCR for the expression of ARF (d) or INK4a (e). GAPDH mRNA was used as an internal standard in each sample, and the ∆∆ C t method was used to determine the relative expression level. Bars represent the mean ± SEM . Data were analyzed by a one‐way ANOVA and Steel–Dwass post hoc analysis. * p

    Article Snippet: 4.6 Real‐time PCR analysis The total RNA was isolated from lung tissues using the PureLink® RNA Mini kit (Thermo Fischer Scientific) and reverse‐transcribed using the PrimeScript RT reagent kit with a gDNA eraser (TAKARA BIO) according to the manufacturer's instructions.

    Techniques: Expressing, Mouse Assay, In Vivo Imaging, Luciferase, Activity Assay, Software, Real-time Polymerase Chain Reaction