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Roche human gingival fibroblasts hgf
Primary <t>HGF</t> express TLR-9 mRNA. RT-PCR for TLR-9, TLR-4, and GAPDH was performed on cDNA from primary HGF, HEK 293, and HEK 293 cells stably transfected with hTLR-9. Genomic DNA contamination was excluded by testing <t>RNA</t> without reverse transcription (−). Colors are inverted.
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1) Product Images from "DNA from Periodontopathogenic Bacteria Is Immunostimulatory for Mouse and Human Immune Cells"

Article Title: DNA from Periodontopathogenic Bacteria Is Immunostimulatory for Mouse and Human Immune Cells

Journal: Infection and Immunity

doi: 10.1128/IAI.71.2.850-856.2003

Primary HGF express TLR-9 mRNA. RT-PCR for TLR-9, TLR-4, and GAPDH was performed on cDNA from primary HGF, HEK 293, and HEK 293 cells stably transfected with hTLR-9. Genomic DNA contamination was excluded by testing RNA without reverse transcription (−). Colors are inverted.
Figure Legend Snippet: Primary HGF express TLR-9 mRNA. RT-PCR for TLR-9, TLR-4, and GAPDH was performed on cDNA from primary HGF, HEK 293, and HEK 293 cells stably transfected with hTLR-9. Genomic DNA contamination was excluded by testing RNA without reverse transcription (−). Colors are inverted.

Techniques Used: Reverse Transcription Polymerase Chain Reaction, Stable Transfection, Transfection

2) Product Images from "Long noncoding RNA AVAN promotes antiviral innate immunity by interacting with TRIM25 and enhancing the transcription of FOXO3a"

Article Title: Long noncoding RNA AVAN promotes antiviral innate immunity by interacting with TRIM25 and enhancing the transcription of FOXO3a

Journal: bioRxiv

doi: 10.1101/623132

AVAN direct binds to TRIM25 and enhances the antivirus immune response (A) RNA pull-down of AVAN -associated proteins using biotinylated AVAN or antisense probes. Isolated proteins were resolved by SDS-PAGE followed by silver staining. (B) Pull-down western blot showing that AVAN can bind directly to TRIM25. (C) ChIRP followed by western blot show that AVAN can bind to TRIM25. (D and E) Exogenous (D) and endogenous (E) RIP of TRIM25 in BJ501 infected cells using anti-TRIM25 or anti-IgG antibodies. The relative enrichment fold of AVAN was calculated by qRT-PCR. (F) AVAN pull-down western blot with lysates of A549 cells transfected with Flag, Flag-TRIM25, Flag-SPRY, Flag-B Box/CCD or Flag-Ring. (G) Truncated AVAN pull-down, truncates (upper panel) were obtained via in vitro transcription and incubated with BJ501-infected A549 lysates for RNA pulldown. (H and I) TRIM25 co-immunoprecipitation with proteins from lysates of BJ501-infected A549 cells transfected with AVAN s or siRNAs, followed by immunoblotting. Anti-TRIM25 and anti-RIG-I antibodies were used for immunoprecipitated. (J and K) Immunoblot analysis of endogenous RIG-I ubiquitylation in BJ501-infected A549 cells transfected with AVAN s or siRNAs. Anti-RIG-I antibody was used for immunoprecipitated. (L) Immunoblot analysis of proteins immunoprecipitated with anti-Flag from lysates of BJ501-infected A549 cells transfected with AVAN , HA-Ub and Flag-tagged RIG-I. (M and N) IFN-alpha (M) and IFN-beta (N) expression upon AVAN transfection in A549 cells that were infected by BJ501 or not (MOI=1) at 24h post-infection, and then individually knock down RIG-I or TRIM25, analyzed by qRT-PCR (n=3; means ± SEM; *p
Figure Legend Snippet: AVAN direct binds to TRIM25 and enhances the antivirus immune response (A) RNA pull-down of AVAN -associated proteins using biotinylated AVAN or antisense probes. Isolated proteins were resolved by SDS-PAGE followed by silver staining. (B) Pull-down western blot showing that AVAN can bind directly to TRIM25. (C) ChIRP followed by western blot show that AVAN can bind to TRIM25. (D and E) Exogenous (D) and endogenous (E) RIP of TRIM25 in BJ501 infected cells using anti-TRIM25 or anti-IgG antibodies. The relative enrichment fold of AVAN was calculated by qRT-PCR. (F) AVAN pull-down western blot with lysates of A549 cells transfected with Flag, Flag-TRIM25, Flag-SPRY, Flag-B Box/CCD or Flag-Ring. (G) Truncated AVAN pull-down, truncates (upper panel) were obtained via in vitro transcription and incubated with BJ501-infected A549 lysates for RNA pulldown. (H and I) TRIM25 co-immunoprecipitation with proteins from lysates of BJ501-infected A549 cells transfected with AVAN s or siRNAs, followed by immunoblotting. Anti-TRIM25 and anti-RIG-I antibodies were used for immunoprecipitated. (J and K) Immunoblot analysis of endogenous RIG-I ubiquitylation in BJ501-infected A549 cells transfected with AVAN s or siRNAs. Anti-RIG-I antibody was used for immunoprecipitated. (L) Immunoblot analysis of proteins immunoprecipitated with anti-Flag from lysates of BJ501-infected A549 cells transfected with AVAN , HA-Ub and Flag-tagged RIG-I. (M and N) IFN-alpha (M) and IFN-beta (N) expression upon AVAN transfection in A549 cells that were infected by BJ501 or not (MOI=1) at 24h post-infection, and then individually knock down RIG-I or TRIM25, analyzed by qRT-PCR (n=3; means ± SEM; *p

Techniques Used: Isolation, SDS Page, Silver Staining, Western Blot, Infection, Quantitative RT-PCR, Transfection, In Vitro, Incubation, Immunoprecipitation, Expressing

3) Product Images from "Influence of copper on expression of nirS, norB and nosZ and the transcription and activity of NIR, NOR and N2OR in the denitrifying soil bacteria Pseudomonas stutzeri"

Article Title: Influence of copper on expression of nirS, norB and nosZ and the transcription and activity of NIR, NOR and N2OR in the denitrifying soil bacteria Pseudomonas stutzeri

Journal: Microbial Biotechnology

doi: 10.1111/1751-7915.12352

Expression ratios of the Pseudomonas stutzeri nirS , norB and nosZ at 5 days post‐inoculation. Expression ratios were calculated and normalized against reference genes fdxA, ropD and gyrB . Expression ratios are the difference in gene expression of Pseudomonas stutzeri cultured in basal salt solution with different copper concentration relative to the RNA expression in culture incubated without the presence of copper. Error bars are the SEM for all sample replicates.
Figure Legend Snippet: Expression ratios of the Pseudomonas stutzeri nirS , norB and nosZ at 5 days post‐inoculation. Expression ratios were calculated and normalized against reference genes fdxA, ropD and gyrB . Expression ratios are the difference in gene expression of Pseudomonas stutzeri cultured in basal salt solution with different copper concentration relative to the RNA expression in culture incubated without the presence of copper. Error bars are the SEM for all sample replicates.

Techniques Used: Expressing, Cell Culture, Concentration Assay, RNA Expression, Incubation

4) Product Images from "Identification of Functional Toxin/Immunity Genes Linked to Contact-Dependent Growth Inhibition (CDI) and Rearrangement Hotspot (Rhs) Systems"

Article Title: Identification of Functional Toxin/Immunity Genes Linked to Contact-Dependent Growth Inhibition (CDI) and Rearrangement Hotspot (Rhs) Systems

Journal: PLoS Genetics

doi: 10.1371/journal.pgen.1002217

The tRNase activity of CdiA-CT o1 EC93 is blocked by the binding of CdiI o1 EC93 . A) Analysis of CdiA-CT/CdiI binding. Purified CdiA-CT and CdiI-His 6 proteins were mixed at equimolar ratios then purified by Ni 2+ -affinity chromatography. Input samples represent the protein mixtures prior to chromatography. Unbound fractions contain proteins that failed to bind the affinity resin. Bound proteins were eluted from the affinity resin with imidazole. All fractions were analyzed by SDS-PAGE. B) Northern blot analysis of CdiA-CT UPEC536 and CdiA-CT o1 EC93 tRNase activity. S100 fractions containing cellular tRNA was treated with purified CdiA-CT and/or CdiI-His 6 proteins and then analyzed by Northern blot hybridization using probes specific for tRNA 1B Ala and tRNA His .
Figure Legend Snippet: The tRNase activity of CdiA-CT o1 EC93 is blocked by the binding of CdiI o1 EC93 . A) Analysis of CdiA-CT/CdiI binding. Purified CdiA-CT and CdiI-His 6 proteins were mixed at equimolar ratios then purified by Ni 2+ -affinity chromatography. Input samples represent the protein mixtures prior to chromatography. Unbound fractions contain proteins that failed to bind the affinity resin. Bound proteins were eluted from the affinity resin with imidazole. All fractions were analyzed by SDS-PAGE. B) Northern blot analysis of CdiA-CT UPEC536 and CdiA-CT o1 EC93 tRNase activity. S100 fractions containing cellular tRNA was treated with purified CdiA-CT and/or CdiI-His 6 proteins and then analyzed by Northern blot hybridization using probes specific for tRNA 1B Ala and tRNA His .

Techniques Used: Activity Assay, Binding Assay, Purification, Affinity Chromatography, Chromatography, SDS Page, Northern Blot, Hybridization

E. coli EC93 contains an orphan cdiA-CT/cdiI module. The CDI region of E. coli EC93 is depicted, with cdiB , cdiA and cdiI genes shown in yellow, green and purple, respectively. The cdiA coding region upstream of the encoded VENN motif is shown in light green, and the cdiA-CT sequence is shown in dark green. The orphan cdiA-CT fragment ( cdiA-CT o1 ) and orphan cdiI ( cdiI o1 ) are dark green and purple, respectively. The nucleotide sequence of the cdiI - cdiA-CT o1 junction and the predicted reading frames are shown in detail. Sequences similar to transposable element genes are shown in red.
Figure Legend Snippet: E. coli EC93 contains an orphan cdiA-CT/cdiI module. The CDI region of E. coli EC93 is depicted, with cdiB , cdiA and cdiI genes shown in yellow, green and purple, respectively. The cdiA coding region upstream of the encoded VENN motif is shown in light green, and the cdiA-CT sequence is shown in dark green. The orphan cdiA-CT fragment ( cdiA-CT o1 ) and orphan cdiI ( cdiI o1 ) are dark green and purple, respectively. The nucleotide sequence of the cdiI - cdiA-CT o1 junction and the predicted reading frames are shown in detail. Sequences similar to transposable element genes are shown in red.

Techniques Used: Sequencing

The EC93 orphan cdiA-CT/cdiI module is functional in contact-dependent growth inhibition (CDI). A) The wild-type EC93 and orphan chimera CDI systems are shown schematically. The cdiA-CT EC93 /cdiI EC93 region was deleted and orphan module fused onto the cdiA EC93 gene at the VENN encoding sequence. B) Growth competitions. CDI + inhibitor cells were co-cultured with target cells expressing either CdiI EC93 or orphan CdiI o1 EC93 immunity proteins. Viable target cells were quantified by plating on selective media to determine the number of colony forming units (cfu) per milliliter.
Figure Legend Snippet: The EC93 orphan cdiA-CT/cdiI module is functional in contact-dependent growth inhibition (CDI). A) The wild-type EC93 and orphan chimera CDI systems are shown schematically. The cdiA-CT EC93 /cdiI EC93 region was deleted and orphan module fused onto the cdiA EC93 gene at the VENN encoding sequence. B) Growth competitions. CDI + inhibitor cells were co-cultured with target cells expressing either CdiI EC93 or orphan CdiI o1 EC93 immunity proteins. Viable target cells were quantified by plating on selective media to determine the number of colony forming units (cfu) per milliliter.

Techniques Used: Functional Assay, Inhibition, Sequencing, Cell Culture, Expressing

The EC93 orphan region is transcribed. RNA from E. coli EC93, EC93 Δ cdiA-CT EC93 Δ cdiI EC93 , and EC93 Δ cdiA-CT o1 EC93 Δ cdiI o1 EC93 was subjected to quantitative RT-PCR. The primer binding sites within the cdi locus are depicted schematically as arrows. The relative expression levels represent the mean ± SEM for three independently isolated RNA samples.
Figure Legend Snippet: The EC93 orphan region is transcribed. RNA from E. coli EC93, EC93 Δ cdiA-CT EC93 Δ cdiI EC93 , and EC93 Δ cdiA-CT o1 EC93 Δ cdiI o1 EC93 was subjected to quantitative RT-PCR. The primer binding sites within the cdi locus are depicted schematically as arrows. The relative expression levels represent the mean ± SEM for three independently isolated RNA samples.

Techniques Used: Quantitative RT-PCR, Binding Assay, Expressing, Isolation

CdiA-CT o1 EC93 inhibits the growth of E. coli cells. A) Growth curves of E. coli Δ sspB cells expressing CdiA-CT o1 EC93 /CdiI o1 EC93 -DAS. Degradation of CdiI o1 EC93 -DAS was initiated by the addition of L-arabinose to induce SspB synthesis. Control cells express SspB(Δ47), which does not deliver CdiI o1 EC93 -DAS to the ClpXP protease. Growth curves with square symbols represent control strains expressing SspB or SspB(Δ47), but not CdiA-CT o1 EC93 /CdiI o1 EC93 -DAS. B) Analysis of in vivo CdiA-CT o1 EC93 tRNase activity. Total RNA was isolated from cells expressing CdiA-CT o1 EC93 /CdiI o1 EC93 -DAS at varying times after L-arabinose induction. Samples were run on polyacrylamide gels followed by staining with ethidium bromide (EtBr) or Northern blot analysis using probes specific for tRNA 1B Ala and tRNA His .
Figure Legend Snippet: CdiA-CT o1 EC93 inhibits the growth of E. coli cells. A) Growth curves of E. coli Δ sspB cells expressing CdiA-CT o1 EC93 /CdiI o1 EC93 -DAS. Degradation of CdiI o1 EC93 -DAS was initiated by the addition of L-arabinose to induce SspB synthesis. Control cells express SspB(Δ47), which does not deliver CdiI o1 EC93 -DAS to the ClpXP protease. Growth curves with square symbols represent control strains expressing SspB or SspB(Δ47), but not CdiA-CT o1 EC93 /CdiI o1 EC93 -DAS. B) Analysis of in vivo CdiA-CT o1 EC93 tRNase activity. Total RNA was isolated from cells expressing CdiA-CT o1 EC93 /CdiI o1 EC93 -DAS at varying times after L-arabinose induction. Samples were run on polyacrylamide gels followed by staining with ethidium bromide (EtBr) or Northern blot analysis using probes specific for tRNA 1B Ala and tRNA His .

Techniques Used: Expressing, In Vivo, Activity Assay, Isolation, Staining, Northern Blot

The EC93 orphan region produces functional CdiI o1 immunity protein. EC93 expressing chimeric CdiA EC93 -CT o1 EC93 was used as an inhibitor strain in growth competition experiments. Inhibitor cells were co-cultured with wild-type EC93, EC93 deleted for the orphan region (Δ cdiA-CT o1 EC93 Δ cdiI o1 EC93 ), and EC93 Δ cdiA-CT o1 EC93 Δ cdiI o1 EC93 cells complemented with a plasmid-borne copy of cdiI o1 EC93 ( cdiI o1 + ). Viable target cells were quantified by plating on selective media to determine the number of colony forming units (cfu) per milliliter.
Figure Legend Snippet: The EC93 orphan region produces functional CdiI o1 immunity protein. EC93 expressing chimeric CdiA EC93 -CT o1 EC93 was used as an inhibitor strain in growth competition experiments. Inhibitor cells were co-cultured with wild-type EC93, EC93 deleted for the orphan region (Δ cdiA-CT o1 EC93 Δ cdiI o1 EC93 ), and EC93 Δ cdiA-CT o1 EC93 Δ cdiI o1 EC93 cells complemented with a plasmid-borne copy of cdiI o1 EC93 ( cdiI o1 + ). Viable target cells were quantified by plating on selective media to determine the number of colony forming units (cfu) per milliliter.

Techniques Used: Functional Assay, Expressing, Cell Culture, Plasmid Preparation

5) Product Images from "Long noncoding RNA AFAP1-AS1 facilitates tumor growth through enhancer of zeste homolog 2 in colorectal cancer"

Article Title: Long noncoding RNA AFAP1-AS1 facilitates tumor growth through enhancer of zeste homolog 2 in colorectal cancer

Journal: American Journal of Cancer Research

doi:

Association of AFAP1-AS1 and Polycomb Repressive Complex 2. (A) RIP enrichment was determined as RNA associated with EZH2 IP relative to an input control. (B) RIP experiments were performed using the EZH2 antibody to immunoprecipitate (IP). (C) Biotinylated AFAP1-AS1 was incubated with nuclear extracts (SW480 and HCT116 cells), targeted with streptavidin beads, and washed, and associated proteins were resolved in a gel. Western blotting analysis of the specific association of EZH2 and AFAP1-AS1 (n = 3). (D, E) RNAs corresponding to different fragments of AFAP1-AS1 were treated as in (C), and associated EZH2 was detected by western blotting (n = 3). Error bars ± SD. *, P
Figure Legend Snippet: Association of AFAP1-AS1 and Polycomb Repressive Complex 2. (A) RIP enrichment was determined as RNA associated with EZH2 IP relative to an input control. (B) RIP experiments were performed using the EZH2 antibody to immunoprecipitate (IP). (C) Biotinylated AFAP1-AS1 was incubated with nuclear extracts (SW480 and HCT116 cells), targeted with streptavidin beads, and washed, and associated proteins were resolved in a gel. Western blotting analysis of the specific association of EZH2 and AFAP1-AS1 (n = 3). (D, E) RNAs corresponding to different fragments of AFAP1-AS1 were treated as in (C), and associated EZH2 was detected by western blotting (n = 3). Error bars ± SD. *, P

Techniques Used: Incubation, Western Blot

6) Product Images from "Abnormal Production of Pro- and Anti-Inflammatory Cytokines by Lupus Monocytes in Response to Apoptotic Cells"

Article Title: Abnormal Production of Pro- and Anti-Inflammatory Cytokines by Lupus Monocytes in Response to Apoptotic Cells

Journal: PLoS ONE

doi: 10.1371/journal.pone.0017495

Production of TGF-β and TNF-α by live or apoptotic Jurkat cells, non-stimulated monocytes and monocytes plus live Jurkat cells. Monocytes from healthy donors (A B) or patients with SLE (C D) were incubated alone or in the presence of live Jurkat cells. In addition, live or apoptotic Jurkat cells were incubated alone. After overnight incubation, production of TGF-β (A C) and TNF-α (B D) were determined in the supernatants by ELISA.
Figure Legend Snippet: Production of TGF-β and TNF-α by live or apoptotic Jurkat cells, non-stimulated monocytes and monocytes plus live Jurkat cells. Monocytes from healthy donors (A B) or patients with SLE (C D) were incubated alone or in the presence of live Jurkat cells. In addition, live or apoptotic Jurkat cells were incubated alone. After overnight incubation, production of TGF-β (A C) and TNF-α (B D) were determined in the supernatants by ELISA.

Techniques Used: Incubation, Enzyme-linked Immunosorbent Assay

Pearson's correlation between phagocytosis of apoptotic cells and TNF-α production by SLE monocytes. Control ( Figure S1 ) or SLE monocytes were co-incubated with CFSE-labeled apoptotic Jurkat cells. The percentage of cells positive for both CD14 and CFSE was used to quantify phagocytosis of apoptotic Jurkat cells by monocytes. Only data from SLE monocytes is shown. Statistical analysis was performed with Stata 10 software (Stata Corporation, College Station, TX).
Figure Legend Snippet: Pearson's correlation between phagocytosis of apoptotic cells and TNF-α production by SLE monocytes. Control ( Figure S1 ) or SLE monocytes were co-incubated with CFSE-labeled apoptotic Jurkat cells. The percentage of cells positive for both CD14 and CFSE was used to quantify phagocytosis of apoptotic Jurkat cells by monocytes. Only data from SLE monocytes is shown. Statistical analysis was performed with Stata 10 software (Stata Corporation, College Station, TX).

Techniques Used: Incubation, Labeling, Software

TNF-α production by lupus monocytes in response to apoptotic cells is sensitive to RNase and DNase treatment. Monocytes from six consecutive SLE patients were co-incubated with apoptotic Jurkat cells (UVJ) in the absence or presence of 5 µg/ml of RNase, 16 u/ml of DNase, or both nucleases; and TNF-α production was determined by ELISA in the supernatants.
Figure Legend Snippet: TNF-α production by lupus monocytes in response to apoptotic cells is sensitive to RNase and DNase treatment. Monocytes from six consecutive SLE patients were co-incubated with apoptotic Jurkat cells (UVJ) in the absence or presence of 5 µg/ml of RNase, 16 u/ml of DNase, or both nucleases; and TNF-α production was determined by ELISA in the supernatants.

Techniques Used: Incubation, Enzyme-linked Immunosorbent Assay

Monocytes from patients with SLE have prominent production of TNF-α, but decreased production of TGF-β. Monocytes obtained from healthy donors (n = 13) or patients with SLE (n = 47) were co-incubated in the presence of apoptotic Jurkat cells. After overnight incubation, production of TGF-β ( A ) and TNF-α ( B ) were determined in the supernatants by ELISA. Data is shown as boxplots. Comparison of mean cytokine secretion between groups was performed by Students t-test.
Figure Legend Snippet: Monocytes from patients with SLE have prominent production of TNF-α, but decreased production of TGF-β. Monocytes obtained from healthy donors (n = 13) or patients with SLE (n = 47) were co-incubated in the presence of apoptotic Jurkat cells. After overnight incubation, production of TGF-β ( A ) and TNF-α ( B ) were determined in the supernatants by ELISA. Data is shown as boxplots. Comparison of mean cytokine secretion between groups was performed by Students t-test.

Techniques Used: Incubation, Enzyme-linked Immunosorbent Assay

7) Product Images from "Inhibition of HIV-1 infection in humanized mice and metabolic stability of protein phosphatase-1-targeting small molecule 1E7-03"

Article Title: Inhibition of HIV-1 infection in humanized mice and metabolic stability of protein phosphatase-1-targeting small molecule 1E7-03

Journal: Oncotarget

doi: 10.18632/oncotarget.19999

Antiviral efficacy of 1E7-03 in HIV-1 89. 6-infected NSG mice Mice were treated with 3 mg/kg of 1E7-03 or 1.5 mg/kg F07#13 by a single dose intraperitoneal (i.p.) injection After 48 hrs, mice were sacrificed; plasma samples were collected and tested for levels of HIV-1 TAR RNA (A) and HIV-1 TAR-gag RNA (B) . For quantitative analysis of HIV-1 RNA, total RNA was isolated from blood specimens, treated with RNase-free DNase I and reverse transcribed. Real-time PCR reactions were carried out in triplicates. Each data point represents the blood from a single animal. For all figures, * p
Figure Legend Snippet: Antiviral efficacy of 1E7-03 in HIV-1 89. 6-infected NSG mice Mice were treated with 3 mg/kg of 1E7-03 or 1.5 mg/kg F07#13 by a single dose intraperitoneal (i.p.) injection After 48 hrs, mice were sacrificed; plasma samples were collected and tested for levels of HIV-1 TAR RNA (A) and HIV-1 TAR-gag RNA (B) . For quantitative analysis of HIV-1 RNA, total RNA was isolated from blood specimens, treated with RNase-free DNase I and reverse transcribed. Real-time PCR reactions were carried out in triplicates. Each data point represents the blood from a single animal. For all figures, * p

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

8) Product Images from "E4-Ubiquitin ligase Ufd2 stabilizes Yap8 and modulates arsenic stress responses independent of the U-box motif"

Article Title: E4-Ubiquitin ligase Ufd2 stabilizes Yap8 and modulates arsenic stress responses independent of the U-box motif

Journal: Biology Open

doi: 10.1242/bio.010405

Ufd2 mediates Yap8 stabilization. (A) Yap8 levels are reduced in ufd2 mutant cells compared to the wild type strain. BY4742 wild type (WT) and ufd2 mutant strains expressing Yap8-HA were incubated with 1.5 mM As(III), harvested at the indicated time-points and subjected to immunoblotting using anti-HA and anti-Pgk1 antibodies. The graph represents relative Yap8 levels (AU, Arbitrary Units). (B) Yap8 is destabilized in the ufd2 mutant. The same strains were first exposed to 1.5 mM As(III) for 90 min, washed and subsequently treated with 0.1 mg/ml cycloheximide (CHX) up to 120 min prior to immunoblotting with the antibodies indicated above. The graph represents the percentage of remaining Yap8 protein after CHX addition. Estimated Yap8 half-life is 98 min in the WT strain and 37 min in the ufd2 mutant. (C) Mps1 stability is increased in ufd2 and Ufd2 U-boxΔ mutant cells in comparison to WT strain. BY4741 WT, ufd2 and Ufd2 U-boxΔ mutant strains carrying the GAL1 promoter MPS1-c-myc construct were induced with galactose before being challenged with glucose and 0.1 mg/ml CHX. Cells were harvested at the indicated time-points and subjected to immunoblotting using anti- c -myc and anti-Pgk1 antibodies. The graph represents the percentage of remaining Mps1 protein after CHX addition. A representative experiment is shown. (D) Epistasis analyses of YAP8 and UFD2 . Exponential phase BY4742 WT, yap8 , ufd2 and yap8ufd2 cells were serially diluted and spotted onto MM media supplemented or not with increasing concentrations of As(V) (up to 2 mM; upper panel) or As(III) (up to 1.5 mM; lower panel). Growth was recorded after 2 days incubation at 30°C. A representative experiment is shown. (E) ACR3 expression is similar in the double yap8ufd2 and single yap8 mutants. The same strains referred in D were challenged with 1.5 mM As(III) for 90 min and ACR3 mRNA levels were determined by qRT-PCR (AU, Arbitrary Units). Values represent the mean±s.d. of three biological replicates and statistical differences denoted as * P
Figure Legend Snippet: Ufd2 mediates Yap8 stabilization. (A) Yap8 levels are reduced in ufd2 mutant cells compared to the wild type strain. BY4742 wild type (WT) and ufd2 mutant strains expressing Yap8-HA were incubated with 1.5 mM As(III), harvested at the indicated time-points and subjected to immunoblotting using anti-HA and anti-Pgk1 antibodies. The graph represents relative Yap8 levels (AU, Arbitrary Units). (B) Yap8 is destabilized in the ufd2 mutant. The same strains were first exposed to 1.5 mM As(III) for 90 min, washed and subsequently treated with 0.1 mg/ml cycloheximide (CHX) up to 120 min prior to immunoblotting with the antibodies indicated above. The graph represents the percentage of remaining Yap8 protein after CHX addition. Estimated Yap8 half-life is 98 min in the WT strain and 37 min in the ufd2 mutant. (C) Mps1 stability is increased in ufd2 and Ufd2 U-boxΔ mutant cells in comparison to WT strain. BY4741 WT, ufd2 and Ufd2 U-boxΔ mutant strains carrying the GAL1 promoter MPS1-c-myc construct were induced with galactose before being challenged with glucose and 0.1 mg/ml CHX. Cells were harvested at the indicated time-points and subjected to immunoblotting using anti- c -myc and anti-Pgk1 antibodies. The graph represents the percentage of remaining Mps1 protein after CHX addition. A representative experiment is shown. (D) Epistasis analyses of YAP8 and UFD2 . Exponential phase BY4742 WT, yap8 , ufd2 and yap8ufd2 cells were serially diluted and spotted onto MM media supplemented or not with increasing concentrations of As(V) (up to 2 mM; upper panel) or As(III) (up to 1.5 mM; lower panel). Growth was recorded after 2 days incubation at 30°C. A representative experiment is shown. (E) ACR3 expression is similar in the double yap8ufd2 and single yap8 mutants. The same strains referred in D were challenged with 1.5 mM As(III) for 90 min and ACR3 mRNA levels were determined by qRT-PCR (AU, Arbitrary Units). Values represent the mean±s.d. of three biological replicates and statistical differences denoted as * P

Techniques Used: Mutagenesis, Expressing, Incubation, Construct, Quantitative RT-PCR

Ubiquitin proteasome pathway (UPP) enzymes Ubc4, Rad23 and Dsk2 do not interfere with Yap8 stability in arsenic-exposed cells. BY4742 wild type (WT), ubc4 (A), rad23 (B) and dsk2 (C) mutant strains expressing Yap8-HA were first exposed to 1.5 mM As(III) for 90 min, washed and subsequently treated with 0.1 mg/ml cycloheximide (CHX) up to 120 min prior to immunoblotting using anti-HA and anti-Pgk1 antibodies. The graphs represent the percentage of remaining Yap8 protein after CHX addition. Representative experiments are shown. (D) ACR3 mRNA levels remain unaltered in ubc4 , rad23 and dsk2 mutant cells. The same strains were challenged with 1.5 mM As(III) for 90 min and ACR3 mRNA levels were determined by qRT-PCR (AU, Arbitrary Units). Values represent the mean±s.d. of three biological replicates. No significant statistical differences were observed.
Figure Legend Snippet: Ubiquitin proteasome pathway (UPP) enzymes Ubc4, Rad23 and Dsk2 do not interfere with Yap8 stability in arsenic-exposed cells. BY4742 wild type (WT), ubc4 (A), rad23 (B) and dsk2 (C) mutant strains expressing Yap8-HA were first exposed to 1.5 mM As(III) for 90 min, washed and subsequently treated with 0.1 mg/ml cycloheximide (CHX) up to 120 min prior to immunoblotting using anti-HA and anti-Pgk1 antibodies. The graphs represent the percentage of remaining Yap8 protein after CHX addition. Representative experiments are shown. (D) ACR3 mRNA levels remain unaltered in ubc4 , rad23 and dsk2 mutant cells. The same strains were challenged with 1.5 mM As(III) for 90 min and ACR3 mRNA levels were determined by qRT-PCR (AU, Arbitrary Units). Values represent the mean±s.d. of three biological replicates. No significant statistical differences were observed.

Techniques Used: Mutagenesis, Expressing, Quantitative RT-PCR

Ufd2 U-box motif is not required for Yap8 stabilization. (A) Yap8 levels are unaffected in the Ufd2 U-boxΔ mutant strain compared to the wild type strain. BY4741 wild type (WT), ufd2 and Ufd2 U-boxΔ strains expressing Yap8-HA were incubated with 1.5 mM As(III), harvested at the indicated time-points and subjected to immunoblotting using anti-HA and anti-Pgk1 antibodies. The graph represents relative Yap8 levels (AU, Arbitrary Units). A representative experiment is shown; SD, control. (B) Yap8 stability is similar in WT and Ufd2 U-boxΔ mutant strains. The same strains were first exposed to 1.5 mM As(III) for 90 min, washed and subsequently treated with 0.1 mg/ml cycloheximide (CHX) up to 90 min prior to immunoblotting, as indicated above. The graph represents the percentage of remaining Yap8 protein after CHX addition. Estimated Yap8 half-life is 63 min in the WT strain, 25 min in ufd2 and 67 min in Ufd2 U-boxΔ . (C) ACR3 mRNA levels are unaltered in the Ufd2 U-boxΔ . The same strains were challenged with 1.5 mM As(III) for 90 min and ACR3 mRNA levels were determined by qRT-PCR (AU, Arbitrary Units). Values represent the mean±s.d. of three biological replicates and statistical differences denoted as * P
Figure Legend Snippet: Ufd2 U-box motif is not required for Yap8 stabilization. (A) Yap8 levels are unaffected in the Ufd2 U-boxΔ mutant strain compared to the wild type strain. BY4741 wild type (WT), ufd2 and Ufd2 U-boxΔ strains expressing Yap8-HA were incubated with 1.5 mM As(III), harvested at the indicated time-points and subjected to immunoblotting using anti-HA and anti-Pgk1 antibodies. The graph represents relative Yap8 levels (AU, Arbitrary Units). A representative experiment is shown; SD, control. (B) Yap8 stability is similar in WT and Ufd2 U-boxΔ mutant strains. The same strains were first exposed to 1.5 mM As(III) for 90 min, washed and subsequently treated with 0.1 mg/ml cycloheximide (CHX) up to 90 min prior to immunoblotting, as indicated above. The graph represents the percentage of remaining Yap8 protein after CHX addition. Estimated Yap8 half-life is 63 min in the WT strain, 25 min in ufd2 and 67 min in Ufd2 U-boxΔ . (C) ACR3 mRNA levels are unaltered in the Ufd2 U-boxΔ . The same strains were challenged with 1.5 mM As(III) for 90 min and ACR3 mRNA levels were determined by qRT-PCR (AU, Arbitrary Units). Values represent the mean±s.d. of three biological replicates and statistical differences denoted as * P

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

Ufd2 mediates arsenic tolerance. (A) ufd2 cells are sensitive to arsenic stress. Exponential phase BY4742 wild type (WT) and the ufd2 mutant were serially diluted and spotted onto SC media supplemented or not with 1.5 mM As(III) or 2 mM As(V) or 1.5 mM As(III). SD, control. Growth was recorded after 2 days incubation at 30°C. A representative experiment is shown. Cell growth was also monitored by means of growth curves. Exponential phase BY4742 WT and ufd2 mutant cells were exposed or not to 2 mM As(V) or 1.5 mM As(III) for 22 h and OD 600 was monitored in intervals of 1 h. The curves represent the mean±s.d. of three biological replicates. (B) UFD2 is induced in cells injured with arsenic. BY4742 cells were challenged or not with 1.5 mM As(III) or 2 mM As(V) or 1.5 mM As(III) and UFD2 mRNA levels were determined by qRT-PCR (AU, Arbitrary Units). Values represent the mean±s.d. of three biological replicates and statistical differences denoted as *** P
Figure Legend Snippet: Ufd2 mediates arsenic tolerance. (A) ufd2 cells are sensitive to arsenic stress. Exponential phase BY4742 wild type (WT) and the ufd2 mutant were serially diluted and spotted onto SC media supplemented or not with 1.5 mM As(III) or 2 mM As(V) or 1.5 mM As(III). SD, control. Growth was recorded after 2 days incubation at 30°C. A representative experiment is shown. Cell growth was also monitored by means of growth curves. Exponential phase BY4742 WT and ufd2 mutant cells were exposed or not to 2 mM As(V) or 1.5 mM As(III) for 22 h and OD 600 was monitored in intervals of 1 h. The curves represent the mean±s.d. of three biological replicates. (B) UFD2 is induced in cells injured with arsenic. BY4742 cells were challenged or not with 1.5 mM As(III) or 2 mM As(V) or 1.5 mM As(III) and UFD2 mRNA levels were determined by qRT-PCR (AU, Arbitrary Units). Values represent the mean±s.d. of three biological replicates and statistical differences denoted as *** P

Techniques Used: Mutagenesis, Incubation, Quantitative RT-PCR

9) Product Images from "LINC01714 Enhances Gemcitabine Sensitivity by Modulating FOXO3 Phosphorylation in Cholangiocarcinoma"

Article Title: LINC01714 Enhances Gemcitabine Sensitivity by Modulating FOXO3 Phosphorylation in Cholangiocarcinoma

Journal: Molecular Therapy. Nucleic Acids

doi: 10.1016/j.omtn.2019.11.028

LINC01714 Is Clinically Associated with CCA Patient Outcome (A) The expression level (FPKM value) of LINC01714 between paired CCA tumor and adjacent normal samples quantified from TCGA RNA-seq data. (B) Kaplan-Meier analysis of the association between LINC01714 expression level and the overall survival of CAA patients in the TCGA dataset. (C) Boxplot shows the relative expression of LINC01714 (70 matched CCA tumor/non-tumor samples in cohort 1). (D) Kaplan-Meier plot indicates the survival difference between CCA patients that show high and low expression of LINC01714. High expression and low expression in groups of CCA patients were divided by the median level of LINC01714 expression. (E) The genomic copy numbers of LINC01714 in tumor and non-tumor samples. (F) Kaplan-Meier plot indicates the survival difference between CCA patients that show high and low copy numbers of LINC01714. High and low copy number groups of CCA patients were divided by the median level of LINC01714 genomic copy number. The RNA levels of LINC01714 in (C) and (D) were quantified by quantitative real-time PCR. Values are indicated as the median with interquartile range in (A), (C), and (E). Statistical analyses were performed using the Student’s t test, as in (A), (C), and (E), and the log-rank test, as in (B), (D), and (F).
Figure Legend Snippet: LINC01714 Is Clinically Associated with CCA Patient Outcome (A) The expression level (FPKM value) of LINC01714 between paired CCA tumor and adjacent normal samples quantified from TCGA RNA-seq data. (B) Kaplan-Meier analysis of the association between LINC01714 expression level and the overall survival of CAA patients in the TCGA dataset. (C) Boxplot shows the relative expression of LINC01714 (70 matched CCA tumor/non-tumor samples in cohort 1). (D) Kaplan-Meier plot indicates the survival difference between CCA patients that show high and low expression of LINC01714. High expression and low expression in groups of CCA patients were divided by the median level of LINC01714 expression. (E) The genomic copy numbers of LINC01714 in tumor and non-tumor samples. (F) Kaplan-Meier plot indicates the survival difference between CCA patients that show high and low copy numbers of LINC01714. High and low copy number groups of CCA patients were divided by the median level of LINC01714 genomic copy number. The RNA levels of LINC01714 in (C) and (D) were quantified by quantitative real-time PCR. Values are indicated as the median with interquartile range in (A), (C), and (E). Statistical analyses were performed using the Student’s t test, as in (A), (C), and (E), and the log-rank test, as in (B), (D), and (F).

Techniques Used: Expressing, RNA Sequencing Assay, Cellular Antioxidant Activity Assay, Low Copy Number, Real-time Polymerase Chain Reaction

10) Product Images from "LINC01714 Enhances Gemcitabine Sensitivity by Modulating FOXO3 Phosphorylation in Cholangiocarcinoma"

Article Title: LINC01714 Enhances Gemcitabine Sensitivity by Modulating FOXO3 Phosphorylation in Cholangiocarcinoma

Journal: Molecular Therapy. Nucleic Acids

doi: 10.1016/j.omtn.2019.11.028

LIHC01714 Physically Interacts with FOXO3 in CCA Cells (A) LINC01714 pull-down assay analyzed by SDS-PAGE. (B) Western blot analysis of the FOXO3 protein retrieved from LINC01714 pull-down assay. (C) The quantitative real-time PCR results of RIP assays using an anti-FOXO3 antibody. (D) Immunoblotting detection of FOXO3 protein in the pull-down samples. The full-length sense and antisense biotinylated-LINC01714 (#1 and #5, respectively) and truncated biotinylated-LINC01714 sequences (#2 includes 1–287 bp, #3 includes 1–195 bp, and #4 includes 1–101 bp) were analyzed. β-actin serves as input control. (E) RIP assays for different domains of FOXO3 protein. Quantitative real-time PCR was used to determine the enrichment of LINC01714 binding with each FOXO3 domain. (F) Western blot analysis of FOXO3 protein level in LINC01714- or si-LIHC01714-transfected cells. (G) The FOXO3 protein levels were measured in LINC01714, si-LINC01714, or control cells. Cells were treated with MG132 (50 μmol/L) for 0, 4, 8, or 16 h before protein harvest. Values are indicated as mean ± SEM in (C) and (E). **p
Figure Legend Snippet: LIHC01714 Physically Interacts with FOXO3 in CCA Cells (A) LINC01714 pull-down assay analyzed by SDS-PAGE. (B) Western blot analysis of the FOXO3 protein retrieved from LINC01714 pull-down assay. (C) The quantitative real-time PCR results of RIP assays using an anti-FOXO3 antibody. (D) Immunoblotting detection of FOXO3 protein in the pull-down samples. The full-length sense and antisense biotinylated-LINC01714 (#1 and #5, respectively) and truncated biotinylated-LINC01714 sequences (#2 includes 1–287 bp, #3 includes 1–195 bp, and #4 includes 1–101 bp) were analyzed. β-actin serves as input control. (E) RIP assays for different domains of FOXO3 protein. Quantitative real-time PCR was used to determine the enrichment of LINC01714 binding with each FOXO3 domain. (F) Western blot analysis of FOXO3 protein level in LINC01714- or si-LIHC01714-transfected cells. (G) The FOXO3 protein levels were measured in LINC01714, si-LINC01714, or control cells. Cells were treated with MG132 (50 μmol/L) for 0, 4, 8, or 16 h before protein harvest. Values are indicated as mean ± SEM in (C) and (E). **p

Techniques Used: Pull Down Assay, SDS Page, Western Blot, Real-time Polymerase Chain Reaction, Binding Assay, Transfection

LINC01714 Regulates the Phosphorylation Level of FOXO3 in CCA Cells (A) Immunoblotting analysis of the FOXO3 distribution between cytoplasm and nucleus in LINC01714 (+) and LINC01714 (−) HuCCT1 cells. GAPDH and lamin B1 serve as controls in cytoplasm and nucleus, respectively. (B) Barplots show the statistical data of FOXO3 distribution between cytoplasm and nucleus in HuCCT1 cells. (C) Immunoblotting results show the phosphorylation status of FOXO3-S318 and FOXO3-S253 in HuCCT1 cells with or without LINC01714 transfection. (D) Immunofluorescence staining of HuCCT1 cells after transfection with LINC01714 or vector control. (E) Immunoblotting analysis for the phosphorylation status of AKT, ERK, and BAD proteins in HuCCT1 with LINC01714, LINC01714+siFOXO3, or control vector transfection. (F) Representative immunohistochemistry (IHC) images of FOXO3 expression levels in tumor and non-tumor samples. Barplots show the mRNA levels of LINC01714 in corresponding tumor and non-tumor samples. (G) Kaplan-Meier analysis of the influence of the LINC01714-FOXO3 combination on CCA patient survival. Values are indicated as mean ± SEM in (B) and (F). **p
Figure Legend Snippet: LINC01714 Regulates the Phosphorylation Level of FOXO3 in CCA Cells (A) Immunoblotting analysis of the FOXO3 distribution between cytoplasm and nucleus in LINC01714 (+) and LINC01714 (−) HuCCT1 cells. GAPDH and lamin B1 serve as controls in cytoplasm and nucleus, respectively. (B) Barplots show the statistical data of FOXO3 distribution between cytoplasm and nucleus in HuCCT1 cells. (C) Immunoblotting results show the phosphorylation status of FOXO3-S318 and FOXO3-S253 in HuCCT1 cells with or without LINC01714 transfection. (D) Immunofluorescence staining of HuCCT1 cells after transfection with LINC01714 or vector control. (E) Immunoblotting analysis for the phosphorylation status of AKT, ERK, and BAD proteins in HuCCT1 with LINC01714, LINC01714+siFOXO3, or control vector transfection. (F) Representative immunohistochemistry (IHC) images of FOXO3 expression levels in tumor and non-tumor samples. Barplots show the mRNA levels of LINC01714 in corresponding tumor and non-tumor samples. (G) Kaplan-Meier analysis of the influence of the LINC01714-FOXO3 combination on CCA patient survival. Values are indicated as mean ± SEM in (B) and (F). **p

Techniques Used: Transfection, Immunofluorescence, Staining, Plasmid Preparation, Immunohistochemistry, Expressing

LINC01714 Is Clinically Associated with CCA Patient Outcome (A) The expression level (FPKM value) of LINC01714 between paired CCA tumor and adjacent normal samples quantified from TCGA RNA-seq data. (B) Kaplan-Meier analysis of the association between LINC01714 expression level and the overall survival of CAA patients in the TCGA dataset. (C) Boxplot shows the relative expression of LINC01714 (70 matched CCA tumor/non-tumor samples in cohort 1). (D) Kaplan-Meier plot indicates the survival difference between CCA patients that show high and low expression of LINC01714. High expression and low expression in groups of CCA patients were divided by the median level of LINC01714 expression. (E) The genomic copy numbers of LINC01714 in tumor and non-tumor samples. (F) Kaplan-Meier plot indicates the survival difference between CCA patients that show high and low copy numbers of LINC01714. High and low copy number groups of CCA patients were divided by the median level of LINC01714 genomic copy number. The RNA levels of LINC01714 in (C) and (D) were quantified by quantitative real-time PCR. Values are indicated as the median with interquartile range in (A), (C), and (E). Statistical analyses were performed using the Student’s t test, as in (A), (C), and (E), and the log-rank test, as in (B), (D), and (F).
Figure Legend Snippet: LINC01714 Is Clinically Associated with CCA Patient Outcome (A) The expression level (FPKM value) of LINC01714 between paired CCA tumor and adjacent normal samples quantified from TCGA RNA-seq data. (B) Kaplan-Meier analysis of the association between LINC01714 expression level and the overall survival of CAA patients in the TCGA dataset. (C) Boxplot shows the relative expression of LINC01714 (70 matched CCA tumor/non-tumor samples in cohort 1). (D) Kaplan-Meier plot indicates the survival difference between CCA patients that show high and low expression of LINC01714. High expression and low expression in groups of CCA patients were divided by the median level of LINC01714 expression. (E) The genomic copy numbers of LINC01714 in tumor and non-tumor samples. (F) Kaplan-Meier plot indicates the survival difference between CCA patients that show high and low copy numbers of LINC01714. High and low copy number groups of CCA patients were divided by the median level of LINC01714 genomic copy number. The RNA levels of LINC01714 in (C) and (D) were quantified by quantitative real-time PCR. Values are indicated as the median with interquartile range in (A), (C), and (E). Statistical analyses were performed using the Student’s t test, as in (A), (C), and (E), and the log-rank test, as in (B), (D), and (F).

Techniques Used: Expressing, RNA Sequencing Assay, Cellular Antioxidant Activity Assay, Low Copy Number, Real-time Polymerase Chain Reaction

LINC01714 Suppresses Proliferation Migration and Invasion of CCA Tumor Cells In Vitro (A) Cell viability assays for CCLP1 cells transfected with LIHC01714 siRNAs or mock controls. (B) Cell migration assays for CCLP1 cells transfected with LIHC01714 siRNAs or mock controls. (C) Cell invasion assays for CCLP1 cells transfected with LIHC01714 siRNAs or mock controls. (D) LINC01714 suppressed the CCA tumor growth in the nude mouse model bearing subcutaneous tumor xenografts from the LINC01714-transfected HuCCT1 cell line. (E) Representative images of H E staining in the metastatic liver loci and statistical data comparing the LINC01714 group with the vector control group. (F) Representative images of H E staining in the metastatic lung loci and statistical data comparing the LINC01714 group with the vector control group. Values are indicated as median with interquartile range in (D) and as mean ± SEM in (A)–(C), (E), and (F). **p
Figure Legend Snippet: LINC01714 Suppresses Proliferation Migration and Invasion of CCA Tumor Cells In Vitro (A) Cell viability assays for CCLP1 cells transfected with LIHC01714 siRNAs or mock controls. (B) Cell migration assays for CCLP1 cells transfected with LIHC01714 siRNAs or mock controls. (C) Cell invasion assays for CCLP1 cells transfected with LIHC01714 siRNAs or mock controls. (D) LINC01714 suppressed the CCA tumor growth in the nude mouse model bearing subcutaneous tumor xenografts from the LINC01714-transfected HuCCT1 cell line. (E) Representative images of H E staining in the metastatic liver loci and statistical data comparing the LINC01714 group with the vector control group. (F) Representative images of H E staining in the metastatic lung loci and statistical data comparing the LINC01714 group with the vector control group. Values are indicated as median with interquartile range in (D) and as mean ± SEM in (A)–(C), (E), and (F). **p

Techniques Used: Migration, In Vitro, Transfection, Staining, Plasmid Preparation

LIHC01714 Is a Candidate Therapeutic Target for CCA (A) IC 50 analysis for gemcitabine-treated CCLP1 cells with si-LINC01714 and corresponding control transfection. (B) IC 50 analysis for gemcitabine-treated HuCCT1 cells with LINC01714 and corresponding control transfection. (C) LINC01714 suppressed the liver metastasis of CCA tumor in the nude mouse model bearing subcutaneous tumor xenografts from gemcitabine-treated CCLP1 cell lines. (D) Thermographic assessment of liver or lung tumor in LINC01714 overexpression and control mouse groups. (E) Representative data of H E staining in the tumor foci of the liver or lung samples obtained from nude mice after injection with LINC01714-overexpression cells. (F) Survival analysis between mouse groups with gemcitabine-treated CCLP1 cell lines with LINC01714 or control transfection. (G) IC 50 analysis for gemcitabine-treated CCLP1 cells with siLINC01714+FOXO3 and corresponding control transfection (Student’s t test p
Figure Legend Snippet: LIHC01714 Is a Candidate Therapeutic Target for CCA (A) IC 50 analysis for gemcitabine-treated CCLP1 cells with si-LINC01714 and corresponding control transfection. (B) IC 50 analysis for gemcitabine-treated HuCCT1 cells with LINC01714 and corresponding control transfection. (C) LINC01714 suppressed the liver metastasis of CCA tumor in the nude mouse model bearing subcutaneous tumor xenografts from gemcitabine-treated CCLP1 cell lines. (D) Thermographic assessment of liver or lung tumor in LINC01714 overexpression and control mouse groups. (E) Representative data of H E staining in the tumor foci of the liver or lung samples obtained from nude mice after injection with LINC01714-overexpression cells. (F) Survival analysis between mouse groups with gemcitabine-treated CCLP1 cell lines with LINC01714 or control transfection. (G) IC 50 analysis for gemcitabine-treated CCLP1 cells with siLINC01714+FOXO3 and corresponding control transfection (Student’s t test p

Techniques Used: Transfection, Over Expression, Staining, Mouse Assay, Injection

11) Product Images from "Role of ?B in Regulating the Compatible Solute Uptake Systems of Listeria monocytogenes: Osmotic Induction of opuC Is ?B Dependent"

Article Title: Role of ?B in Regulating the Compatible Solute Uptake Systems of Listeria monocytogenes: Osmotic Induction of opuC Is ?B Dependent

Journal: Applied and Environmental Microbiology

doi: 10.1128/AEM.69.4.2015-2022.2003

RT-PCR analysis of compatible solute transporter transcript levels. Transcript levels from either the wild type (10403S) or the Δ sigB mutant were assessed, as indicated. Prior to preparation of total cellular RNA, the cells were grown in DM in either the presence or absence of NaCl, as indicated. The products were run on ethidium bromide-stained gels and photographed under UV illumination. The cDNA concentration was normalized against the amount of product generated by primers against the 16S rRNA gene. The number of PCR cycles used in each experiment is indicated in parentheses. The results shown are representative of those from two independent experiments, each performed in duplicate.
Figure Legend Snippet: RT-PCR analysis of compatible solute transporter transcript levels. Transcript levels from either the wild type (10403S) or the Δ sigB mutant were assessed, as indicated. Prior to preparation of total cellular RNA, the cells were grown in DM in either the presence or absence of NaCl, as indicated. The products were run on ethidium bromide-stained gels and photographed under UV illumination. The cDNA concentration was normalized against the amount of product generated by primers against the 16S rRNA gene. The number of PCR cycles used in each experiment is indicated in parentheses. The results shown are representative of those from two independent experiments, each performed in duplicate.

Techniques Used: Reverse Transcription Polymerase Chain Reaction, Mutagenesis, Staining, Concentration Assay, Generated, Polymerase Chain Reaction

12) Product Images from "linc-HOXA1 is a noncoding RNA that represses Hoxa1 transcription in cis"

Article Title: linc-HOXA1 is a noncoding RNA that represses Hoxa1 transcription in cis

Journal: Genes & Development

doi: 10.1101/gad.217018.113

Identification and characterization of linc-HOXA1 as a noncoding RNA. ( A ) Illustration of the genomic location and transcript structure of the linc-HOXA1 gene and its three isoforms. ( B ) Cumulative probability distribution of coding potential as measured
Figure Legend Snippet: Identification and characterization of linc-HOXA1 as a noncoding RNA. ( A ) Illustration of the genomic location and transcript structure of the linc-HOXA1 gene and its three isoforms. ( B ) Cumulative probability distribution of coding potential as measured

Techniques Used:

Knockdown of linc-HOXA1 increases Hoxa1 levels. ( A ) Bar graphs showing changes in average number of linc-HOXA1 and Hoxa1 RNA molecules per cell upon treatment with siRNA or Isis antisense oligonucleotides targeting either linc-HOXA1 ( top row) or Hoxa1
Figure Legend Snippet: Knockdown of linc-HOXA1 increases Hoxa1 levels. ( A ) Bar graphs showing changes in average number of linc-HOXA1 and Hoxa1 RNA molecules per cell upon treatment with siRNA or Isis antisense oligonucleotides targeting either linc-HOXA1 ( top row) or Hoxa1

Techniques Used:

Overexpression of linc-HOXA1 isoforms does not alter Hoxa1 abundance. ( A ) Bar plots showing the number of the various linc-HOXA1 RNA isoforms overexpressed in embryonic stem cells (and a vector containing GFP as a control) ( left ) and the resultant number
Figure Legend Snippet: Overexpression of linc-HOXA1 isoforms does not alter Hoxa1 abundance. ( A ) Bar plots showing the number of the various linc-HOXA1 RNA isoforms overexpressed in embryonic stem cells (and a vector containing GFP as a control) ( left ) and the resultant number

Techniques Used: Over Expression, Plasmid Preparation

linc-HOXA1 and Hoxa1 anti-correlate at the single-cell level. ( A ) Micrographs of three representative mouse embryonic stem cells expressing linc-HOXA1 (red) and Hoxa1 (blue). The arrow points to the location of an active transcription site. ( B ) Average
Figure Legend Snippet: linc-HOXA1 and Hoxa1 anti-correlate at the single-cell level. ( A ) Micrographs of three representative mouse embryonic stem cells expressing linc-HOXA1 (red) and Hoxa1 (blue). The arrow points to the location of an active transcription site. ( B ) Average

Techniques Used: Expressing

Detection of individual RNA isoforms of linc-HOXA1 in single cells. ( A ) Raw micrographs of a single cell containing all three isoforms. (Three left panels) We designed probes specifically targeting exons 3c, 3b, and 3a, each of which revealed single spots
Figure Legend Snippet: Detection of individual RNA isoforms of linc-HOXA1 in single cells. ( A ) Raw micrographs of a single cell containing all three isoforms. (Three left panels) We designed probes specifically targeting exons 3c, 3b, and 3a, each of which revealed single spots

Techniques Used:

PURB binds to linc-HOXA1 in vitro and in vivo and mediates repression of Hoxa1 transcription. ( A ) Illustration of linc-HOXA1 gene structure showing the location of the GAA purine-rich region. ( B ) Protein products visualized by gel electrophoresis that
Figure Legend Snippet: PURB binds to linc-HOXA1 in vitro and in vivo and mediates repression of Hoxa1 transcription. ( A ) Illustration of linc-HOXA1 gene structure showing the location of the GAA purine-rich region. ( B ) Protein products visualized by gel electrophoresis that

Techniques Used: In Vitro, In Vivo, Nucleic Acid Electrophoresis

13) Product Images from "linc-HOXA1 is a noncoding RNA that represses Hoxa1 transcription in cis"

Article Title: linc-HOXA1 is a noncoding RNA that represses Hoxa1 transcription in cis

Journal: Genes & Development

doi: 10.1101/gad.217018.113

Identification and characterization of linc-HOXA1 as a noncoding RNA. ( A ) Illustration of the genomic location and transcript structure of the linc-HOXA1 gene and its three isoforms. ( B ) Cumulative probability distribution of coding potential as measured
Figure Legend Snippet: Identification and characterization of linc-HOXA1 as a noncoding RNA. ( A ) Illustration of the genomic location and transcript structure of the linc-HOXA1 gene and its three isoforms. ( B ) Cumulative probability distribution of coding potential as measured

Techniques Used:

Overexpression of linc-HOXA1 isoforms does not alter Hoxa1 abundance. ( A ) Bar plots showing the number of the various linc-HOXA1 RNA isoforms overexpressed in embryonic stem cells (and a vector containing GFP as a control) ( left ) and the resultant number
Figure Legend Snippet: Overexpression of linc-HOXA1 isoforms does not alter Hoxa1 abundance. ( A ) Bar plots showing the number of the various linc-HOXA1 RNA isoforms overexpressed in embryonic stem cells (and a vector containing GFP as a control) ( left ) and the resultant number

Techniques Used: Over Expression, Plasmid Preparation

Detection of individual RNA isoforms of linc-HOXA1 in single cells. ( A ) Raw micrographs of a single cell containing all three isoforms. (Three left panels) We designed probes specifically targeting exons 3c, 3b, and 3a, each of which revealed single spots
Figure Legend Snippet: Detection of individual RNA isoforms of linc-HOXA1 in single cells. ( A ) Raw micrographs of a single cell containing all three isoforms. (Three left panels) We designed probes specifically targeting exons 3c, 3b, and 3a, each of which revealed single spots

Techniques Used:

14) Product Images from "Acid-Induced Gene Expression in Helicobacter pylori: Study in Genomic Scale by Microarray"

Article Title: Acid-Induced Gene Expression in Helicobacter pylori: Study in Genomic Scale by Microarray

Journal: Infection and Immunity

doi: 10.1128/IAI.69.3.1679-1686.2001

Semiquantification of HP1037 ORF mRNA by PCR. Total RNAs were isolated at pH 7.2 (A) and pH 5.5 (B) separately. One microgram from each was reverse transcribed with antisense primer HP1037-r. Tenfold serial dilutions of cDNA were made, and the end point was determined by negativity of PCR. (−), RNA templates not subjected to the RT reaction served as negative control to exclude the possibility of contamination of genomic DNA; 1×, 1 μg of total RNA used as template; 10×, 100×, and 1000×, sample was at a 10-, 100-, or 1,000-fold serial dilution, respectively.
Figure Legend Snippet: Semiquantification of HP1037 ORF mRNA by PCR. Total RNAs were isolated at pH 7.2 (A) and pH 5.5 (B) separately. One microgram from each was reverse transcribed with antisense primer HP1037-r. Tenfold serial dilutions of cDNA were made, and the end point was determined by negativity of PCR. (−), RNA templates not subjected to the RT reaction served as negative control to exclude the possibility of contamination of genomic DNA; 1×, 1 μg of total RNA used as template; 10×, 100×, and 1000×, sample was at a 10-, 100-, or 1,000-fold serial dilution, respectively.

Techniques Used: Polymerase Chain Reaction, Isolation, Negative Control, Serial Dilution

15) Product Images from "PIWI Proteins Are Dispensable for Mouse Somatic Development and Reprogramming of Fibroblasts into Pluripotent Stem Cells"

Article Title: PIWI Proteins Are Dispensable for Mouse Somatic Development and Reprogramming of Fibroblasts into Pluripotent Stem Cells

Journal: PLoS ONE

doi: 10.1371/journal.pone.0097821

Expression of Piwi transcripts. qRT-PCR comparison of piwi expression in mouse cells (A) and human cells (B). RNA was isolated from mouse ESCs (CCE) and embryonic fibroblasts (MEF) and human ESCs (H1 and H7), human foreskin keratinocytes, human foreskin fibroblasts. The ratios of individual piwi genes/eukaryotic 18S rRNA are shown for both panels.
Figure Legend Snippet: Expression of Piwi transcripts. qRT-PCR comparison of piwi expression in mouse cells (A) and human cells (B). RNA was isolated from mouse ESCs (CCE) and embryonic fibroblasts (MEF) and human ESCs (H1 and H7), human foreskin keratinocytes, human foreskin fibroblasts. The ratios of individual piwi genes/eukaryotic 18S rRNA are shown for both panels.

Techniques Used: Expressing, Quantitative RT-PCR, Isolation

16) Product Images from "Silencing of IFN-stimulated gene transcription is regulated by histone H1 and its chaperone TAF-I"

Article Title: Silencing of IFN-stimulated gene transcription is regulated by histone H1 and its chaperone TAF-I

Journal: Nucleic Acids Research

doi: 10.1093/nar/gku485

TAF-I negatively regulates ISG transcription. ( A ) Expression level of TAF-I in TAF-I KD cells. HeLa S3 cells were transfected with EGFP siRNA expression vector used as a control (siCont, lanes 1–3) or with TAF-I siRNA expression vector (siTAF-I, lane 4). Cell lysates were subjected to western blot analysis using anti-TAF-I and anti-β-actin antibodies. ( B ) and ( C ) Effects of TAF-I KD in ISG transcription. Total RNA was prepared from siCont- and siTAF-I-transfected cells treated with or without IFN-β for 3 h (B) or for indicated periods (C) and subjected to qRT-PCR using specific primer sets for ISG mRNAs, ISG15 pre-mRNA and GAPDH mRNA. The amount of ISG mRNA was normalized as a relative amount of GAPDH mRNA. The amounts of ISG15 mRNA and ISG15 pre-mRNA in TAF-I KD cells relative to those of control cells were shown in the right panel of (C). Error bars represent standard deviation ( n ≥ 3). * P
Figure Legend Snippet: TAF-I negatively regulates ISG transcription. ( A ) Expression level of TAF-I in TAF-I KD cells. HeLa S3 cells were transfected with EGFP siRNA expression vector used as a control (siCont, lanes 1–3) or with TAF-I siRNA expression vector (siTAF-I, lane 4). Cell lysates were subjected to western blot analysis using anti-TAF-I and anti-β-actin antibodies. ( B ) and ( C ) Effects of TAF-I KD in ISG transcription. Total RNA was prepared from siCont- and siTAF-I-transfected cells treated with or without IFN-β for 3 h (B) or for indicated periods (C) and subjected to qRT-PCR using specific primer sets for ISG mRNAs, ISG15 pre-mRNA and GAPDH mRNA. The amount of ISG mRNA was normalized as a relative amount of GAPDH mRNA. The amounts of ISG15 mRNA and ISG15 pre-mRNA in TAF-I KD cells relative to those of control cells were shown in the right panel of (C). Error bars represent standard deviation ( n ≥ 3). * P

Techniques Used: Expressing, Transfection, Plasmid Preparation, Western Blot, Quantitative RT-PCR, Standard Deviation

Histone H1 negatively regulates ISG transcription. ( A ) Expression level of histone H1 in histone H1 KD cells. HeLa S3 cells were transfected with EGFP siRNA expression vector used as a control (siCont, lanes 1–3) or histone H1 siRNA expression vector (siH1, lane 4). Cell lysates were subjected to western blot analysis using anti-histone H1.2 (H1) and anti-β-actin antibodies. ( B ) Effects of histone H1 KD on ISG transcription. Total RNA was prepared from siCont- and siH1-transfected cells treated with or without IFN-β for 3 h and subjected to qRT-PCR using specific primer sets for each ISG mRNA and GAPDH mRNA. The amount of ISG mRNA was normalized as relative amounts of GAPDH mRNA. Error bars represent standard deviation ( n ≥ 3). * P
Figure Legend Snippet: Histone H1 negatively regulates ISG transcription. ( A ) Expression level of histone H1 in histone H1 KD cells. HeLa S3 cells were transfected with EGFP siRNA expression vector used as a control (siCont, lanes 1–3) or histone H1 siRNA expression vector (siH1, lane 4). Cell lysates were subjected to western blot analysis using anti-histone H1.2 (H1) and anti-β-actin antibodies. ( B ) Effects of histone H1 KD on ISG transcription. Total RNA was prepared from siCont- and siH1-transfected cells treated with or without IFN-β for 3 h and subjected to qRT-PCR using specific primer sets for each ISG mRNA and GAPDH mRNA. The amount of ISG mRNA was normalized as relative amounts of GAPDH mRNA. Error bars represent standard deviation ( n ≥ 3). * P

Techniques Used: Expressing, Transfection, Plasmid Preparation, Western Blot, Quantitative RT-PCR, Standard Deviation

TAF-I and histone H1 regulate the chromatin structure of ISG promoters. ( A ) Nucleosome digestion by MNase. HeLa S3 cells were transfected with EGFP siRNA expression vector (siCont, lanes 1–4), TAF-I siRNA expression vector (siTAF-I, lanes 5–8) and histone H1 siRNA expression vector (siH1, lanes 9–12), and then treated with or without IFN-β for indicated periods followed by the fixation with formaldehyde. MNase digestion was carried out as described in the Materials and Methods section, and then genomic DNAs were purified, separated by electrophoresis in 1% agarose gel and visualized by ethidium bromide staining. Lane M shows DNA size markers. ( B ) Increase of the MNase sensitivity of the ISG15 promoter in TAF-I KD and H1 KD cells. MNase-digested DNA was prepared as shown in (A) and subjected to qRT-PCR using specific primer sets for the ISG15 and GAPDH promoters. The amount of the ISG15 promoter DNA was normalized by that of the GAPDH promoter DNA and is shown as a relative amount to that from IFN-untreated control cells. Error bars represent standard deviation ( n ≥ 3). * P
Figure Legend Snippet: TAF-I and histone H1 regulate the chromatin structure of ISG promoters. ( A ) Nucleosome digestion by MNase. HeLa S3 cells were transfected with EGFP siRNA expression vector (siCont, lanes 1–4), TAF-I siRNA expression vector (siTAF-I, lanes 5–8) and histone H1 siRNA expression vector (siH1, lanes 9–12), and then treated with or without IFN-β for indicated periods followed by the fixation with formaldehyde. MNase digestion was carried out as described in the Materials and Methods section, and then genomic DNAs were purified, separated by electrophoresis in 1% agarose gel and visualized by ethidium bromide staining. Lane M shows DNA size markers. ( B ) Increase of the MNase sensitivity of the ISG15 promoter in TAF-I KD and H1 KD cells. MNase-digested DNA was prepared as shown in (A) and subjected to qRT-PCR using specific primer sets for the ISG15 and GAPDH promoters. The amount of the ISG15 promoter DNA was normalized by that of the GAPDH promoter DNA and is shown as a relative amount to that from IFN-untreated control cells. Error bars represent standard deviation ( n ≥ 3). * P

Techniques Used: Transfection, Expressing, Plasmid Preparation, Purification, Electrophoresis, Agarose Gel Electrophoresis, Staining, Quantitative RT-PCR, Standard Deviation

TAF-I regulates the amounts of the transcription factors and histone H1 on ISG promoters. ( A ) Promoter binding of transcription factors in TAF-I KD cells. siCont- and siTAF-I-transfected cells were treated with or without IFN-β for indicated periods, and cell lysates were subjected to ChIP assays using antibodies specific for STAT1 (left), STAT2 (middle) and Pol II (right) followed by qRT-PCR using a specific primer set for the ISG15 promoter. The amount of DNA co-immunoprecipitated with each antibody was shown as % of input. Error bars represent standard deviation ( n ≥ 3). * P
Figure Legend Snippet: TAF-I regulates the amounts of the transcription factors and histone H1 on ISG promoters. ( A ) Promoter binding of transcription factors in TAF-I KD cells. siCont- and siTAF-I-transfected cells were treated with or without IFN-β for indicated periods, and cell lysates were subjected to ChIP assays using antibodies specific for STAT1 (left), STAT2 (middle) and Pol II (right) followed by qRT-PCR using a specific primer set for the ISG15 promoter. The amount of DNA co-immunoprecipitated with each antibody was shown as % of input. Error bars represent standard deviation ( n ≥ 3). * P

Techniques Used: Binding Assay, Transfection, Chromatin Immunoprecipitation, Quantitative RT-PCR, Immunoprecipitation, Standard Deviation

The acidic domain of TAF-I is essential for ISG transcriptional regulation. ( A ) IFN-responsive STAT phosphorylation in TAF-I KD cells. Cell lysates were prepared from siCont- (lanes 1–4) and siTAF-I-transfected (lanes 5–8) cells treated with or without IFN-β for indicated periods, and subjected to western blot analysis using anti-tyrosine phosphorylated (pY)-STAT1, anti-pY-STAT2, anti-serine phosphorylated (pS)-STAT1, anti-STAT1, anti-STAT2, anti-IRF9, anti-TAF-I and anti-β-actin antibodies. ( B ) Dispensability of TAF-I in ISRE-dependent transcription. HEK293 cells were transfected with EGFP siRNA expression vector used as a control (siCont) or with TAF-I siRNA expression vector (siTAF-I) together with pISRE-TA- Luc and pSEAP-control, and then cells were treated with or without IFN-β for 6 h followed by the luciferase assay. The luciferase activity was normalized with the SEAP activity and represented as fold-induction relative to that from IFN-treated control cells. Error bars represent standard deviation ( n ≥ 3). ‘n/s’ indicates ‘not significant’. ( C ) Schematic representation of TAF-I proteins. The N-terminal regions specific for TAF-Iα (aa 1–37) and TAF-Iβ (aa 1–24) and the C-terminal acidic region for TAF-Iα (aa 239–290) and TAF-Iβ (aa 226–227) are indicated. ( D ) Expression level of Flag-TAF-Is in TAF-I KD cells. Cell lysates were prepared from HeLa S3 cells transfected with EGFP siRNA expression vector used as a control (lanes 1–3) or TAF-I siRNA expression vector (siTAF-I, lanes 4–8) together with expression vectors expressing Flag-tagged TAF-Iα (lane 5), Flag-tagged TAF-IαΔC (lane 6), Flag-tagged TAF-Iβ (lane 7), Flag-tagged TAF-IβΔC (lane 8) or empty vector (lane 4), and cell lysates were subjected to western blot analysis using anti-TAF-I, anti-Flag and anti-β-actin antibodies. ( E ) Transcription rescue experiments by Flag-TAF-I. Cells were prepared as shown in (D) and treated with or without IFN-β for 3 h. Total RNA was subjected to qRT-PCR using specific primer sets for ISG15 mRNA (left), ISG56 mRNA (right) and GAPDH mRNA. The amount of ISG mRNA was normalized as a relative amount of GAPDH mRNA. Error bars represent standard deviation ( n ≥ 3). * P
Figure Legend Snippet: The acidic domain of TAF-I is essential for ISG transcriptional regulation. ( A ) IFN-responsive STAT phosphorylation in TAF-I KD cells. Cell lysates were prepared from siCont- (lanes 1–4) and siTAF-I-transfected (lanes 5–8) cells treated with or without IFN-β for indicated periods, and subjected to western blot analysis using anti-tyrosine phosphorylated (pY)-STAT1, anti-pY-STAT2, anti-serine phosphorylated (pS)-STAT1, anti-STAT1, anti-STAT2, anti-IRF9, anti-TAF-I and anti-β-actin antibodies. ( B ) Dispensability of TAF-I in ISRE-dependent transcription. HEK293 cells were transfected with EGFP siRNA expression vector used as a control (siCont) or with TAF-I siRNA expression vector (siTAF-I) together with pISRE-TA- Luc and pSEAP-control, and then cells were treated with or without IFN-β for 6 h followed by the luciferase assay. The luciferase activity was normalized with the SEAP activity and represented as fold-induction relative to that from IFN-treated control cells. Error bars represent standard deviation ( n ≥ 3). ‘n/s’ indicates ‘not significant’. ( C ) Schematic representation of TAF-I proteins. The N-terminal regions specific for TAF-Iα (aa 1–37) and TAF-Iβ (aa 1–24) and the C-terminal acidic region for TAF-Iα (aa 239–290) and TAF-Iβ (aa 226–227) are indicated. ( D ) Expression level of Flag-TAF-Is in TAF-I KD cells. Cell lysates were prepared from HeLa S3 cells transfected with EGFP siRNA expression vector used as a control (lanes 1–3) or TAF-I siRNA expression vector (siTAF-I, lanes 4–8) together with expression vectors expressing Flag-tagged TAF-Iα (lane 5), Flag-tagged TAF-IαΔC (lane 6), Flag-tagged TAF-Iβ (lane 7), Flag-tagged TAF-IβΔC (lane 8) or empty vector (lane 4), and cell lysates were subjected to western blot analysis using anti-TAF-I, anti-Flag and anti-β-actin antibodies. ( E ) Transcription rescue experiments by Flag-TAF-I. Cells were prepared as shown in (D) and treated with or without IFN-β for 3 h. Total RNA was subjected to qRT-PCR using specific primer sets for ISG15 mRNA (left), ISG56 mRNA (right) and GAPDH mRNA. The amount of ISG mRNA was normalized as a relative amount of GAPDH mRNA. Error bars represent standard deviation ( n ≥ 3). * P

Techniques Used: Transfection, Western Blot, Expressing, Plasmid Preparation, Luciferase, Activity Assay, Standard Deviation, Quantitative RT-PCR

Dissociation of TAF-I from ISG promoters. ( A ) Dynamics of TAF-I at ISG promoters in an IFN-dependent manner. HeLa S3 cells were transfected with TAF-I siRNA expression vector (lanes 1–4) together with empty vector (lane 4) or Flag-tagged TAF-Iα expression vector (lanes 1–3) and then treated with or without IFN-β for indicated periods. Cells were, then, subjected to ChIP assays using the agarose-conjugated antibody against Flag followed by qRT-PCR using specific primer sets for each ISG promoter. The amount of DNA co-immunoprecipitated with each antibody was shown as % of input. Error bars represent standard deviation ( n ≥ 3). * P
Figure Legend Snippet: Dissociation of TAF-I from ISG promoters. ( A ) Dynamics of TAF-I at ISG promoters in an IFN-dependent manner. HeLa S3 cells were transfected with TAF-I siRNA expression vector (lanes 1–4) together with empty vector (lane 4) or Flag-tagged TAF-Iα expression vector (lanes 1–3) and then treated with or without IFN-β for indicated periods. Cells were, then, subjected to ChIP assays using the agarose-conjugated antibody against Flag followed by qRT-PCR using specific primer sets for each ISG promoter. The amount of DNA co-immunoprecipitated with each antibody was shown as % of input. Error bars represent standard deviation ( n ≥ 3). * P

Techniques Used: Transfection, Expressing, Plasmid Preparation, Chromatin Immunoprecipitation, Quantitative RT-PCR, Immunoprecipitation, Standard Deviation

17) Product Images from "OmpR-Mediated Transcriptional Regulation and Function of Two Heme Receptor Proteins of Yersinia enterocolitica Bio-Serotype 2/O:9"

Article Title: OmpR-Mediated Transcriptional Regulation and Function of Two Heme Receptor Proteins of Yersinia enterocolitica Bio-Serotype 2/O:9

Journal: Frontiers in Cellular and Infection Microbiology

doi: 10.3389/fcimb.2018.00333

Influence of temperature on hemR1 expression. (A) Potential FourU thermometer RNA secondary structures in the 5′-UTRs of Y. enterocolitica Ye9 hemR1 and S. dysenteriae shuA , composed of a stretch of FourU and the ribosomal binding site (RBS), revealed by Mfold analysis ( http://mfold.rna.albany.edu ). Boxes indicate the location of the FourU motif (shaded) and putative RBS, the translation start codon is marked red, and unpaired nucleotides within the predicted secondary structure are in green. (B) Temperature-dependent HemR1 expression in Y. enterocolitica examined by immunoblotting. The analyzed samples were cell lysates of the fur mutant grown at 26 or 37°C in LB medium. The top panel shows the Western blot probed with a polyclonal antibody against HemR1, the bottom panel shows the Coomassie blue-stained gel as a loading control. The percentage values on the blot, indicating the HemR1 band intensities relative to that of cells grown at 26°C, were determined using Amersham Imager 600 Analysis Software V1.0.0 (GE Healthcare). (C) Expression of a hemR1 ′-′ gfp translational fusion at different temperatures, monitored by fluorescence intensity. E. coli BW25113 harboring the reporter plasmid pFX-P lac -hemR1 or control plasmids pFX-0 and pFX-1 were grown in LB medium at 26 or 37°C. The GFP fluorescence intensity (RFU) of overnight cultures was determined. The data represent the averages ± SD from at least three experiments with duplicate cultures. Significance was calculated using one-way ANOVA [ P > 0.05, * P
Figure Legend Snippet: Influence of temperature on hemR1 expression. (A) Potential FourU thermometer RNA secondary structures in the 5′-UTRs of Y. enterocolitica Ye9 hemR1 and S. dysenteriae shuA , composed of a stretch of FourU and the ribosomal binding site (RBS), revealed by Mfold analysis ( http://mfold.rna.albany.edu ). Boxes indicate the location of the FourU motif (shaded) and putative RBS, the translation start codon is marked red, and unpaired nucleotides within the predicted secondary structure are in green. (B) Temperature-dependent HemR1 expression in Y. enterocolitica examined by immunoblotting. The analyzed samples were cell lysates of the fur mutant grown at 26 or 37°C in LB medium. The top panel shows the Western blot probed with a polyclonal antibody against HemR1, the bottom panel shows the Coomassie blue-stained gel as a loading control. The percentage values on the blot, indicating the HemR1 band intensities relative to that of cells grown at 26°C, were determined using Amersham Imager 600 Analysis Software V1.0.0 (GE Healthcare). (C) Expression of a hemR1 ′-′ gfp translational fusion at different temperatures, monitored by fluorescence intensity. E. coli BW25113 harboring the reporter plasmid pFX-P lac -hemR1 or control plasmids pFX-0 and pFX-1 were grown in LB medium at 26 or 37°C. The GFP fluorescence intensity (RFU) of overnight cultures was determined. The data represent the averages ± SD from at least three experiments with duplicate cultures. Significance was calculated using one-way ANOVA [ P > 0.05, * P

Techniques Used: Expressing, Binding Assay, Mutagenesis, Western Blot, Staining, Software, Fluorescence, Plasmid Preparation

sqRT-PCR analysis of the hemPRSTUV- 1 and hemPRST -2 gene clusters of Y. enterocolitica Ye9. (A,C) Scheme showing primers used in sqRT-PCR analysis of hem clusters 1 and 2. The sequences of the following primers are listed in Table S2 . 1, RThPR1F; 2, RThPR1R; 3, RThRS1F; 4, RThRS1R; 5, RThPV1R; 6, RThPR2F; 7, RThPR2R; 8, RThRS2F; 9, RThRS2R; 10, RThPT2R. (B,D) Agarose gel electrophoresis of sqRT-PCR products from hem clusters 1 and 2. Total RNA isolated from strain Ye9 grown in iron-depleted LBD medium (LB with the iron chelator 2,2′-dipyridyl) at 37°C was DNase I treated and then reverse transcribed with random hexamers. The obtained cDNA was used as a template in PCR reaction with pairs of primers shown in (A) and (C) . RNA was used as the template in negative control reactions. M, GeneRuler 1 kb DNA Ladder.
Figure Legend Snippet: sqRT-PCR analysis of the hemPRSTUV- 1 and hemPRST -2 gene clusters of Y. enterocolitica Ye9. (A,C) Scheme showing primers used in sqRT-PCR analysis of hem clusters 1 and 2. The sequences of the following primers are listed in Table S2 . 1, RThPR1F; 2, RThPR1R; 3, RThRS1F; 4, RThRS1R; 5, RThPV1R; 6, RThPR2F; 7, RThPR2R; 8, RThRS2F; 9, RThRS2R; 10, RThPT2R. (B,D) Agarose gel electrophoresis of sqRT-PCR products from hem clusters 1 and 2. Total RNA isolated from strain Ye9 grown in iron-depleted LBD medium (LB with the iron chelator 2,2′-dipyridyl) at 37°C was DNase I treated and then reverse transcribed with random hexamers. The obtained cDNA was used as a template in PCR reaction with pairs of primers shown in (A) and (C) . RNA was used as the template in negative control reactions. M, GeneRuler 1 kb DNA Ladder.

Techniques Used: Polymerase Chain Reaction, Agarose Gel Electrophoresis, Isolation, Negative Control

18) Product Images from "OmpR-Mediated Transcriptional Regulation and Function of Two Heme Receptor Proteins of Yersinia enterocolitica Bio-Serotype 2/O:9"

Article Title: OmpR-Mediated Transcriptional Regulation and Function of Two Heme Receptor Proteins of Yersinia enterocolitica Bio-Serotype 2/O:9

Journal: Frontiers in Cellular and Infection Microbiology

doi: 10.3389/fcimb.2018.00333

Influence of temperature on hemR1 expression. (A) Potential FourU thermometer RNA secondary structures in the 5′-UTRs of Y. enterocolitica Ye9 hemR1 and S. dysenteriae shuA ). Boxes indicate the location of the FourU motif (shaded) and putative RBS, the translation start codon is marked red, and unpaired nucleotides within the predicted secondary structure are in green. (B) Temperature-dependent HemR1 expression in Y. enterocolitica examined by immunoblotting. The analyzed samples were cell lysates of the fur mutant grown at 26 or 37°C in LB medium. The top panel shows the Western blot probed with a polyclonal antibody against HemR1, the bottom panel shows the Coomassie blue-stained gel as a loading control. The percentage values on the blot, indicating the HemR1 band intensities relative to that of cells grown at 26°C, were determined using Amersham Imager 600 Analysis Software V1.0.0 (GE Healthcare). (C) Expression of a hemR1 ′-′ gfp translational fusion at different temperatures, monitored by fluorescence intensity. E. coli BW25113 harboring the reporter plasmid pFX-P lac -hemR1 or control plasmids pFX-0 and pFX-1 were grown in LB medium at 26 or 37°C. The GFP fluorescence intensity (RFU) of overnight cultures was determined. The data represent the averages ± SD from at least three experiments with duplicate cultures. Significance was calculated using one-way ANOVA [ P > 0.05, * P
Figure Legend Snippet: Influence of temperature on hemR1 expression. (A) Potential FourU thermometer RNA secondary structures in the 5′-UTRs of Y. enterocolitica Ye9 hemR1 and S. dysenteriae shuA ). Boxes indicate the location of the FourU motif (shaded) and putative RBS, the translation start codon is marked red, and unpaired nucleotides within the predicted secondary structure are in green. (B) Temperature-dependent HemR1 expression in Y. enterocolitica examined by immunoblotting. The analyzed samples were cell lysates of the fur mutant grown at 26 or 37°C in LB medium. The top panel shows the Western blot probed with a polyclonal antibody against HemR1, the bottom panel shows the Coomassie blue-stained gel as a loading control. The percentage values on the blot, indicating the HemR1 band intensities relative to that of cells grown at 26°C, were determined using Amersham Imager 600 Analysis Software V1.0.0 (GE Healthcare). (C) Expression of a hemR1 ′-′ gfp translational fusion at different temperatures, monitored by fluorescence intensity. E. coli BW25113 harboring the reporter plasmid pFX-P lac -hemR1 or control plasmids pFX-0 and pFX-1 were grown in LB medium at 26 or 37°C. The GFP fluorescence intensity (RFU) of overnight cultures was determined. The data represent the averages ± SD from at least three experiments with duplicate cultures. Significance was calculated using one-way ANOVA [ P > 0.05, * P

Techniques Used: Expressing, Mutagenesis, Western Blot, Staining, Software, Fluorescence, Plasmid Preparation

19) Product Images from "A yeast model for target-primed (non-LTR) retrotransposition"

Article Title: A yeast model for target-primed (non-LTR) retrotransposition

Journal: BMC Genomics

doi: 10.1186/1471-2164-8-263

Expression and splicing of tagged Zorro 3 RNA . (A) Map of tagged Zorro3 element showing primer locations. URNAR2 straddles the intron insertion site. (B) RT-PCR of tagged Zorro3 RNA. Genomic DNA and total RNA were separately extracted from a C. albicans CAI4 derivative stably transformed with a plasmid carrying a marked Zorro3 element. The RNA was reverse transcribed using oligonucleotide ZRNAR2 as a primer. PCRs were then performed using oligonucleotides URNAR1 and ZRNAR1 as primers. Templates in the reactions were as follows: lane 1, total RNA reverse transcribed with primer ZRNAR2; lane 2, total RNA not reverse transcribed; lane 3, genomic DNA; lane 4, no template. The intron-containing PCR amplicon obtained using primers URNAR1 and ZRNAR1 would be 364 bp long. Amplification of reverse transcribed RNA from which the intron had been precisely spliced would produce an amplicon of 280 bp. Primer URNAR2 was used (in combination with ZRNAR1) to quantify the tagged Zorro3 RNA (see text).
Figure Legend Snippet: Expression and splicing of tagged Zorro 3 RNA . (A) Map of tagged Zorro3 element showing primer locations. URNAR2 straddles the intron insertion site. (B) RT-PCR of tagged Zorro3 RNA. Genomic DNA and total RNA were separately extracted from a C. albicans CAI4 derivative stably transformed with a plasmid carrying a marked Zorro3 element. The RNA was reverse transcribed using oligonucleotide ZRNAR2 as a primer. PCRs were then performed using oligonucleotides URNAR1 and ZRNAR1 as primers. Templates in the reactions were as follows: lane 1, total RNA reverse transcribed with primer ZRNAR2; lane 2, total RNA not reverse transcribed; lane 3, genomic DNA; lane 4, no template. The intron-containing PCR amplicon obtained using primers URNAR1 and ZRNAR1 would be 364 bp long. Amplification of reverse transcribed RNA from which the intron had been precisely spliced would produce an amplicon of 280 bp. Primer URNAR2 was used (in combination with ZRNAR1) to quantify the tagged Zorro3 RNA (see text).

Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction, Stable Transfection, Transformation Assay, Plasmid Preparation, Polymerase Chain Reaction, Amplification

20) Product Images from "CCDC26 knockdown enhances resistance of gastrointestinal stromal tumor cells to imatinib by interacting with c-KIT"

Article Title: CCDC26 knockdown enhances resistance of gastrointestinal stromal tumor cells to imatinib by interacting with c-KIT

Journal: American Journal of Translational Research

doi:

CCDC26 expression in GIST cells and its relationship with imatinib sensitivity. A. Real-time PCR detection of CCDC26 expression in GIST cell lines. B. CCK-8 assay of GIST cell viability in the presence of imatinib (0, 20, 40, 60, 80 μM). C, D. GIST cell viability in the presence of IC50 of imatinib (GIST-882, 56.06 μM; GIST-T1, 41.08 μM) for 0 h, 6 h, 24 h, and 48 h (** P
Figure Legend Snippet: CCDC26 expression in GIST cells and its relationship with imatinib sensitivity. A. Real-time PCR detection of CCDC26 expression in GIST cell lines. B. CCK-8 assay of GIST cell viability in the presence of imatinib (0, 20, 40, 60, 80 μM). C, D. GIST cell viability in the presence of IC50 of imatinib (GIST-882, 56.06 μM; GIST-T1, 41.08 μM) for 0 h, 6 h, 24 h, and 48 h (** P

Techniques Used: Expressing, Real-time Polymerase Chain Reaction, CCK-8 Assay

The relationship between CCDC26 and c-KIT. A. Western blot determination of c-KIT expression levels following RNA pull-down assay in CCDC26 RNA group (Target), control RNA group (Untreated), and blank group (None). (L, total protein; FT, flow-through; E, eluate). B, C. The effect of CCDC26 siRNA on c-KIT mRNA expression in GIST cells; GAPDH was the internal control (** P
Figure Legend Snippet: The relationship between CCDC26 and c-KIT. A. Western blot determination of c-KIT expression levels following RNA pull-down assay in CCDC26 RNA group (Target), control RNA group (Untreated), and blank group (None). (L, total protein; FT, flow-through; E, eluate). B, C. The effect of CCDC26 siRNA on c-KIT mRNA expression in GIST cells; GAPDH was the internal control (** P

Techniques Used: Western Blot, Expressing, Pull Down Assay, Flow Cytometry

Effect of CCDC26 on GIST cell viability, proliferation, and apoptosis in vitro . A, B. CCK-8 assay of the viability of GIST cells treated with CCDC26 siRNA or control siRNA in the presence of imatinib (0, 20, 40, 60, 80 μM). C, D. EdU assay of cell proliferation rate under IC50 of imatinib in GIST cells treated with CCDC26 siRNA or control siRNA. The number of EdU-positive cells was counted. E. Flow cytometry analysis of apoptosis in GIST cells transfected with control siRNA or CCDC26 siRNA (* P
Figure Legend Snippet: Effect of CCDC26 on GIST cell viability, proliferation, and apoptosis in vitro . A, B. CCK-8 assay of the viability of GIST cells treated with CCDC26 siRNA or control siRNA in the presence of imatinib (0, 20, 40, 60, 80 μM). C, D. EdU assay of cell proliferation rate under IC50 of imatinib in GIST cells treated with CCDC26 siRNA or control siRNA. The number of EdU-positive cells was counted. E. Flow cytometry analysis of apoptosis in GIST cells transfected with control siRNA or CCDC26 siRNA (* P

Techniques Used: In Vitro, CCK-8 Assay, EdU Assay, Flow Cytometry, Cytometry, Transfection

c-KIT knockdown reversed CCDC26 inhibition-mediated imatinib resistance. A. Western blotting validation of the interference efficiency of c-KIT siRNA. B, C. CCK-8 assay of the viability of GIST cells treated with c-KIT siRNA or both c-KIT siRNA and CCDC26 siRNA in the presence of imatinib (0, 20, 40, 60, 80 μM).
Figure Legend Snippet: c-KIT knockdown reversed CCDC26 inhibition-mediated imatinib resistance. A. Western blotting validation of the interference efficiency of c-KIT siRNA. B, C. CCK-8 assay of the viability of GIST cells treated with c-KIT siRNA or both c-KIT siRNA and CCDC26 siRNA in the presence of imatinib (0, 20, 40, 60, 80 μM).

Techniques Used: Inhibition, Western Blot, CCK-8 Assay

Effect of c-KIT on GIST cell viability, proliferation, and apoptosis in vitro . A, B. CCK-8 assay of the viability of GIST cells treated with c-KIT siRNA, Negative siRNA, or Control in the presence of imatinib (0, 20, 40, 60, 80 μM). C, D. EdU assay of cell proliferation rate under IC50 of imatinib in GIST cells treated with c-KIT siRNA or control siRNA. The number of EDU-positive cells was counted. E. Flow cytometry analysis of apoptosis in GIST cells transfected with control siRNA or CCDC26 siRNA (* P
Figure Legend Snippet: Effect of c-KIT on GIST cell viability, proliferation, and apoptosis in vitro . A, B. CCK-8 assay of the viability of GIST cells treated with c-KIT siRNA, Negative siRNA, or Control in the presence of imatinib (0, 20, 40, 60, 80 μM). C, D. EdU assay of cell proliferation rate under IC50 of imatinib in GIST cells treated with c-KIT siRNA or control siRNA. The number of EDU-positive cells was counted. E. Flow cytometry analysis of apoptosis in GIST cells transfected with control siRNA or CCDC26 siRNA (* P

Techniques Used: In Vitro, CCK-8 Assay, EdU Assay, Flow Cytometry, Cytometry, Transfection

21) Product Images from "Long noncoding RNA CNALPTC1 promotes cell proliferation and migration of papillary thyroid cancer via sponging miR-30 family"

Article Title: Long noncoding RNA CNALPTC1 promotes cell proliferation and migration of papillary thyroid cancer via sponging miR-30 family

Journal: American Journal of Cancer Research

doi:

CNALPTC1 exerts oncogenic activity in PTC via sponging miR-30 family. A. After transfection of CNALPTC1 or CNALPTC1-mut overexpression plasmids, or co-transfection of CNALPTC1 overexpression plasmids and miR-30 mimics mix into TPC-1 cells, cell proliferation was determined by CCK-8 assays. B. After transfection of CNALPTC1 or CNALPTC1-mut overexpression plasmids, or co-transfection of CNALPTC1 overexpression plasmids and miR-30 mimics mix into TPC-1 cells, cell proliferation was determined by EdU incorporation assays. The red colour indicates EdU-positive nuclei. Scale bars = 100 µm. C. After transfection of CNALPTC1 or CNALPTC1-mut overexpression plasmids, or co-transfection of CNALPTC1 overexpression plasmids and miR-30 mimics mix into TPC-1 cells, cell apoptosis was determined by TUNEL assays. The green colour indicates TUNEL-positive or apoptotic cells. Scale bars = 100 µm. D. After transfection of CNALPTC1 or CNALPTC1-mut overexpression plasmids, or co-transfection of CNALPTC1 overexpression plasmids and miR-30 mimics mix into TPC-1 cells, cell migration was determined by transwell assays. Represent images are shown. Scale bars = 100 µm. Results are shown as mean ± s.d. of 3 independent experiments. *P
Figure Legend Snippet: CNALPTC1 exerts oncogenic activity in PTC via sponging miR-30 family. A. After transfection of CNALPTC1 or CNALPTC1-mut overexpression plasmids, or co-transfection of CNALPTC1 overexpression plasmids and miR-30 mimics mix into TPC-1 cells, cell proliferation was determined by CCK-8 assays. B. After transfection of CNALPTC1 or CNALPTC1-mut overexpression plasmids, or co-transfection of CNALPTC1 overexpression plasmids and miR-30 mimics mix into TPC-1 cells, cell proliferation was determined by EdU incorporation assays. The red colour indicates EdU-positive nuclei. Scale bars = 100 µm. C. After transfection of CNALPTC1 or CNALPTC1-mut overexpression plasmids, or co-transfection of CNALPTC1 overexpression plasmids and miR-30 mimics mix into TPC-1 cells, cell apoptosis was determined by TUNEL assays. The green colour indicates TUNEL-positive or apoptotic cells. Scale bars = 100 µm. D. After transfection of CNALPTC1 or CNALPTC1-mut overexpression plasmids, or co-transfection of CNALPTC1 overexpression plasmids and miR-30 mimics mix into TPC-1 cells, cell migration was determined by transwell assays. Represent images are shown. Scale bars = 100 µm. Results are shown as mean ± s.d. of 3 independent experiments. *P

Techniques Used: Activity Assay, Transfection, Over Expression, Cotransfection, CCK-8 Assay, TUNEL Assay, Migration

CNALPTC1 up-regulates miR-30 targets BCL9 , SNAI1 , and VIM expression. A. Luciferase activities of pmirGLO, pmirGLO-BCL9, pmirGLO-SNAI1, or pmirGLO-VIM upon transfection of CNALPTC1 or miR-30 binding site mutated CNALPTC1 (CNALPTC1-mut) overexpression plasmids, or co-transfection of CNALPTC1 overexpression plasmids and miR-30 mimics mix. Results are presented as the relative ratio of firefly luciferase activity to renilla luciferase activity. B. Luciferase activities of pmirGLO, pmirGLO-BCL9, pmirGLO-SNAI1, or pmirGLO-VIM in CNALPTC1 stably depleted and control TPC-1 cells. Results are presented as the relative ratio of firefly luciferase activity to renilla luciferase activity. C. After transfection of CNALPTC1 or CNALPTC1-mut overexpression plasmids, or co-transfection of CNALPTC1 overexpression plasmids and miR-30 mimics mix into TPC-1 cells, BCL9 , SNAI1 , and VIM RNA expression levels were determined by qRT-PCR. D. BCL9 , SNAI1 , and VIM RNA expression levels were determined by qRT-PCR in CNALPTC1 stably depleted and control TPC-1 cells. E. After transfection of CNALPTC1 or CNALPTC1-mut overexpression plasmids, or co-transfection of CNALPTC1 overexpression plasmids and miR-30 mimics mix into TPC-1 cells, BCL9 , SNAI1 , and VIM protein expression levels were determined by western blot. F. BCL9 , SNAI1 , and VIM protein expression levels were determined by western blot in CNALPTC1 stably depleted and control TPC-1 cells. Results are shown as mean ± s.d. of 3 independent experiments. **P
Figure Legend Snippet: CNALPTC1 up-regulates miR-30 targets BCL9 , SNAI1 , and VIM expression. A. Luciferase activities of pmirGLO, pmirGLO-BCL9, pmirGLO-SNAI1, or pmirGLO-VIM upon transfection of CNALPTC1 or miR-30 binding site mutated CNALPTC1 (CNALPTC1-mut) overexpression plasmids, or co-transfection of CNALPTC1 overexpression plasmids and miR-30 mimics mix. Results are presented as the relative ratio of firefly luciferase activity to renilla luciferase activity. B. Luciferase activities of pmirGLO, pmirGLO-BCL9, pmirGLO-SNAI1, or pmirGLO-VIM in CNALPTC1 stably depleted and control TPC-1 cells. Results are presented as the relative ratio of firefly luciferase activity to renilla luciferase activity. C. After transfection of CNALPTC1 or CNALPTC1-mut overexpression plasmids, or co-transfection of CNALPTC1 overexpression plasmids and miR-30 mimics mix into TPC-1 cells, BCL9 , SNAI1 , and VIM RNA expression levels were determined by qRT-PCR. D. BCL9 , SNAI1 , and VIM RNA expression levels were determined by qRT-PCR in CNALPTC1 stably depleted and control TPC-1 cells. E. After transfection of CNALPTC1 or CNALPTC1-mut overexpression plasmids, or co-transfection of CNALPTC1 overexpression plasmids and miR-30 mimics mix into TPC-1 cells, BCL9 , SNAI1 , and VIM protein expression levels were determined by western blot. F. BCL9 , SNAI1 , and VIM protein expression levels were determined by western blot in CNALPTC1 stably depleted and control TPC-1 cells. Results are shown as mean ± s.d. of 3 independent experiments. **P

Techniques Used: Expressing, Luciferase, Transfection, Binding Assay, Over Expression, Cotransfection, Activity Assay, Stable Transfection, RNA Expression, Quantitative RT-PCR, Western Blot

CNALPTC1 sponges and down-regulates miR-30 family. A. Schematic outlining the predicted binding site of miR-30 family on CNALPTC1. B. The specific bindings of miR-30 family to CNALPTC1 or miR-30 binding site mutated CNALPTC1 (CNALPTC1-mut) were determined by MS2 based RIP assays, followed by qRT-PCR. C. TPC-1 cell lysates were incubated with biotin-labeled CNALPTC1 or CNALPTC1-mut; after pull-down, microRNAs were extracted and determined by qRT-PCR. D. CNALPTC1 or miR-30 binding site mutated CNALPTC1 (CNALPTC1-mut) overexpression plasmids were transfected into TPC-1 cells. Forty-eight hours later, miR-30 family expression levels were determined by qRT-PCR. E. Expression levels of miR-30 family were determined by qRT-PCR in CNALPTC1 stably depleted and control TPC-1 cells. Results are shown as mean ± s.d. of 3 independent experiments. **P
Figure Legend Snippet: CNALPTC1 sponges and down-regulates miR-30 family. A. Schematic outlining the predicted binding site of miR-30 family on CNALPTC1. B. The specific bindings of miR-30 family to CNALPTC1 or miR-30 binding site mutated CNALPTC1 (CNALPTC1-mut) were determined by MS2 based RIP assays, followed by qRT-PCR. C. TPC-1 cell lysates were incubated with biotin-labeled CNALPTC1 or CNALPTC1-mut; after pull-down, microRNAs were extracted and determined by qRT-PCR. D. CNALPTC1 or miR-30 binding site mutated CNALPTC1 (CNALPTC1-mut) overexpression plasmids were transfected into TPC-1 cells. Forty-eight hours later, miR-30 family expression levels were determined by qRT-PCR. E. Expression levels of miR-30 family were determined by qRT-PCR in CNALPTC1 stably depleted and control TPC-1 cells. Results are shown as mean ± s.d. of 3 independent experiments. **P

Techniques Used: Binding Assay, Quantitative RT-PCR, Incubation, Labeling, Over Expression, Transfection, Expressing, Stable Transfection

Depletion of CNALPTC1 represses migration of PTC cells. A. Migration of CNALPTC1 stably depleted and control TPC-1 cells were determined by transwell assays. Represent images are shown. Scale bars = 100 µm. B. Migration of CNALPTC1 stably depleted and control IHH-4 cells were determined by transwell assays. Represent images are shown. Scale bars = 100 µm. Results are shown as mean ± s.d. of 3 independent experiments. ***P
Figure Legend Snippet: Depletion of CNALPTC1 represses migration of PTC cells. A. Migration of CNALPTC1 stably depleted and control TPC-1 cells were determined by transwell assays. Represent images are shown. Scale bars = 100 µm. B. Migration of CNALPTC1 stably depleted and control IHH-4 cells were determined by transwell assays. Represent images are shown. Scale bars = 100 µm. Results are shown as mean ± s.d. of 3 independent experiments. ***P

Techniques Used: Migration, Stable Transfection

Depletion of CNALPTC1 promotes apoptosis of PTC cells. A. Apoptosis of CNALPTC1 stably depleted and control TPC-1 cells were determined by TUNEL assays. The green colour indicates TUNEL-positive and apoptotic cells. Scale bars = 100 µm. B. Apoptosis of CNALPTC1 stably depleted and control IHH-4 cells were determined by TUNEL assays. The green colour indicates TUNEL-positive and apoptotic cells. Scale bars = 100 µm. Results are shown as mean ± s.d. of 3 independent experiments. **P
Figure Legend Snippet: Depletion of CNALPTC1 promotes apoptosis of PTC cells. A. Apoptosis of CNALPTC1 stably depleted and control TPC-1 cells were determined by TUNEL assays. The green colour indicates TUNEL-positive and apoptotic cells. Scale bars = 100 µm. B. Apoptosis of CNALPTC1 stably depleted and control IHH-4 cells were determined by TUNEL assays. The green colour indicates TUNEL-positive and apoptotic cells. Scale bars = 100 µm. Results are shown as mean ± s.d. of 3 independent experiments. **P

Techniques Used: Stable Transfection, TUNEL Assay

Depletion of CNALPTC1 inhibits proliferation of PTC cells. A. CNALPTC1 RNA expression levels were determined by qRT-PCR in CNALPTC1 stably depleted and control TPC-1 cells. B. Cell proliferation of CNALPTC1 stably depleted and control TPC-1 cells were determined by CCK-8 assays. C. Cell proliferation of CNALPTC1 stably depleted and control TPC-1 cells were determined by EdU incorporation assays. The red colour indicates EdU-positive nuclei. Scale bars = 100 µm. D. CNALPTC1 RNA expression levels were determined by qRT-PCR in CNALPTC1 stably depleted and control IHH-4 cells. E. Cell proliferation of CNALPTC1 stably depleted and control IHH-4 cells were determined by CCK-8 assays. F. Cell proliferation of CNALPTC1 stably depleted and control IHH-4 cells were determined by EdU incorporation assays. The red colour indicates EdU-positive nuclei. Scale bars = 100 µm. Results are shown as mean ± s.d. of 3 independent experiments. **P
Figure Legend Snippet: Depletion of CNALPTC1 inhibits proliferation of PTC cells. A. CNALPTC1 RNA expression levels were determined by qRT-PCR in CNALPTC1 stably depleted and control TPC-1 cells. B. Cell proliferation of CNALPTC1 stably depleted and control TPC-1 cells were determined by CCK-8 assays. C. Cell proliferation of CNALPTC1 stably depleted and control TPC-1 cells were determined by EdU incorporation assays. The red colour indicates EdU-positive nuclei. Scale bars = 100 µm. D. CNALPTC1 RNA expression levels were determined by qRT-PCR in CNALPTC1 stably depleted and control IHH-4 cells. E. Cell proliferation of CNALPTC1 stably depleted and control IHH-4 cells were determined by CCK-8 assays. F. Cell proliferation of CNALPTC1 stably depleted and control IHH-4 cells were determined by EdU incorporation assays. The red colour indicates EdU-positive nuclei. Scale bars = 100 µm. Results are shown as mean ± s.d. of 3 independent experiments. **P

Techniques Used: RNA Expression, Quantitative RT-PCR, Stable Transfection, CCK-8 Assay

22) Product Images from "Low Expression of lncRNA-GAS5 Is Implicated in Human Primary Varicose Great Saphenous Veins"

Article Title: Low Expression of lncRNA-GAS5 Is Implicated in Human Primary Varicose Great Saphenous Veins

Journal: PLoS ONE

doi: 10.1371/journal.pone.0120550

The expression of lncRNAs-GAS5 by Q-RT-PCR with the increase of tested sample pairs. lncRNA-GAS5 show significantly different expressions consistently with the increasing of tested samples. ΔΔCT show the actual relative expression fold change as 2 ΔΔCT . Values are mean±SE. *: P
Figure Legend Snippet: The expression of lncRNAs-GAS5 by Q-RT-PCR with the increase of tested sample pairs. lncRNA-GAS5 show significantly different expressions consistently with the increasing of tested samples. ΔΔCT show the actual relative expression fold change as 2 ΔΔCT . Values are mean±SE. *: P

Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction

lncRNA-GAS5 affects the proliferation of HSVSMCs. As a proliferation inhibitor, rapamycin was used to inhibit the proliferation abilities of normal HSVSMCs to make the proliferation abilities changes more significant. The concentration of rapamycin used in the study were 0, 100 and 200ng/ml, and the action time of rapamycin were 48 hours. A: Knockdown of lncRNA-GAS5 increased the proliferation of HSVSMCs by CCK-8 assay. Values are mean±SE, N = 3; *, P
Figure Legend Snippet: lncRNA-GAS5 affects the proliferation of HSVSMCs. As a proliferation inhibitor, rapamycin was used to inhibit the proliferation abilities of normal HSVSMCs to make the proliferation abilities changes more significant. The concentration of rapamycin used in the study were 0, 100 and 200ng/ml, and the action time of rapamycin were 48 hours. A: Knockdown of lncRNA-GAS5 increased the proliferation of HSVSMCs by CCK-8 assay. Values are mean±SE, N = 3; *, P

Techniques Used: Concentration Assay, CCK-8 Assay

lncRNA-GAS5 affects the migration of HSVSMCs. Cell scratch test and Transwell were used to measure the migration abilities of HSVSMCs. NC = Negative control group, only control siRNA transfected; GAS5(-) = lncRNA-GAS5 knockdown group transfected with silence siRNA. A: Cell scratch test was used to measure the migration abilities of HSVSMCs. The results showed that the HSVSMCs have the best migration abilities in the first 24 hours. Values are mean±SE, N = 4. B: The migration abilities of HSVSMCs measured by Transwell. After transfected by lncRNA-GAS5 siRNA for 48 hours, the HSVSMCs were passage into the Transwell Inserts. Then 4 hours, 7 hours, 10 hours later, the migration HSVSMCs were photographed and counted, respectively. Knockdown of lncRNA-GAS5 expression promotes migration of HSVSMCs. Optical microscope images under 200x magnification. C: The migration abilities of HSVSMCs were reflected indirectly by the new migration cells counting with Transwell. Silencing of lncRNA-GAS5 expression increses migration ability of HSVSMCs. Values are mean±SE, N = 10; *, P
Figure Legend Snippet: lncRNA-GAS5 affects the migration of HSVSMCs. Cell scratch test and Transwell were used to measure the migration abilities of HSVSMCs. NC = Negative control group, only control siRNA transfected; GAS5(-) = lncRNA-GAS5 knockdown group transfected with silence siRNA. A: Cell scratch test was used to measure the migration abilities of HSVSMCs. The results showed that the HSVSMCs have the best migration abilities in the first 24 hours. Values are mean±SE, N = 4. B: The migration abilities of HSVSMCs measured by Transwell. After transfected by lncRNA-GAS5 siRNA for 48 hours, the HSVSMCs were passage into the Transwell Inserts. Then 4 hours, 7 hours, 10 hours later, the migration HSVSMCs were photographed and counted, respectively. Knockdown of lncRNA-GAS5 expression promotes migration of HSVSMCs. Optical microscope images under 200x magnification. C: The migration abilities of HSVSMCs were reflected indirectly by the new migration cells counting with Transwell. Silencing of lncRNA-GAS5 expression increses migration ability of HSVSMCs. Values are mean±SE, N = 10; *, P

Techniques Used: Migration, Negative Control, Transfection, Expressing, Microscopy

lncRNA-GAS5 affects the cell cycle and apoptosis of HSVSMCs. As Negative regulatory factors of Cyclin Dependent Kinase, p27-kip1and p21-cip1 can delay the process of cell cycle acting as antagonist to Cyclin. Therefore, they were chosen to reflected cell cycle changes indirectly. A: The p27-kip1and p21-cip1 mRNA expression levels were measured by Q-RT-PCR with knockdown of lncRNA-GAS5 in HSVSMCs. Values are mean±SE, N = 3; *, P
Figure Legend Snippet: lncRNA-GAS5 affects the cell cycle and apoptosis of HSVSMCs. As Negative regulatory factors of Cyclin Dependent Kinase, p27-kip1and p21-cip1 can delay the process of cell cycle acting as antagonist to Cyclin. Therefore, they were chosen to reflected cell cycle changes indirectly. A: The p27-kip1and p21-cip1 mRNA expression levels were measured by Q-RT-PCR with knockdown of lncRNA-GAS5 in HSVSMCs. Values are mean±SE, N = 3; *, P

Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction

The effects of lncRNA-GAS5 mediated by Annexin A2. A2 = Annexin A2; Mock = control group, only empty plasmid transfected; NC = Negative control group, only control siRNA transfected; A2 siRNA = Annexin A2 knockdown group transfected with silence siRNA; A2 over = Annexin A2 overexpression group transfected with Annexin A2 expression plasmid; GAS5 down = lncRNA-GAS5 knockdown group transfected with silence siRNA. A: Silver stains for protein gels obtained by lncRNA-GAS5 RNA Pulldown. Bio-GAS5 (sense): treatment group; Bio-GAS5 (antisense): control groups; GAS5 (sense): negative control groups. The GAS5- specific-binding protein gels (in Red box) was identified by MALDI-TOF-MS. Results show that Annexin A2 isoform 2 is a direct binding protein to lncRNA-GAS5. N = 2. B: The Annexin A2 expression level was knockdowned in HSVSMCs effectively by siRNA. Values are mean±SE, N = 3; *, P
Figure Legend Snippet: The effects of lncRNA-GAS5 mediated by Annexin A2. A2 = Annexin A2; Mock = control group, only empty plasmid transfected; NC = Negative control group, only control siRNA transfected; A2 siRNA = Annexin A2 knockdown group transfected with silence siRNA; A2 over = Annexin A2 overexpression group transfected with Annexin A2 expression plasmid; GAS5 down = lncRNA-GAS5 knockdown group transfected with silence siRNA. A: Silver stains for protein gels obtained by lncRNA-GAS5 RNA Pulldown. Bio-GAS5 (sense): treatment group; Bio-GAS5 (antisense): control groups; GAS5 (sense): negative control groups. The GAS5- specific-binding protein gels (in Red box) was identified by MALDI-TOF-MS. Results show that Annexin A2 isoform 2 is a direct binding protein to lncRNA-GAS5. N = 2. B: The Annexin A2 expression level was knockdowned in HSVSMCs effectively by siRNA. Values are mean±SE, N = 3; *, P

Techniques Used: Plasmid Preparation, Transfection, Negative Control, Over Expression, Expressing, Binding Assay, Mass Spectrometry

Knockdown and over-expression of lncRNA-GAS5 in HSVSMCs. A: The lncRNA-GAS5 expression level was knockdowned by siRNA. Three siRNAs knockdowned the lncRNA-GAS5 expression level in HSVSMCs effectively. mean±SE, N = 3; P
Figure Legend Snippet: Knockdown and over-expression of lncRNA-GAS5 in HSVSMCs. A: The lncRNA-GAS5 expression level was knockdowned by siRNA. Three siRNAs knockdowned the lncRNA-GAS5 expression level in HSVSMCs effectively. mean±SE, N = 3; P

Techniques Used: Over Expression, Expressing

23) Product Images from "LncRNA DLEU1 contributes to colorectal cancer progression via activation of KPNA3"

Article Title: LncRNA DLEU1 contributes to colorectal cancer progression via activation of KPNA3

Journal: Molecular Cancer

doi: 10.1186/s12943-018-0873-2

DLEU1 interacts with SMARCA1 in CRC cells. a The expression of DLEU1 in cytoplasm and nucleus of HCT8 cells was measured by qRT-PCR. U6 serves as a nuclear control. GAPDH serves as a cytoplasmic control. b SMARCA1 was a potential interactive candidate of DLEU1. Biotin-labeled DLEU1 and intron control were incubated with HCT8 cell lysates, and the enriched products were eluted and separated by SDS-PAGE electrophoresis and silver staining. The differential band appearing in DLEU1 lane was analyzed by mass spectrum. c DLEU1 associated with SMARCA1 as shown by RNA pulldown and Western blot. Biotin-labeled DLEU1 and intron control were added into HCT8 cell lysates, and pulldown assays were performed. d DLEU1 was enriched by SMARCA1 in HCT8 and SW480 cell lysates. e SMARCA1 enriched DLEU1 in HCT8 cell lysates. SMARCA1 antibody was added into cell lysates and enriched RNAs were isolated. Then enriched DLEU1 was analyzed by PCR. f DLEU1 co-localized with SMARCA1 in HCT8 cells as shown by RNA FISH. Green, DLEU1; Red, SMARCA1; Blue, DAPI. Scale bar, 10 μm. g the region of nt 1~ 400 in DLEU1 was important for the interaction with SMARCA1. h DLEU1 (nt 1~ 400) associated with SMARCA1 directly as shown by RNA EMSA assays. i The region of nt 700~ 1050 is indispensable for the function of DLEU1 in colorectal cancer. Overexpression of DLEU1 with deletion of nt 1~ 400 cannot promoted proliferation and metastasis in CC. *** P
Figure Legend Snippet: DLEU1 interacts with SMARCA1 in CRC cells. a The expression of DLEU1 in cytoplasm and nucleus of HCT8 cells was measured by qRT-PCR. U6 serves as a nuclear control. GAPDH serves as a cytoplasmic control. b SMARCA1 was a potential interactive candidate of DLEU1. Biotin-labeled DLEU1 and intron control were incubated with HCT8 cell lysates, and the enriched products were eluted and separated by SDS-PAGE electrophoresis and silver staining. The differential band appearing in DLEU1 lane was analyzed by mass spectrum. c DLEU1 associated with SMARCA1 as shown by RNA pulldown and Western blot. Biotin-labeled DLEU1 and intron control were added into HCT8 cell lysates, and pulldown assays were performed. d DLEU1 was enriched by SMARCA1 in HCT8 and SW480 cell lysates. e SMARCA1 enriched DLEU1 in HCT8 cell lysates. SMARCA1 antibody was added into cell lysates and enriched RNAs were isolated. Then enriched DLEU1 was analyzed by PCR. f DLEU1 co-localized with SMARCA1 in HCT8 cells as shown by RNA FISH. Green, DLEU1; Red, SMARCA1; Blue, DAPI. Scale bar, 10 μm. g the region of nt 1~ 400 in DLEU1 was important for the interaction with SMARCA1. h DLEU1 (nt 1~ 400) associated with SMARCA1 directly as shown by RNA EMSA assays. i The region of nt 700~ 1050 is indispensable for the function of DLEU1 in colorectal cancer. Overexpression of DLEU1 with deletion of nt 1~ 400 cannot promoted proliferation and metastasis in CC. *** P

Techniques Used: Expressing, Quantitative RT-PCR, Labeling, Incubation, SDS Page, Electrophoresis, Silver Staining, Western Blot, Isolation, Polymerase Chain Reaction, Fluorescence In Situ Hybridization, Over Expression

24) Product Images from "Genome-Wide Prediction and Validation of Intergenic Enhancers in Arabidopsis Using Open Chromatin Signatures [OPEN]"

Article Title: Genome-Wide Prediction and Validation of Intergenic Enhancers in Arabidopsis Using Open Chromatin Signatures [OPEN]

Journal: The Plant Cell

doi: 10.1105/tpc.15.00537

DNase I-PCR Assay of the DNase I Sensitivity of a Transgenic Enhancer Sequence. (A) . (B) A diagram illustrating the design of a PCR primer that allows the transgenic L3 locus, but not the endogenous L3, to be specifically amplified. (C) DNase I-PCR shows differential DNase I sensitivity of chromatin in selected genomic regions. The DNase I sensitivity of the transgenic L3 locus was similar to that of the open chromatin control ACTIN7 .
Figure Legend Snippet: DNase I-PCR Assay of the DNase I Sensitivity of a Transgenic Enhancer Sequence. (A) . (B) A diagram illustrating the design of a PCR primer that allows the transgenic L3 locus, but not the endogenous L3, to be specifically amplified. (C) DNase I-PCR shows differential DNase I sensitivity of chromatin in selected genomic regions. The DNase I sensitivity of the transgenic L3 locus was similar to that of the open chromatin control ACTIN7 .

Techniques Used: Polymerase Chain Reaction, Transgenic Assay, Sequencing, Amplification

25) Product Images from "A Simple and Effective Method for High Quality Co-Extraction of Genomic DNA and Total RNA from Low Biomass Ectocarpus siliculosus, the Model Brown Alga"

Article Title: A Simple and Effective Method for High Quality Co-Extraction of Genomic DNA and Total RNA from Low Biomass Ectocarpus siliculosus, the Model Brown Alga

Journal: PLoS ONE

doi: 10.1371/journal.pone.0096470

Summary of nucleic acids extraction from E. siliculosus brown alga. High yields of good quality DNA and RNA are isolated from as little as 25 Steps 1–5: Harvested tissue is immediately homogenised using commercial 3 mm solid-glass beads in the presence of 1 mL EB containing 100 mM Tris-HCl, 150 mM NaCl, 5 mM DTT and 1% sarkosyl. These stages allow the lysis of the cell wall, the release of highest amount of nucleic acids, the inactivation of cellular nucleases, and the removal of most of the polysaccharides and other insoluble material. Steps 6–10: Simultaneous presence of absolute ethanol and potassium acetate aids polysaccharide precipitation. Moreover proteins, lipids, pigments and cell debris are removed through extraction of the aqueous phase with chloroform. Steps 11–12: Nucleic acids are then recovered by precipitation with 0.8 V of isopropanol and 0.1 V of 3 M sodium acetate (pH 5.2) in the presence of 1% 2-mercaptoetanol at −80°C. During the precipitation step, salts and other solutes are separated from nucleic acids that form a white precipitate collected by centrifugation. The excess of isopropanol and 2-mercaptoetanol are removed through washing the pellet with 75% ethanol. Step 13: All traces of ethanol are removed, the nucleic acid pellet is dried and resuspended in nuclease-free water. After RNase or DNase treatment the superfluous quantities of proteins, polysaccharides, lipids, and cell debris were removed from the extracted DNA and RNA through double extended purification treatment with phenol:chloroform:isoamyl alcohol.
Figure Legend Snippet: Summary of nucleic acids extraction from E. siliculosus brown alga. High yields of good quality DNA and RNA are isolated from as little as 25 Steps 1–5: Harvested tissue is immediately homogenised using commercial 3 mm solid-glass beads in the presence of 1 mL EB containing 100 mM Tris-HCl, 150 mM NaCl, 5 mM DTT and 1% sarkosyl. These stages allow the lysis of the cell wall, the release of highest amount of nucleic acids, the inactivation of cellular nucleases, and the removal of most of the polysaccharides and other insoluble material. Steps 6–10: Simultaneous presence of absolute ethanol and potassium acetate aids polysaccharide precipitation. Moreover proteins, lipids, pigments and cell debris are removed through extraction of the aqueous phase with chloroform. Steps 11–12: Nucleic acids are then recovered by precipitation with 0.8 V of isopropanol and 0.1 V of 3 M sodium acetate (pH 5.2) in the presence of 1% 2-mercaptoetanol at −80°C. During the precipitation step, salts and other solutes are separated from nucleic acids that form a white precipitate collected by centrifugation. The excess of isopropanol and 2-mercaptoetanol are removed through washing the pellet with 75% ethanol. Step 13: All traces of ethanol are removed, the nucleic acid pellet is dried and resuspended in nuclease-free water. After RNase or DNase treatment the superfluous quantities of proteins, polysaccharides, lipids, and cell debris were removed from the extracted DNA and RNA through double extended purification treatment with phenol:chloroform:isoamyl alcohol.

Techniques Used: Isolation, Lysis, Centrifugation, Purification

26) Product Images from "Promotion of mitochondrial biogenesis by necdin protects neurons against mitochondrial insults"

Article Title: Promotion of mitochondrial biogenesis by necdin protects neurons against mitochondrial insults

Journal: Nature Communications

doi: 10.1038/ncomms10943

Necdin stabilizes PGC-1α by inhibiting its ubiquitin-proteasomal degradation. ( a ) Forebrain extracts from E14.5 wild-type (WT) and necdin-null (KO) mice were immunoprecipitated with anti-necdin IgG and control preimmune IgG for endogenous binding between necdin and PGC-1α. PCNA, negative control. ( b ) Necdin and PGC-1α expressed in cDNA-transfected HEK293A cells were immunoprecipitated and immunoblotted. ( c ) Diagram of the PGC-1α deletion mutants. ( d ) Necdin and 6Myc-tagged PGC-1α deletion mutants in transfected HEK293A cells were immunoprecipitated and immunoblotted. ( e ) Bacterially synthesized MBP-fused PGC-1α deletion mutants were incubated with His-tagged necdin (His-Necdin). Bound His-Necdin was detected by immunoblotting. Recombinant proteins were stained with Coomassie Brilliant Blue (CBB). Results are summarized in c (right). ( f ) MTCO1 levels in HEK293A cells transfected with PGC-1α and necdin cDNAs were detected 72 h post transfection. ( g , h ) PGC-1α levels in transfected HEK293A were analysed 24 h post transfection after treating with CHX for 60 min (CHX+) ( g ) or MG132 for 3 h (MG132+) ( h ) before harvest. ( i ) HEK293A cells were transfected with cDNAs for PGC-1α, V5-tagged Rnf34 (Rnf34-V5) and necdin, and incubated for 24 h. PGC-1α, V5, necdin and β-tubulin were detected by immunoblotting. ( j , k ) Effects of necdin on PGC-1α degradation and PGC-1α ubiquitination mediated by Rnf34 ( j ) or Fbxw7 ( k ) were analysed using HEK293A cells transfected with cDNAs for PGC-1α, necdin, V5-tagged Rnf34 (Rnf34-V5), V5-tagged Fbxw7 (Fbxw7-V5) and FLAG-tagged ubiquitin (FLAG-Ub). Ubiquitination of immunoprecipitated PGC-1α was detected with anti-FLAG antibody. CT, C-terminal; IB, immunoblotted; IM, intermediate; IP, immunoprecipitated; KO, knockout; NT, N-terminal; RR, RNA recognition; TA, transcriptional activation; TR, transcriptional repression.
Figure Legend Snippet: Necdin stabilizes PGC-1α by inhibiting its ubiquitin-proteasomal degradation. ( a ) Forebrain extracts from E14.5 wild-type (WT) and necdin-null (KO) mice were immunoprecipitated with anti-necdin IgG and control preimmune IgG for endogenous binding between necdin and PGC-1α. PCNA, negative control. ( b ) Necdin and PGC-1α expressed in cDNA-transfected HEK293A cells were immunoprecipitated and immunoblotted. ( c ) Diagram of the PGC-1α deletion mutants. ( d ) Necdin and 6Myc-tagged PGC-1α deletion mutants in transfected HEK293A cells were immunoprecipitated and immunoblotted. ( e ) Bacterially synthesized MBP-fused PGC-1α deletion mutants were incubated with His-tagged necdin (His-Necdin). Bound His-Necdin was detected by immunoblotting. Recombinant proteins were stained with Coomassie Brilliant Blue (CBB). Results are summarized in c (right). ( f ) MTCO1 levels in HEK293A cells transfected with PGC-1α and necdin cDNAs were detected 72 h post transfection. ( g , h ) PGC-1α levels in transfected HEK293A were analysed 24 h post transfection after treating with CHX for 60 min (CHX+) ( g ) or MG132 for 3 h (MG132+) ( h ) before harvest. ( i ) HEK293A cells were transfected with cDNAs for PGC-1α, V5-tagged Rnf34 (Rnf34-V5) and necdin, and incubated for 24 h. PGC-1α, V5, necdin and β-tubulin were detected by immunoblotting. ( j , k ) Effects of necdin on PGC-1α degradation and PGC-1α ubiquitination mediated by Rnf34 ( j ) or Fbxw7 ( k ) were analysed using HEK293A cells transfected with cDNAs for PGC-1α, necdin, V5-tagged Rnf34 (Rnf34-V5), V5-tagged Fbxw7 (Fbxw7-V5) and FLAG-tagged ubiquitin (FLAG-Ub). Ubiquitination of immunoprecipitated PGC-1α was detected with anti-FLAG antibody. CT, C-terminal; IB, immunoblotted; IM, intermediate; IP, immunoprecipitated; KO, knockout; NT, N-terminal; RR, RNA recognition; TA, transcriptional activation; TR, transcriptional repression.

Techniques Used: Pyrolysis Gas Chromatography, Mouse Assay, Immunoprecipitation, Binding Assay, Negative Control, Transfection, Synthesized, Incubation, Recombinant, Staining, Knock-Out, Activation Assay

27) Product Images from "Transcription factor binding sites in the pol gene intragenic regulatory region of HIV-1 are important for virus infectivity"

Article Title: Transcription factor binding sites in the pol gene intragenic regulatory region of HIV-1 are important for virus infectivity

Journal: Nucleic Acids Research

doi: 10.1093/nar/gki720

Mutations in the HS7 binding sites do not affect viral particle formation. ( A ) Equivalent amounts of viral particles (assessed by p24 ELISA assay) from the wild-type and each HS7 mutant virus stocks were pelleted by ultracentrifugation. After lysis of viral particles, HIV-1 RNA was detected by RNase protection analysis with an antisense riboprobe which protects two bands (200 and 83 nt) corresponding to the HIV-1 3′- and 5′-LTR, respectively. The undigested HIV probe is shown for reference. Lane Marker (M) contains pSK-/ApaII markers, whose sizes (in nt) are indicated on the left-hand side. Intensities of RNA bands were quantified by radioimaging (InstantImager). ( B ) Equivalent amounts of viral particles (assessed by p24 ELISA assay) from the wild-type and each HS7 mutant virus stocks were pelleted by ultracentrifugation. The ultracentrifuged viral stocks were lysed in Laemmli buffer, analyzed by western blotting with anti-HIV-1 immunoglobulin, and detected by enhanced chemiluminescence with a horseradish peroxidase-conjugated goat anti-human IgG. The bands corresponding to the HIV-1 reverse transcriptase, integrase, p24 protein and protease are indicated. MW, molecular mass (indicated in kDa).
Figure Legend Snippet: Mutations in the HS7 binding sites do not affect viral particle formation. ( A ) Equivalent amounts of viral particles (assessed by p24 ELISA assay) from the wild-type and each HS7 mutant virus stocks were pelleted by ultracentrifugation. After lysis of viral particles, HIV-1 RNA was detected by RNase protection analysis with an antisense riboprobe which protects two bands (200 and 83 nt) corresponding to the HIV-1 3′- and 5′-LTR, respectively. The undigested HIV probe is shown for reference. Lane Marker (M) contains pSK-/ApaII markers, whose sizes (in nt) are indicated on the left-hand side. Intensities of RNA bands were quantified by radioimaging (InstantImager). ( B ) Equivalent amounts of viral particles (assessed by p24 ELISA assay) from the wild-type and each HS7 mutant virus stocks were pelleted by ultracentrifugation. The ultracentrifuged viral stocks were lysed in Laemmli buffer, analyzed by western blotting with anti-HIV-1 immunoglobulin, and detected by enhanced chemiluminescence with a horseradish peroxidase-conjugated goat anti-human IgG. The bands corresponding to the HIV-1 reverse transcriptase, integrase, p24 protein and protease are indicated. MW, molecular mass (indicated in kDa).

Techniques Used: Binding Assay, Enzyme-linked Immunosorbent Assay, Mutagenesis, Lysis, Marker, Western Blot

28) Product Images from "Long intergenic non-coding RNA APOC1P1-3 inhibits apoptosis by decreasing α-tubulin acetylation in breast cancer"

Article Title: Long intergenic non-coding RNA APOC1P1-3 inhibits apoptosis by decreasing α-tubulin acetylation in breast cancer

Journal: Cell Death & Disease

doi: 10.1038/cddis.2016.142

APOC1P1-3 can bind to tubulin and modify its acetylation levels. ( a ) RNA pull-down to detect the specific combining proteins of APOC1P1-3 , silver stain of the SDS-PAGE gel showed that there was a specific bond between 34 and 55 kDa. The bond was cut to mass spectrometry analysis and was identified as tubulin. ( b ) Western blot to validate the mass spectrometry results in MDA-MB-231 and MCF7 cell lines. ( c ) Relative RIP experiments were performed with anti-tubulin antibodies on extracts from MDA-MB-231 and MCF7 cells, respectively, with IgG as a negative control. The purified RNA was used for qPCR analysis, and the enrichment of APOC1P1-3 was normalized to input. * P
Figure Legend Snippet: APOC1P1-3 can bind to tubulin and modify its acetylation levels. ( a ) RNA pull-down to detect the specific combining proteins of APOC1P1-3 , silver stain of the SDS-PAGE gel showed that there was a specific bond between 34 and 55 kDa. The bond was cut to mass spectrometry analysis and was identified as tubulin. ( b ) Western blot to validate the mass spectrometry results in MDA-MB-231 and MCF7 cell lines. ( c ) Relative RIP experiments were performed with anti-tubulin antibodies on extracts from MDA-MB-231 and MCF7 cells, respectively, with IgG as a negative control. The purified RNA was used for qPCR analysis, and the enrichment of APOC1P1-3 was normalized to input. * P

Techniques Used: Silver Staining, SDS Page, Mass Spectrometry, Western Blot, Multiple Displacement Amplification, Negative Control, Purification, Real-time Polymerase Chain Reaction

29) Product Images from "The expression of the human neuronal ?3 Na+,K+-ATPase subunit gene is regulated by the activity of the Sp1 and NF-Y transcription factors"

Article Title: The expression of the human neuronal ?3 Na+,K+-ATPase subunit gene is regulated by the activity of the Sp1 and NF-Y transcription factors

Journal:

doi: 10.1042/BJ20041294

DNase I footprinting analysis of the −204/+70 region of the α3 promoter
Figure Legend Snippet: DNase I footprinting analysis of the −204/+70 region of the α3 promoter

Techniques Used: Footprinting

30) Product Images from "The heterogeneous ribonuclear protein C interacts with the hepatitis delta virus small antigen"

Article Title: The heterogeneous ribonuclear protein C interacts with the hepatitis delta virus small antigen

Journal: Virology Journal

doi: 10.1186/1743-422X-8-358

Co-localization of hnRNPC with HDV antigens and RNA in human liver cells . (A-C) Double indirect immunofluorescence was used to detect HDAg and hnRNPC proteins in cultured Huh7-D12 cells. Cells were fixed with 3.7% formaldehyde, permeabilized with 0.5% Triton X-100, and incubated with an anti-HDAg antibody (A, green staining) and an anti-hnRNPC antibody (B, red staining). Combined in situ hybridization and immunofluorescence was performed to detect HDV RNA and the hnRNPC protein. After fixation and permeabilization, cells were hybridized with a digoxigenin-labeled probe to detect HDV RNA (D, green staining) and with an anti-hnRNPC antibody (E, red staining). Overlaps of images are shown in panels C and F. (G) 10 individual cells from each experiment were analyzed using the ImageJ software and the JaCoP plugin to determine the Mander's overlap coefficient.
Figure Legend Snippet: Co-localization of hnRNPC with HDV antigens and RNA in human liver cells . (A-C) Double indirect immunofluorescence was used to detect HDAg and hnRNPC proteins in cultured Huh7-D12 cells. Cells were fixed with 3.7% formaldehyde, permeabilized with 0.5% Triton X-100, and incubated with an anti-HDAg antibody (A, green staining) and an anti-hnRNPC antibody (B, red staining). Combined in situ hybridization and immunofluorescence was performed to detect HDV RNA and the hnRNPC protein. After fixation and permeabilization, cells were hybridized with a digoxigenin-labeled probe to detect HDV RNA (D, green staining) and with an anti-hnRNPC antibody (E, red staining). Overlaps of images are shown in panels C and F. (G) 10 individual cells from each experiment were analyzed using the ImageJ software and the JaCoP plugin to determine the Mander's overlap coefficient.

Techniques Used: Immunofluorescence, Cell Culture, Incubation, Staining, In Situ Hybridization, Labeling, Software

31) Product Images from "Isolation of DNA after Extraction of RNA To Detect the Presence of Borrelia burgdorferi and Expression of Host Cellular Genes from the Same Tissue Sample"

Article Title: Isolation of DNA after Extraction of RNA To Detect the Presence of Borrelia burgdorferi and Expression of Host Cellular Genes from the Same Tissue Sample

Journal: Journal of Clinical Microbiology

doi:

Gel analysis of PCR and RT PCR products prepared from DNA and RNA, respectively, from tissues in experiment 2. (A) PCR of DNAs for OspB. Lanes: 1, heart; 2, midbrain; 3, spinal cord (thoracic); 4, cerebellum; 5, heart plus midbrain; 6, heart plus cerebellum; 7, no-DNA control. (B) RT PCR of RNAs. Lanes: 1, heart, GAPDH; 2, heart, TGF-β1; 3, midbrain, GAPDH; 4, midbrain, TGF-β1; 5, spinal cord (thoracic), GAPDH; 6, spinal cord (thoracic), TGF-β1; 7, cerebellum, GAPDH; 8, cerebellum, TGF-β1; 9, no-cDNA control; M, DNA size markers.
Figure Legend Snippet: Gel analysis of PCR and RT PCR products prepared from DNA and RNA, respectively, from tissues in experiment 2. (A) PCR of DNAs for OspB. Lanes: 1, heart; 2, midbrain; 3, spinal cord (thoracic); 4, cerebellum; 5, heart plus midbrain; 6, heart plus cerebellum; 7, no-DNA control. (B) RT PCR of RNAs. Lanes: 1, heart, GAPDH; 2, heart, TGF-β1; 3, midbrain, GAPDH; 4, midbrain, TGF-β1; 5, spinal cord (thoracic), GAPDH; 6, spinal cord (thoracic), TGF-β1; 7, cerebellum, GAPDH; 8, cerebellum, TGF-β1; 9, no-cDNA control; M, DNA size markers.

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

Gel analysis of PCR and RT PCR products prepared from DNA and RNA, respectively, from tissues in experiment 1. (A) PCR of DNAs for OspB. Lanes: 1, heart (apex); 2, spinal cord (lumbar); 3, bladder; 4, temporal lobe; 5, pons; 6, heart (apex) plus temporal lobe; 7, heart (apex) plus pons; 8, no-DNA control. (B) RT PCR of RNAs. Lanes: 1, heart (apex), GAPDH; 2, heart (apex), TGF-β1; 3, spinal cord (lumbar), GAPDH; 4, spinal cord (lumbar), TGF-β1; 5, bladder, GAPDH; 6, bladder, TGF-β1; 7, temporal lobe, GAPDH; 8, temporal lobe, TGF-β1; 9, pons, GAPDH; 10, pons, TGF-β1; 11, no-cDNA control; M, DNA size markers.
Figure Legend Snippet: Gel analysis of PCR and RT PCR products prepared from DNA and RNA, respectively, from tissues in experiment 1. (A) PCR of DNAs for OspB. Lanes: 1, heart (apex); 2, spinal cord (lumbar); 3, bladder; 4, temporal lobe; 5, pons; 6, heart (apex) plus temporal lobe; 7, heart (apex) plus pons; 8, no-DNA control. (B) RT PCR of RNAs. Lanes: 1, heart (apex), GAPDH; 2, heart (apex), TGF-β1; 3, spinal cord (lumbar), GAPDH; 4, spinal cord (lumbar), TGF-β1; 5, bladder, GAPDH; 6, bladder, TGF-β1; 7, temporal lobe, GAPDH; 8, temporal lobe, TGF-β1; 9, pons, GAPDH; 10, pons, TGF-β1; 11, no-cDNA control; M, DNA size markers.

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

32) Product Images from "LncRNA DLEU1 contributes to colorectal cancer progression via activation of KPNA3"

Article Title: LncRNA DLEU1 contributes to colorectal cancer progression via activation of KPNA3

Journal: Molecular Cancer

doi: 10.1186/s12943-018-0873-2

DLEU1 interacts with SMARCA1 in CRC cells. a The expression of DLEU1 in cytoplasm and nucleus of HCT8 cells was measured by qRT-PCR. U6 serves as a nuclear control. GAPDH serves as a cytoplasmic control. b SMARCA1 was a potential interactive candidate of DLEU1. Biotin-labeled DLEU1 and intron control were incubated with HCT8 cell lysates, and the enriched products were eluted and separated by SDS-PAGE electrophoresis and silver staining. The differential band appearing in DLEU1 lane was analyzed by mass spectrum. c DLEU1 associated with SMARCA1 as shown by RNA pulldown and Western blot. Biotin-labeled DLEU1 and intron control were added into HCT8 cell lysates, and pulldown assays were performed. d DLEU1 was enriched by SMARCA1 in HCT8 and SW480 cell lysates. e SMARCA1 enriched DLEU1 in HCT8 cell lysates. SMARCA1 antibody was added into cell lysates and enriched RNAs were isolated. Then enriched DLEU1 was analyzed by PCR. f DLEU1 co-localized with SMARCA1 in HCT8 cells as shown by RNA FISH. Green, DLEU1; Red, SMARCA1; Blue, DAPI. Scale bar, 10 μm. g the region of nt 1~ 400 in DLEU1 was important for the interaction with SMARCA1. h DLEU1 (nt 1~ 400) associated with SMARCA1 directly as shown by RNA EMSA assays. i The region of nt 700~ 1050 is indispensable for the function of DLEU1 in colorectal cancer. Overexpression of DLEU1 with deletion of nt 1~ 400 cannot promoted proliferation and metastasis in CC. *** P
Figure Legend Snippet: DLEU1 interacts with SMARCA1 in CRC cells. a The expression of DLEU1 in cytoplasm and nucleus of HCT8 cells was measured by qRT-PCR. U6 serves as a nuclear control. GAPDH serves as a cytoplasmic control. b SMARCA1 was a potential interactive candidate of DLEU1. Biotin-labeled DLEU1 and intron control were incubated with HCT8 cell lysates, and the enriched products were eluted and separated by SDS-PAGE electrophoresis and silver staining. The differential band appearing in DLEU1 lane was analyzed by mass spectrum. c DLEU1 associated with SMARCA1 as shown by RNA pulldown and Western blot. Biotin-labeled DLEU1 and intron control were added into HCT8 cell lysates, and pulldown assays were performed. d DLEU1 was enriched by SMARCA1 in HCT8 and SW480 cell lysates. e SMARCA1 enriched DLEU1 in HCT8 cell lysates. SMARCA1 antibody was added into cell lysates and enriched RNAs were isolated. Then enriched DLEU1 was analyzed by PCR. f DLEU1 co-localized with SMARCA1 in HCT8 cells as shown by RNA FISH. Green, DLEU1; Red, SMARCA1; Blue, DAPI. Scale bar, 10 μm. g the region of nt 1~ 400 in DLEU1 was important for the interaction with SMARCA1. h DLEU1 (nt 1~ 400) associated with SMARCA1 directly as shown by RNA EMSA assays. i The region of nt 700~ 1050 is indispensable for the function of DLEU1 in colorectal cancer. Overexpression of DLEU1 with deletion of nt 1~ 400 cannot promoted proliferation and metastasis in CC. *** P

Techniques Used: Expressing, Quantitative RT-PCR, Labeling, Incubation, SDS Page, Electrophoresis, Silver Staining, Western Blot, Isolation, Polymerase Chain Reaction, Fluorescence In Situ Hybridization, Over Expression

33) Product Images from "Long non-coding RNA MUC5B-AS1 promotes metastasis through mutually regulating MUC5B expression in lung adenocarcinoma"

Article Title: Long non-coding RNA MUC5B-AS1 promotes metastasis through mutually regulating MUC5B expression in lung adenocarcinoma

Journal: Cell Death & Disease

doi: 10.1038/s41419-018-0472-6

MUC5B-AS1 increases the stability of MUC5B mRNA by forming a protective RNA duplex. a Schematic representation of the PCR amplification regions for overlapping (OL) and non-overlapping (non-OL) regions of MUC5B. We designed two pairs of primers to amplify the OL regions (OL1 and OL2) and non-OL (non-OL1 and non-OL2) regions of MUC5B, respectively. F forward primer, R reverse primer. b RT-PCR products of OL and non-OL regions of MUC5B. Total RNA samples were treated with RNAse A + T cocktail and then cleaned up RNA using RNeasy kits. RT-PCR was conducted using the primers to detect the OL and non-OL regions of the MUC5B mRNA. OL and non-OL regions of KRT7-AS were used as a positive control. c Stability of MUC5B mRNA over 12 h was measured by qRT-PCR relative to time 0 h after blocking new RNA synthesis with Actinomycin D (1 μg/mL; indicated with black arrow). H1299 cells with MUC5B-AS1 or empty vector stable expression were treated with 1 μg/mL ActD, and then harvested cells for RNA purification at 12 h after addition of ActD. Then, MUC5B mRNA stability were subsequently measured by qRT-PCR and were normalized against a synthesized exogenous reference λ polyA + RNA. Student’s t -test, * P
Figure Legend Snippet: MUC5B-AS1 increases the stability of MUC5B mRNA by forming a protective RNA duplex. a Schematic representation of the PCR amplification regions for overlapping (OL) and non-overlapping (non-OL) regions of MUC5B. We designed two pairs of primers to amplify the OL regions (OL1 and OL2) and non-OL (non-OL1 and non-OL2) regions of MUC5B, respectively. F forward primer, R reverse primer. b RT-PCR products of OL and non-OL regions of MUC5B. Total RNA samples were treated with RNAse A + T cocktail and then cleaned up RNA using RNeasy kits. RT-PCR was conducted using the primers to detect the OL and non-OL regions of the MUC5B mRNA. OL and non-OL regions of KRT7-AS were used as a positive control. c Stability of MUC5B mRNA over 12 h was measured by qRT-PCR relative to time 0 h after blocking new RNA synthesis with Actinomycin D (1 μg/mL; indicated with black arrow). H1299 cells with MUC5B-AS1 or empty vector stable expression were treated with 1 μg/mL ActD, and then harvested cells for RNA purification at 12 h after addition of ActD. Then, MUC5B mRNA stability were subsequently measured by qRT-PCR and were normalized against a synthesized exogenous reference λ polyA + RNA. Student’s t -test, * P

Techniques Used: Polymerase Chain Reaction, Amplification, Reverse Transcription Polymerase Chain Reaction, Positive Control, Quantitative RT-PCR, Blocking Assay, Plasmid Preparation, Expressing, Purification, Synthesized

Identification of an overlapping antisense lncRNA at the MUC5B gene locus. a The localizations of MUC5B-AS1 and MUC5B on the UCSC genome browser. The schema was not drawn to scale. MUC5B-AS1 is located at chromosomal 11p15.5, and composes of two exons. MUC5B-AS1 is an antisense lncRNA, embedded on the opposite DNA strand of the MUC5B gene within its 31st exon. Green blocks indicate exons, and red blocks are overlapping regions. Primers of MUC5B-AS1 and MUC5B are also indicated in the schema: F primer forward primer, R primer reverse primer. The forward primer of MUC5B-AS1 spans the exon1–exon2 junction to avoid the non-specific amplification of MUC5B mRNA or genomic DNA. b Upper chart: ORF prediction of MUC5B-AS1 sequence. Three potential ORFs that might code peptides of 33–118 amino acids are present in the MUC5B-AS1 sequence. Lower chart: coding potentials of lncRNAs (MUC5B-AS1, MALAT1, TUG1) and mRNA (MUC5B, GAPDH, ACTB) were calculated using CPAT and CPC. c Localization of MUC5B-AS1 by RNA-FISH. Blue, DAPI-stained nuclei; red, Cy3-labeled positive hybridization signals (scale bar, 20 μm). The U6 and 18S were used as positive control. d Expression analysis of MUC5B-AS1 in lung adenocarcinoma tissues ( n = 72) and paired adjacent normal lung tissues ( n = 72). The ΔCt was used to show the expression level of MUC5B-AS1 (ΔCt = Ct MUC5B-AS1 –Ct β-actin ). Lower ΔCt values indicate higher expression. Normal vs. tumor tissues, Student’s t -test
Figure Legend Snippet: Identification of an overlapping antisense lncRNA at the MUC5B gene locus. a The localizations of MUC5B-AS1 and MUC5B on the UCSC genome browser. The schema was not drawn to scale. MUC5B-AS1 is located at chromosomal 11p15.5, and composes of two exons. MUC5B-AS1 is an antisense lncRNA, embedded on the opposite DNA strand of the MUC5B gene within its 31st exon. Green blocks indicate exons, and red blocks are overlapping regions. Primers of MUC5B-AS1 and MUC5B are also indicated in the schema: F primer forward primer, R primer reverse primer. The forward primer of MUC5B-AS1 spans the exon1–exon2 junction to avoid the non-specific amplification of MUC5B mRNA or genomic DNA. b Upper chart: ORF prediction of MUC5B-AS1 sequence. Three potential ORFs that might code peptides of 33–118 amino acids are present in the MUC5B-AS1 sequence. Lower chart: coding potentials of lncRNAs (MUC5B-AS1, MALAT1, TUG1) and mRNA (MUC5B, GAPDH, ACTB) were calculated using CPAT and CPC. c Localization of MUC5B-AS1 by RNA-FISH. Blue, DAPI-stained nuclei; red, Cy3-labeled positive hybridization signals (scale bar, 20 μm). The U6 and 18S were used as positive control. d Expression analysis of MUC5B-AS1 in lung adenocarcinoma tissues ( n = 72) and paired adjacent normal lung tissues ( n = 72). The ΔCt was used to show the expression level of MUC5B-AS1 (ΔCt = Ct MUC5B-AS1 –Ct β-actin ). Lower ΔCt values indicate higher expression. Normal vs. tumor tissues, Student’s t -test

Techniques Used: Amplification, Sequencing, Fluorescence In Situ Hybridization, Staining, Labeling, Hybridization, Positive Control, Expressing

34) Product Images from "LncRNA epigenetic landscape analysis identifies EPIC1 as an oncogenic lncRNA that interacts with MYC and promotes cell cycle progression in cancer"

Article Title: LncRNA epigenetic landscape analysis identifies EPIC1 as an oncogenic lncRNA that interacts with MYC and promotes cell cycle progression in cancer

Journal: Cancer cell

doi: 10.1016/j.ccell.2018.03.006

EPIC1 is a nuclear lncRNA regulating MYC targets expression (A) qRT-PCR analysis of EPIC1 expression (top) and Western blot (bottom) of subcellular fractionation in MCF-7cells. GAPDH and U6 RNA served as a marker for cytoplasmic and nuclear gene localization, respectively. SNRP70 and GAPDH served as a specific nuclear and cytoplasmic marker to whole cell lysates (WCL), cytoplasmic (Cyto), and nuclear fractionation (Nuc). Error bars indicate mean ± SD, n = 3 for technical replicates. (B) Schematic of the identification of EPIC1 correlated genes in breast tumors from TCGA (yellow), and genes potentially regulated by EPIC1 in MCF-7 cells (green). (C) Co-expression analysis showing that EPIC1 expression is associated with 2005 genes in 559 patients with breast cancer (BRCA). Each column represents one patient. (D) GSEA analysis of the EPIC1 -related pathways in 20 cancer types (left panel) and EPIC1 knockdown MCF-7 cells (right panel). The heatmap indicates the GSEA scores. (E) Association between the enrichment of MYC targets and EPIC1 expression in breast tumors by GSEA analysis (D). (F) EPIC1 -regulated gene expression by qRT-PCR analysis (top) and RNA-seq (bottom). Error bars indicate mean ± SD, n = 3 for technical replicates. (G) Western blot of MYC-regulated targets in MCF-7 (left) and ZR-75-1 (right) cells treated with EPIC1 and MYC siRNAs. .
Figure Legend Snippet: EPIC1 is a nuclear lncRNA regulating MYC targets expression (A) qRT-PCR analysis of EPIC1 expression (top) and Western blot (bottom) of subcellular fractionation in MCF-7cells. GAPDH and U6 RNA served as a marker for cytoplasmic and nuclear gene localization, respectively. SNRP70 and GAPDH served as a specific nuclear and cytoplasmic marker to whole cell lysates (WCL), cytoplasmic (Cyto), and nuclear fractionation (Nuc). Error bars indicate mean ± SD, n = 3 for technical replicates. (B) Schematic of the identification of EPIC1 correlated genes in breast tumors from TCGA (yellow), and genes potentially regulated by EPIC1 in MCF-7 cells (green). (C) Co-expression analysis showing that EPIC1 expression is associated with 2005 genes in 559 patients with breast cancer (BRCA). Each column represents one patient. (D) GSEA analysis of the EPIC1 -related pathways in 20 cancer types (left panel) and EPIC1 knockdown MCF-7 cells (right panel). The heatmap indicates the GSEA scores. (E) Association between the enrichment of MYC targets and EPIC1 expression in breast tumors by GSEA analysis (D). (F) EPIC1 -regulated gene expression by qRT-PCR analysis (top) and RNA-seq (bottom). Error bars indicate mean ± SD, n = 3 for technical replicates. (G) Western blot of MYC-regulated targets in MCF-7 (left) and ZR-75-1 (right) cells treated with EPIC1 and MYC siRNAs. .

Techniques Used: Expressing, Quantitative RT-PCR, Western Blot, Fractionation, Marker, RNA Sequencing Assay

35) Product Images from "A Spontaneous Translational Fusion of Bacillus cereus PlcR and PapR Activates Transcription of PlcR-Dependent Genes in Bacillus anthracis via Binding with a Specific Palindromic Sequence "

Article Title: A Spontaneous Translational Fusion of Bacillus cereus PlcR and PapR Activates Transcription of PlcR-Dependent Genes in Bacillus anthracis via Binding with a Specific Palindromic Sequence

Journal: Infection and Immunity

doi: 10.1128/IAI.72.10.5814-5823.2004

Electrophoretic analysis of PlcR species. Lysates of B. anthracis SdT2 and SdT12 were probed with C-PlcR antiserum 1451. (A) Lysates made in the presence of 10 mM EDTA. (B) Lysates made in the presence of 10 mM magnesium acetate and then treated with DNase I for 30 min at 37°C. All the samples were made to contain 2% SDS and were heated for 5 min at 95°C before electrophoresis. Putative identities shown in the right margin were determined only on the basis of molecular mass. M, molecular mass markers.
Figure Legend Snippet: Electrophoretic analysis of PlcR species. Lysates of B. anthracis SdT2 and SdT12 were probed with C-PlcR antiserum 1451. (A) Lysates made in the presence of 10 mM EDTA. (B) Lysates made in the presence of 10 mM magnesium acetate and then treated with DNase I for 30 min at 37°C. All the samples were made to contain 2% SDS and were heated for 5 min at 95°C before electrophoresis. Putative identities shown in the right margin were determined only on the basis of molecular mass. M, molecular mass markers.

Techniques Used: Electrophoresis

Stability of PlcR complexes. Whole-cell proteins from 12-h cultures of B. anthracis SdT2 and SdT12 were treated sequentially with DNase I and Triton X-100 or with DNase I and urea. Triton-containing samples were heated in SDS sample buffer, whereas urea-containing samples were loaded directly onto the gels. C-PlcR antiserum 1451 was used for the blotting analysis. M, molecular mass markers.
Figure Legend Snippet: Stability of PlcR complexes. Whole-cell proteins from 12-h cultures of B. anthracis SdT2 and SdT12 were treated sequentially with DNase I and Triton X-100 or with DNase I and urea. Triton-containing samples were heated in SDS sample buffer, whereas urea-containing samples were loaded directly onto the gels. C-PlcR antiserum 1451 was used for the blotting analysis. M, molecular mass markers.

Techniques Used:

36) Product Images from "YjcC, a c-di-GMP Phosphodiesterase Protein, Regulates the Oxidative Stress Response and Virulence of Klebsiella pneumoniae CG43"

Article Title: YjcC, a c-di-GMP Phosphodiesterase Protein, Regulates the Oxidative Stress Response and Virulence of Klebsiella pneumoniae CG43

Journal: PLoS ONE

doi: 10.1371/journal.pone.0066740

The yjcC is paraquat inducible, and SoxRS and RpoS dependent . (A) The putative promoters respectively containing 525 bp (P yjcC1 ), 385 bp (P yjcC2 ) and 415 bp (P yjcC0 ) of yjcC were isolated and cloned into the LacZ reporter plasmid placZ15 (22). (B) The recombinant plasmids placz15, pP yjcC1 , pP yjcC2 and pP yjcC0 were then transformed to K. pneumoniae CG43Z01 and the β-galactosidase activities of the transformants grown to log-phase in LB broth were determined. The results are shown as an average of triplicate samples. Error bars indicate standard deviations. (C) Total RNA of K. pneumoniae CG43S3 was isolated after the bacteria were grown in 2 mM H 2 O 2 or 30 µM of paraquat. Specific primer pairs used to detect the expression of soxR , soxS , rpoS , and yjcC are listed in Table S2 . Relative fold expression was compared with the non-induced condition and determined by the 2 −ΔΔCt method (60). Error bars indicate standard deviation of the mean. Data are representative of three independent experiments. (D) The expression of yjcC was determined in Δ soxRS and Δ rpoS mutant by qRT-PCR. Data are representative of three independent experiments, *, P
Figure Legend Snippet: The yjcC is paraquat inducible, and SoxRS and RpoS dependent . (A) The putative promoters respectively containing 525 bp (P yjcC1 ), 385 bp (P yjcC2 ) and 415 bp (P yjcC0 ) of yjcC were isolated and cloned into the LacZ reporter plasmid placZ15 (22). (B) The recombinant plasmids placz15, pP yjcC1 , pP yjcC2 and pP yjcC0 were then transformed to K. pneumoniae CG43Z01 and the β-galactosidase activities of the transformants grown to log-phase in LB broth were determined. The results are shown as an average of triplicate samples. Error bars indicate standard deviations. (C) Total RNA of K. pneumoniae CG43S3 was isolated after the bacteria were grown in 2 mM H 2 O 2 or 30 µM of paraquat. Specific primer pairs used to detect the expression of soxR , soxS , rpoS , and yjcC are listed in Table S2 . Relative fold expression was compared with the non-induced condition and determined by the 2 −ΔΔCt method (60). Error bars indicate standard deviation of the mean. Data are representative of three independent experiments. (D) The expression of yjcC was determined in Δ soxRS and Δ rpoS mutant by qRT-PCR. Data are representative of three independent experiments, *, P

Techniques Used: Isolation, Clone Assay, Plasmid Preparation, Recombinant, Transformation Assay, Expressing, Standard Deviation, Mutagenesis, Quantitative RT-PCR

qRT-PCR analysis of the expression of mrkA , mrkH , and mrkI . Total RNA of K. pneumoniae CG43S3[pRK415] and CG43S3[pJR1] were isolated after the bacteria were grown overnight in LB supplemented with 12.5 µg/ml tetracycline. Specific primer pairs used to detect the expression of mrkA , mrkH , and mrkJ are listed in Table S2 . Relative fold expression was compared with the non-induced condition and determined by the 2 −ΔΔCt method (60). Error bars indicate standard deviation of the mean. Data are representative of three independent experiments.
Figure Legend Snippet: qRT-PCR analysis of the expression of mrkA , mrkH , and mrkI . Total RNA of K. pneumoniae CG43S3[pRK415] and CG43S3[pJR1] were isolated after the bacteria were grown overnight in LB supplemented with 12.5 µg/ml tetracycline. Specific primer pairs used to detect the expression of mrkA , mrkH , and mrkJ are listed in Table S2 . Relative fold expression was compared with the non-induced condition and determined by the 2 −ΔΔCt method (60). Error bars indicate standard deviation of the mean. Data are representative of three independent experiments.

Techniques Used: Quantitative RT-PCR, Expressing, Isolation, Standard Deviation

37) Product Images from "Sialylated Cervical Mucins Inhibit the Activation of Neutrophils to Form Neutrophil Extracellular Traps in Bovine in vitro Model"

Article Title: Sialylated Cervical Mucins Inhibit the Activation of Neutrophils to Form Neutrophil Extracellular Traps in Bovine in vitro Model

Journal: Frontiers in Immunology

doi: 10.3389/fimmu.2019.02478

Siglecs expressed in bovine neutrophils. (A) Cartoon of the Siglecs expressed by bovine neutrophils and their functional intracellular domains. The number of C2-set Ig-like domains was determined by SMART ( http://smart.embl-heidelberg.de/smart/set_mode.cgi?NORMAL=1 ) based on the following NCBI sequences: Siglec-2: XP_024834447.1; Siglec-4: XP_0248341235.1; Siglec-5: XP_005219620.2; Siglec-8: XP_010813599.1; Siglec-14: XP_019834314.1. (B) Pie chart of the percentage Siglec expression in bovine neutrophils. qPCR raw data was normalized against the reference genes. Siglec-2, -4, -5, -8, and -14 were detected by RT-qPCR and the total of the detected Siglecs was set to 100%. (C) Determined copies of Siglecs per ng RNA used during qPCR. X-axis shows the Siglecs expressed by bovine neutrophils; Y-axis shows the log 10 values. Mean values and standard deviations are displayed in the diagrams (Siglec-2, Siglec-5, Siglec-8 I, Siglec-8 II, n = 3 different animals; Siglec-4, Siglec-14, n = 4 different animals). For Siglec-8, values generated with two different primer pairs are displayed.
Figure Legend Snippet: Siglecs expressed in bovine neutrophils. (A) Cartoon of the Siglecs expressed by bovine neutrophils and their functional intracellular domains. The number of C2-set Ig-like domains was determined by SMART ( http://smart.embl-heidelberg.de/smart/set_mode.cgi?NORMAL=1 ) based on the following NCBI sequences: Siglec-2: XP_024834447.1; Siglec-4: XP_0248341235.1; Siglec-5: XP_005219620.2; Siglec-8: XP_010813599.1; Siglec-14: XP_019834314.1. (B) Pie chart of the percentage Siglec expression in bovine neutrophils. qPCR raw data was normalized against the reference genes. Siglec-2, -4, -5, -8, and -14 were detected by RT-qPCR and the total of the detected Siglecs was set to 100%. (C) Determined copies of Siglecs per ng RNA used during qPCR. X-axis shows the Siglecs expressed by bovine neutrophils; Y-axis shows the log 10 values. Mean values and standard deviations are displayed in the diagrams (Siglec-2, Siglec-5, Siglec-8 I, Siglec-8 II, n = 3 different animals; Siglec-4, Siglec-14, n = 4 different animals). For Siglec-8, values generated with two different primer pairs are displayed.

Techniques Used: Functional Assay, Expressing, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Generated

38) Product Images from "Aflatoxin Biosynthesis Is a Novel Source of Reactive Oxygen Species—A Potential Redox Signal to Initiate Resistance to Oxidative Stress?"

Article Title: Aflatoxin Biosynthesis Is a Novel Source of Reactive Oxygen Species—A Potential Redox Signal to Initiate Resistance to Oxidative Stress?

Journal: Toxins

doi: 10.3390/toxins7051411

Analysis of transcript levels in 7 h germlings. A. parasiticus SU-1, AFS10, and Δ veA were grown on YES solid media and spores were collected at 5 days. Germlings were grown from fresh spores in GMS liquid medium for 7 h, frozen in liquid nitrogen, and stored at −80 °C. RNA was extracted from germlings using grinding and sonication as described in Methods. RT-PCR was performed on total RNA treated with RNAse-free DNAse I with primers specific for each gene ( Table 3 ). PCR products were separated on a 1% agarose gel by electrophoresis. Citrate synthase, a constitutively expressed gene, was used as a positive control. gDNA, genomic DNA.
Figure Legend Snippet: Analysis of transcript levels in 7 h germlings. A. parasiticus SU-1, AFS10, and Δ veA were grown on YES solid media and spores were collected at 5 days. Germlings were grown from fresh spores in GMS liquid medium for 7 h, frozen in liquid nitrogen, and stored at −80 °C. RNA was extracted from germlings using grinding and sonication as described in Methods. RT-PCR was performed on total RNA treated with RNAse-free DNAse I with primers specific for each gene ( Table 3 ). PCR products were separated on a 1% agarose gel by electrophoresis. Citrate synthase, a constitutively expressed gene, was used as a positive control. gDNA, genomic DNA.

Techniques Used: Sonication, Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction, Agarose Gel Electrophoresis, Electrophoresis, Positive Control

39) Product Images from "The role of Arabidopsis aldehyde dehydrogenase genes in response to high temperature and stress combinations"

Article Title: The role of Arabidopsis aldehyde dehydrogenase genes in response to high temperature and stress combinations

Journal: Journal of Experimental Botany

doi: 10.1093/jxb/erx194

Assessment of heat tolerance and expression of ALDH genes in 10-day-old Arabidopsis wild-type seedlings. (A) Seedlings were subjected to 45 °C heat treatment for 0, 3, 6, 12, and 24 h without or with recovery (REC). (B) Seedlings were subjected to a basal heat stress regime (Ba) and to an acquired thermotolerance regime (Ac). REC: recovery (for details see ‘Materials and methods’). (C, D) RNA isolated from 10-day-old seedlings exposed to 0, 1, 3, 6, or 12 h to 45 °C without recovery or followed by 5 d recovery and samples subjected to a Ba or Ac treatment. (C) rRNA bands in total RNAs. (D) ALDH , RD29 , or HSP70 transcripts amplified by RT-PCR. (E) Protein blots of the samples analysed in (C). Equal loading of proteins was monitored by staining the membrane with Ponceau S (data not shown).
Figure Legend Snippet: Assessment of heat tolerance and expression of ALDH genes in 10-day-old Arabidopsis wild-type seedlings. (A) Seedlings were subjected to 45 °C heat treatment for 0, 3, 6, 12, and 24 h without or with recovery (REC). (B) Seedlings were subjected to a basal heat stress regime (Ba) and to an acquired thermotolerance regime (Ac). REC: recovery (for details see ‘Materials and methods’). (C, D) RNA isolated from 10-day-old seedlings exposed to 0, 1, 3, 6, or 12 h to 45 °C without recovery or followed by 5 d recovery and samples subjected to a Ba or Ac treatment. (C) rRNA bands in total RNAs. (D) ALDH , RD29 , or HSP70 transcripts amplified by RT-PCR. (E) Protein blots of the samples analysed in (C). Equal loading of proteins was monitored by staining the membrane with Ponceau S (data not shown).

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

Assessment of heat tolerance and expression of ALDH genes in 4-week-old Arabidopsis plants. (A) Four-week-old plants were subjected to 45 °C heat treatment up to 72 h without or with recovery (REC). (B) Four-week-old plants were subjected to a basal heat stress regime (Ba) and to an acquired thermotolerance regime (Ac) (for details see ‘Materials and methods’). (C, D) RNA isolated from heat-stressed plants (B) shows the rRNA bands in total RNAs (C) and shows ALDH , RD29 or HSP70 transcripts amplified by RT-PCR (D). (E) Protein blots of the samples analysed in (C). Equal loading of proteins was monitored by staining the membrane with Ponceau S (data not shown).
Figure Legend Snippet: Assessment of heat tolerance and expression of ALDH genes in 4-week-old Arabidopsis plants. (A) Four-week-old plants were subjected to 45 °C heat treatment up to 72 h without or with recovery (REC). (B) Four-week-old plants were subjected to a basal heat stress regime (Ba) and to an acquired thermotolerance regime (Ac) (for details see ‘Materials and methods’). (C, D) RNA isolated from heat-stressed plants (B) shows the rRNA bands in total RNAs (C) and shows ALDH , RD29 or HSP70 transcripts amplified by RT-PCR (D). (E) Protein blots of the samples analysed in (C). Equal loading of proteins was monitored by staining the membrane with Ponceau S (data not shown).

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

40) Product Images from "Extracellular Hepatitis B Virus RNAs Are Heterogeneous in Length and Circulate as Capsid-Antibody Complexes in Addition to Virions in Chronic Hepatitis B Patients"

Article Title: Extracellular Hepatitis B Virus RNAs Are Heterogeneous in Length and Circulate as Capsid-Antibody Complexes in Addition to Virions in Chronic Hepatitis B Patients

Journal: Journal of Virology

doi: 10.1128/JVI.00798-18

Analysis of extracellular HBV DNA and RNA by EPA. (A to D) Southern blot analysis of viral DNA strand elongation after EPA treatment. EPA was carried out employing HepAD38 cell culture supernatant and plasma sample from patient 0. Total nucleic acids were extracted via the SDS-proteinase K method. Viral DNA was separated by electrophoresis in TAE or alkaline agarose gels, followed by Southern blot analysis with minus- or plus-strand-specific riboprobes. (E) Northern blot analysis of viral RNA changed upon EPA treatment. Total viral nucleic acids (lanes 3, 5, 7, and 9) or RNA (treated with DNase I) (lanes 4, 6, 8, and 10) were separated by formaldehyde-MOPS agarose gel electrophoresis and subjected to Northern blotting.
Figure Legend Snippet: Analysis of extracellular HBV DNA and RNA by EPA. (A to D) Southern blot analysis of viral DNA strand elongation after EPA treatment. EPA was carried out employing HepAD38 cell culture supernatant and plasma sample from patient 0. Total nucleic acids were extracted via the SDS-proteinase K method. Viral DNA was separated by electrophoresis in TAE or alkaline agarose gels, followed by Southern blot analysis with minus- or plus-strand-specific riboprobes. (E) Northern blot analysis of viral RNA changed upon EPA treatment. Total viral nucleic acids (lanes 3, 5, 7, and 9) or RNA (treated with DNase I) (lanes 4, 6, 8, and 10) were separated by formaldehyde-MOPS agarose gel electrophoresis and subjected to Northern blotting.

Techniques Used: Southern Blot, Cell Culture, Electrophoresis, Northern Blot, Agarose Gel Electrophoresis

Analysis of HBV DNA and RNA change upon entecavir treatment of HepAD38 cells. (A) Change of total cellular HBV RNA level upon entecavir (ETV) treatment. HepAD38 cells were treated with ETV (0.1 μM) for 4 days, and total cellular RNA was analyzed by Northern blotting with ribosomal RNAs serving as loading controls. (B) Change of intracellular nucleocapsid-associated viral RNA (core RNA) and DNA (core DNA) level after ETV treatment. Cytoplasmic core RNA was extracted by the SDS-proteinase K method and analyzed by Northern blotting. Intracellular nucleocapsids were first separated by native agarose gel electrophoresis, and capsid-associated viral DNA (core DNA) was then probed with minus-strand-specific riboprobe. (C to E) Change of extracellular HBV DNA and RNA level upon ETV treatment. Total nucleic acids in HepAD38 cell culture supernatant were extracted and subjected to Southern and Northern blot analyses with specific riboprobes or quantification by PCR. (F to H) CsCl density gradient analysis of viral DNA/RNA level in naked capsids and virions after ETV treatment. HepAD38 cells were left untreated or were treated with ETV, and culture media were concentrated by ultrafiltration, followed by fractionation in CsCl density gradients as described in the legend to Fig. 4 . Viral particles in each fraction were separated by native agarose gel electrophoresis, followed by immunoblotting with anti-HBcAg antibody. Viral DNA and RNA were extracted and subjected to Southern or Northern blot analyses.
Figure Legend Snippet: Analysis of HBV DNA and RNA change upon entecavir treatment of HepAD38 cells. (A) Change of total cellular HBV RNA level upon entecavir (ETV) treatment. HepAD38 cells were treated with ETV (0.1 μM) for 4 days, and total cellular RNA was analyzed by Northern blotting with ribosomal RNAs serving as loading controls. (B) Change of intracellular nucleocapsid-associated viral RNA (core RNA) and DNA (core DNA) level after ETV treatment. Cytoplasmic core RNA was extracted by the SDS-proteinase K method and analyzed by Northern blotting. Intracellular nucleocapsids were first separated by native agarose gel electrophoresis, and capsid-associated viral DNA (core DNA) was then probed with minus-strand-specific riboprobe. (C to E) Change of extracellular HBV DNA and RNA level upon ETV treatment. Total nucleic acids in HepAD38 cell culture supernatant were extracted and subjected to Southern and Northern blot analyses with specific riboprobes or quantification by PCR. (F to H) CsCl density gradient analysis of viral DNA/RNA level in naked capsids and virions after ETV treatment. HepAD38 cells were left untreated or were treated with ETV, and culture media were concentrated by ultrafiltration, followed by fractionation in CsCl density gradients as described in the legend to Fig. 4 . Viral particles in each fraction were separated by native agarose gel electrophoresis, followed by immunoblotting with anti-HBcAg antibody. Viral DNA and RNA were extracted and subjected to Southern or Northern blot analyses.

Techniques Used: Northern Blot, Agarose Gel Electrophoresis, Cell Culture, Polymerase Chain Reaction, Fractionation

Sucrose gradient separation and analysis of viral particles from HepAD38 cell culture supernatant. (A) Distribution of hepatitis B viral particle-associated antigens and DNA/RNA in sucrose gradient. Viral particles prepared from HepAD38 cell culture supernatant (via PEG 8000 precipitation) were layered over a 10% to 60% (wt/wt) sucrose gradient for ultracentrifugation separation. Fractions were collected from top to bottom, and HBsAg level was analyzed by enzyme-linked immunosorbent assay (ELISA). HBsAg and viral DNA and RNA (quantified from gray density of bands in panel B) signals and sucrose density were plotted together. Viral particles were first resolved by native agarose gel electrophoresis, followed by immunoblotting (IB) of HBV envelope and core proteins with anti-HBsAg and anti-HBcAg antibodies. (B) Detection of viral DNA/RNA by Southern or Northern blotting. Total viral nucleic acids were extracted by the SDS-proteinase K method, and viral DNA (extracted from one-tenth of the samples used for Northern blotting) and RNA (treated with DNase I) were detected by Southern and Northern blot analyses with minus- or plus-strand-specific riboprobes, respectively. Symbols of HBsAg particles, empty virions (without nucleic acid), virions (with RC DNA), and naked capsids (empty or with nucleic acids) are depicted on the lower right side of panel A. Blank, no nucleic acids; two centered and gapped circles, RC DNA; straight line, SS DNA; wavy lines, pgRNA; M, markers (50 pg of 1-kb, 2-kb, and 3.2-kb DNA fragments released from plasmids as the DNA ladder or total RNA extracted from HepAD38 cells as the RNA ladder).
Figure Legend Snippet: Sucrose gradient separation and analysis of viral particles from HepAD38 cell culture supernatant. (A) Distribution of hepatitis B viral particle-associated antigens and DNA/RNA in sucrose gradient. Viral particles prepared from HepAD38 cell culture supernatant (via PEG 8000 precipitation) were layered over a 10% to 60% (wt/wt) sucrose gradient for ultracentrifugation separation. Fractions were collected from top to bottom, and HBsAg level was analyzed by enzyme-linked immunosorbent assay (ELISA). HBsAg and viral DNA and RNA (quantified from gray density of bands in panel B) signals and sucrose density were plotted together. Viral particles were first resolved by native agarose gel electrophoresis, followed by immunoblotting (IB) of HBV envelope and core proteins with anti-HBsAg and anti-HBcAg antibodies. (B) Detection of viral DNA/RNA by Southern or Northern blotting. Total viral nucleic acids were extracted by the SDS-proteinase K method, and viral DNA (extracted from one-tenth of the samples used for Northern blotting) and RNA (treated with DNase I) were detected by Southern and Northern blot analyses with minus- or plus-strand-specific riboprobes, respectively. Symbols of HBsAg particles, empty virions (without nucleic acid), virions (with RC DNA), and naked capsids (empty or with nucleic acids) are depicted on the lower right side of panel A. Blank, no nucleic acids; two centered and gapped circles, RC DNA; straight line, SS DNA; wavy lines, pgRNA; M, markers (50 pg of 1-kb, 2-kb, and 3.2-kb DNA fragments released from plasmids as the DNA ladder or total RNA extracted from HepAD38 cells as the RNA ladder).

Techniques Used: Cell Culture, Enzyme-linked Immunosorbent Assay, Agarose Gel Electrophoresis, Northern Blot

Characterization of nucleic acid content within viral particles in plasma sample from patient 0. (A) CsCl density gradient analysis of plasma sample. Plasma from patient 0 was added directly with CsCl salt to a concentration of 21% (wt/wt) or 34% (wt/wt). Two milliliters of the 21% CsCl-plasma mixture was underlayered with 2.9 ml 34% CsCl-plasma mixture, followed by ultracentrifugation. Viral DNA from each fraction was extracted and subjected to Southern blot analysis. (B) Sucrose gradient analysis of concentrated plasma sample. Five hundred microliters of concentrated plasma sample (via ultracentrifugation through a 20% sucrose cushion) was fractionated in a 10% to 60% (wt/wt) sucrose gradient. PreS1 and HBsAg levels were determined by ELISA. Viral DNA and RNA were detected by Southern and Northern blotting with minus- or plus-strand-specific riboprobes. HBsAg, PreS1, and viral DNA and RNA (quantified from gray density of viral DNA/RNA bands, middle and lower) signals and sucrose density were plotted together. (C) Analysis of concentrated plasma sample with lower CsCl density gradient centrifugation. Two hundred fifty microliters of concentrated plasma sample was mixed with 2.2 ml TNE buffer and 2.45 ml of 37% (wt/wt) CsCl-TNE buffer (resulting in a homogenous CsCl solution with density of about 1.18 g/cm 3 ), followed by ultracentrifugation. DNA in viral particle pellets (lane P) stuck to the sidewall of centrifugation tubes and was recovered by digesting with SDS-proteinase K solution. Viral DNA and RNA were subjected to Southern and Northern blot analyses. (D) Analysis of concentrated plasma sample with higher level of CsCl density gradient centrifugation. Two hundred fifty microliters of concentrated plasma sample was mixed with 1 ml of TNE buffer and 1.25 ml of 37% (wt/wt) CsCl-TNE buffer and underlayered with 2.4 ml of 27% (wt/wt) (1.25 g/cm 3 ) CsCl-TNE solution, followed by ultracentrifugation. HBV DNA and RNA was detected by Southern and Northern blotting.
Figure Legend Snippet: Characterization of nucleic acid content within viral particles in plasma sample from patient 0. (A) CsCl density gradient analysis of plasma sample. Plasma from patient 0 was added directly with CsCl salt to a concentration of 21% (wt/wt) or 34% (wt/wt). Two milliliters of the 21% CsCl-plasma mixture was underlayered with 2.9 ml 34% CsCl-plasma mixture, followed by ultracentrifugation. Viral DNA from each fraction was extracted and subjected to Southern blot analysis. (B) Sucrose gradient analysis of concentrated plasma sample. Five hundred microliters of concentrated plasma sample (via ultracentrifugation through a 20% sucrose cushion) was fractionated in a 10% to 60% (wt/wt) sucrose gradient. PreS1 and HBsAg levels were determined by ELISA. Viral DNA and RNA were detected by Southern and Northern blotting with minus- or plus-strand-specific riboprobes. HBsAg, PreS1, and viral DNA and RNA (quantified from gray density of viral DNA/RNA bands, middle and lower) signals and sucrose density were plotted together. (C) Analysis of concentrated plasma sample with lower CsCl density gradient centrifugation. Two hundred fifty microliters of concentrated plasma sample was mixed with 2.2 ml TNE buffer and 2.45 ml of 37% (wt/wt) CsCl-TNE buffer (resulting in a homogenous CsCl solution with density of about 1.18 g/cm 3 ), followed by ultracentrifugation. DNA in viral particle pellets (lane P) stuck to the sidewall of centrifugation tubes and was recovered by digesting with SDS-proteinase K solution. Viral DNA and RNA were subjected to Southern and Northern blot analyses. (D) Analysis of concentrated plasma sample with higher level of CsCl density gradient centrifugation. Two hundred fifty microliters of concentrated plasma sample was mixed with 1 ml of TNE buffer and 1.25 ml of 37% (wt/wt) CsCl-TNE buffer and underlayered with 2.4 ml of 27% (wt/wt) (1.25 g/cm 3 ) CsCl-TNE solution, followed by ultracentrifugation. HBV DNA and RNA was detected by Southern and Northern blotting.

Techniques Used: Concentration Assay, Southern Blot, Enzyme-linked Immunosorbent Assay, Northern Blot, Gradient Centrifugation, Centrifugation

Related Articles

Transfection:

Article Title: DNA from Periodontopathogenic Bacteria Is Immunostimulatory for Mouse and Human Immune Cells
Article Snippet: .. RNA from human gingival fibroblasts (HGF), HEK 293 cells, and HEK 293 cells stably transfected with hTLR-9 (106 cells) was prepared by using a High Pure RNA isolation kit (Roche Molecular Biochemicals), which included treatment with RNase-free DNase I. cDNA was obtained from 5 μg of RNA by using 200 U of Superscript II reverse transcriptase (Life Technologies, Inc.) and 0.5 μg of oligo(dT)12-18 primer (Life Technologies, Inc.). .. PCR amplification of cDNA was performed with Taq polymerase by using primer pairs specific for hTLR-4 (5′-CCA GAG CCG CTG GTG TAT CT and 5′-AGA AGG CGG TAC AGC TCC AC), hTLR-9 (5′-CCA CCC TGG AAG AGC TAA ACC and 5′-GCC GTC CAT GAA TAG GAA GC), and human GAPDH (glyceraldehyde-3-phosphate dehydrogenase) (5′-ACG GAT TTG GTC GTA TTG GGC and 5′-TTG ACG GTG CCA TGG AAT TTG).

Article Title: Hepatitis B Viral DNA Decline at Loss of HBeAg Is Mainly Explained by Reduced cccDNA Load - Down-Regulated Transcription of PgRNA Has Limited Impact
Article Snippet: .. Extraction of DNA and RNA from Cells DNA and RNA were extracted from transfected and non-transfected hepatoma cells in a Magnapure robot (Roche Applied Science, Germany) using the Total NA protocol. .. Harvested cells were washed in 1 mL PBS and after centrifugation at 5000 rpm for 3 min the pellet was re-suspended in 800 µL RLT lysis buffer (Qiagen Sciences, MD, USA) before extraction.

Stable Transfection:

Article Title: DNA from Periodontopathogenic Bacteria Is Immunostimulatory for Mouse and Human Immune Cells
Article Snippet: .. RNA from human gingival fibroblasts (HGF), HEK 293 cells, and HEK 293 cells stably transfected with hTLR-9 (106 cells) was prepared by using a High Pure RNA isolation kit (Roche Molecular Biochemicals), which included treatment with RNase-free DNase I. cDNA was obtained from 5 μg of RNA by using 200 U of Superscript II reverse transcriptase (Life Technologies, Inc.) and 0.5 μg of oligo(dT)12-18 primer (Life Technologies, Inc.). .. PCR amplification of cDNA was performed with Taq polymerase by using primer pairs specific for hTLR-4 (5′-CCA GAG CCG CTG GTG TAT CT and 5′-AGA AGG CGG TAC AGC TCC AC), hTLR-9 (5′-CCA CCC TGG AAG AGC TAA ACC and 5′-GCC GTC CAT GAA TAG GAA GC), and human GAPDH (glyceraldehyde-3-phosphate dehydrogenase) (5′-ACG GAT TTG GTC GTA TTG GGC and 5′-TTG ACG GTG CCA TGG AAT TTG).

Synthesized:

Article Title: Chronic hypoxia‐induced slug promotes invasive behavior of prostate cancer cells by activating expression of ephrin‐B1, et al. Chronic hypoxia‐induced slug promotes invasive behavior of prostate cancer cells by activating expression of ephrin‐B1
Article Snippet: .. 2.5 Real‐time quantitative RT‐PCR First‐strand cDNA was synthesized from the total RNA using ThermoScript RT‐PCR System (Roche, Indianapolis, IN, USA). .. PCR was performed on a LightCycler system (Roche) using LightCycler FastStart DNA Master SYBR Green I reaction mix (Roche) and QuantiTect Primer Assays (QIAGEN, Hilden, Germany).

Isolation:

Article Title: DNA from Periodontopathogenic Bacteria Is Immunostimulatory for Mouse and Human Immune Cells
Article Snippet: .. RNA from human gingival fibroblasts (HGF), HEK 293 cells, and HEK 293 cells stably transfected with hTLR-9 (106 cells) was prepared by using a High Pure RNA isolation kit (Roche Molecular Biochemicals), which included treatment with RNase-free DNase I. cDNA was obtained from 5 μg of RNA by using 200 U of Superscript II reverse transcriptase (Life Technologies, Inc.) and 0.5 μg of oligo(dT)12-18 primer (Life Technologies, Inc.). .. PCR amplification of cDNA was performed with Taq polymerase by using primer pairs specific for hTLR-4 (5′-CCA GAG CCG CTG GTG TAT CT and 5′-AGA AGG CGG TAC AGC TCC AC), hTLR-9 (5′-CCA CCC TGG AAG AGC TAA ACC and 5′-GCC GTC CAT GAA TAG GAA GC), and human GAPDH (glyceraldehyde-3-phosphate dehydrogenase) (5′-ACG GAT TTG GTC GTA TTG GGC and 5′-TTG ACG GTG CCA TGG AAT TTG).

Article Title: Genome-wide analyses reveal the IRE1a-XBP1 pathway promotes T helper cell differentiation by resolving secretory stress and accelerating proliferation
Article Snippet: .. Reverse transcription quantitative PCR (RT-qPCR) Total RNA was isolated from two million cells by SV total RNA isolation kit (Promega). cDNA was prepared by annealing 500 ng RNA with oligo dT as per the manufacturer’s instructions (Transcriptor High Fidelity cDNA Synthesis kit, Roche). ..

Quantitative RT-PCR:

Article Title: Chronic hypoxia‐induced slug promotes invasive behavior of prostate cancer cells by activating expression of ephrin‐B1, et al. Chronic hypoxia‐induced slug promotes invasive behavior of prostate cancer cells by activating expression of ephrin‐B1
Article Snippet: .. 2.5 Real‐time quantitative RT‐PCR First‐strand cDNA was synthesized from the total RNA using ThermoScript RT‐PCR System (Roche, Indianapolis, IN, USA). .. PCR was performed on a LightCycler system (Roche) using LightCycler FastStart DNA Master SYBR Green I reaction mix (Roche) and QuantiTect Primer Assays (QIAGEN, Hilden, Germany).

Article Title: Genome-wide analyses reveal the IRE1a-XBP1 pathway promotes T helper cell differentiation by resolving secretory stress and accelerating proliferation
Article Snippet: .. Reverse transcription quantitative PCR (RT-qPCR) Total RNA was isolated from two million cells by SV total RNA isolation kit (Promega). cDNA was prepared by annealing 500 ng RNA with oligo dT as per the manufacturer’s instructions (Transcriptor High Fidelity cDNA Synthesis kit, Roche). ..

Real-time Polymerase Chain Reaction:

Article Title: Genome-wide analyses reveal the IRE1a-XBP1 pathway promotes T helper cell differentiation by resolving secretory stress and accelerating proliferation
Article Snippet: .. Reverse transcription quantitative PCR (RT-qPCR) Total RNA was isolated from two million cells by SV total RNA isolation kit (Promega). cDNA was prepared by annealing 500 ng RNA with oligo dT as per the manufacturer’s instructions (Transcriptor High Fidelity cDNA Synthesis kit, Roche). ..

other:

Article Title: Extracellular DNA facilitates bacterial adhesion during Burkholderia pseudomallei biofilm formation
Article Snippet: DNase I noticeably lowered eDNA concentrations in biofilm if the enzyme was added into the starting inoculum (0 h).

Reverse Transcription Polymerase Chain Reaction:

Article Title: Chronic hypoxia‐induced slug promotes invasive behavior of prostate cancer cells by activating expression of ephrin‐B1, et al. Chronic hypoxia‐induced slug promotes invasive behavior of prostate cancer cells by activating expression of ephrin‐B1
Article Snippet: .. 2.5 Real‐time quantitative RT‐PCR First‐strand cDNA was synthesized from the total RNA using ThermoScript RT‐PCR System (Roche, Indianapolis, IN, USA). .. PCR was performed on a LightCycler system (Roche) using LightCycler FastStart DNA Master SYBR Green I reaction mix (Roche) and QuantiTect Primer Assays (QIAGEN, Hilden, Germany).

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    Roche ribonuclease free deoxyribonuclease dnase i
    Identification and functional analysis of an evolutionarily conserved GRE/MRE located at −5.3 kb in the mouse (−4.6 kb in the human) Klf9 gene. A, <t>DNAse</t> I protection assay with the hGR-DBD of the evolutionary conserved 179-bp fragment
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    DNase I-PCR Assay of the <t>DNase</t> I Sensitivity of a Transgenic Enhancer Sequence. (A) . (B) A diagram illustrating the design of a PCR primer that allows the transgenic L3 locus, but not the endogenous L3, to be specifically amplified. (C) DNase I-PCR shows differential DNase I sensitivity of chromatin in selected genomic regions. The DNase I sensitivity of the transgenic L3 locus was similar to that of the open chromatin control ACTIN7 .
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    Identification and functional analysis of an evolutionarily conserved GRE/MRE located at −5.3 kb in the mouse (−4.6 kb in the human) Klf9 gene. A, DNAse I protection assay with the hGR-DBD of the evolutionary conserved 179-bp fragment

    Journal: Endocrinology

    Article Title: Molecular Basis for Glucocorticoid Induction of the Kr?ppel-Like Factor 9 Gene in Hippocampal Neurons

    doi: 10.1210/en.2012-1303

    Figure Lengend Snippet: Identification and functional analysis of an evolutionarily conserved GRE/MRE located at −5.3 kb in the mouse (−4.6 kb in the human) Klf9 gene. A, DNAse I protection assay with the hGR-DBD of the evolutionary conserved 179-bp fragment

    Article Snippet: We treated 1 μg of total RNA with 20 U of ribonuclease-free deoxyribonuclease (DNAse) I (Roche, Indianapolis, IN) ( ) before cDNA synthesis with the High Capacity Reverse Transcription kit (Invitrogen) with or without the addition of RT.

    Techniques: Functional Assay

    DNase I-PCR Assay of the DNase I Sensitivity of a Transgenic Enhancer Sequence. (A) . (B) A diagram illustrating the design of a PCR primer that allows the transgenic L3 locus, but not the endogenous L3, to be specifically amplified. (C) DNase I-PCR shows differential DNase I sensitivity of chromatin in selected genomic regions. The DNase I sensitivity of the transgenic L3 locus was similar to that of the open chromatin control ACTIN7 .

    Journal: The Plant Cell

    Article Title: Genome-Wide Prediction and Validation of Intergenic Enhancers in Arabidopsis Using Open Chromatin Signatures [OPEN]

    doi: 10.1105/tpc.15.00537

    Figure Lengend Snippet: DNase I-PCR Assay of the DNase I Sensitivity of a Transgenic Enhancer Sequence. (A) . (B) A diagram illustrating the design of a PCR primer that allows the transgenic L3 locus, but not the endogenous L3, to be specifically amplified. (C) DNase I-PCR shows differential DNase I sensitivity of chromatin in selected genomic regions. The DNase I sensitivity of the transgenic L3 locus was similar to that of the open chromatin control ACTIN7 .

    Article Snippet: A DNase I (RNase-free; 10 U/μL; Roche Applied Science; catalog number 04716728001) dilution series was prepared by step-wise dilution using digestion buffer.

    Techniques: Polymerase Chain Reaction, Transgenic Assay, Sequencing, Amplification

    DNase I footprinting analysis of the −204/+70 region of the α3 promoter

    Journal:

    Article Title: The expression of the human neuronal ?3 Na+,K+-ATPase subunit gene is regulated by the activity of the Sp1 and NF-Y transcription factors

    doi: 10.1042/BJ20041294

    Figure Lengend Snippet: DNase I footprinting analysis of the −204/+70 region of the α3 promoter

    Article Snippet: DNase I (DNase I RNase-free; Hoffmann-La Roche, Basel, Switzerland) was diluted in 1× binding buffer, 100 mM KCl and 20 mM MgCl2 , and used at concentrations of 0.05–0.2 units/μg of DNA without extract and 1.5 units/μg of DNA in the presence of extract.

    Techniques: Footprinting

    Electrophoretic analysis of PlcR species. Lysates of B. anthracis SdT2 and SdT12 were probed with C-PlcR antiserum 1451. (A) Lysates made in the presence of 10 mM EDTA. (B) Lysates made in the presence of 10 mM magnesium acetate and then treated with DNase I for 30 min at 37°C. All the samples were made to contain 2% SDS and were heated for 5 min at 95°C before electrophoresis. Putative identities shown in the right margin were determined only on the basis of molecular mass. M, molecular mass markers.

    Journal: Infection and Immunity

    Article Title: A Spontaneous Translational Fusion of Bacillus cereus PlcR and PapR Activates Transcription of PlcR-Dependent Genes in Bacillus anthracis via Binding with a Specific Palindromic Sequence

    doi: 10.1128/IAI.72.10.5814-5823.2004

    Figure Lengend Snippet: Electrophoretic analysis of PlcR species. Lysates of B. anthracis SdT2 and SdT12 were probed with C-PlcR antiserum 1451. (A) Lysates made in the presence of 10 mM EDTA. (B) Lysates made in the presence of 10 mM magnesium acetate and then treated with DNase I for 30 min at 37°C. All the samples were made to contain 2% SDS and were heated for 5 min at 95°C before electrophoresis. Putative identities shown in the right margin were determined only on the basis of molecular mass. M, molecular mass markers.

    Article Snippet: The lysates from the second group were treated with DNase I (RNase free; Roche Diagnostics GmbH, Mannheim, Germany) for 30 min at 37°C (1 U of the DNase per μl of lysate) and then used for Western immunoblotting experiments.

    Techniques: Electrophoresis

    Stability of PlcR complexes. Whole-cell proteins from 12-h cultures of B. anthracis SdT2 and SdT12 were treated sequentially with DNase I and Triton X-100 or with DNase I and urea. Triton-containing samples were heated in SDS sample buffer, whereas urea-containing samples were loaded directly onto the gels. C-PlcR antiserum 1451 was used for the blotting analysis. M, molecular mass markers.

    Journal: Infection and Immunity

    Article Title: A Spontaneous Translational Fusion of Bacillus cereus PlcR and PapR Activates Transcription of PlcR-Dependent Genes in Bacillus anthracis via Binding with a Specific Palindromic Sequence

    doi: 10.1128/IAI.72.10.5814-5823.2004

    Figure Lengend Snippet: Stability of PlcR complexes. Whole-cell proteins from 12-h cultures of B. anthracis SdT2 and SdT12 were treated sequentially with DNase I and Triton X-100 or with DNase I and urea. Triton-containing samples were heated in SDS sample buffer, whereas urea-containing samples were loaded directly onto the gels. C-PlcR antiserum 1451 was used for the blotting analysis. M, molecular mass markers.

    Article Snippet: The lysates from the second group were treated with DNase I (RNase free; Roche Diagnostics GmbH, Mannheim, Germany) for 30 min at 37°C (1 U of the DNase per μl of lysate) and then used for Western immunoblotting experiments.

    Techniques: