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Roche biotin rna labeling mix
The <t>gdpS</t> mRNA binds to sarS mRNA in vitro . (A) Predicted base pairings between gdpS mRNA and sarS mRNA. The minimum free energy value is given. (B) The ability of gdpS mRNA to bind to sarS mRNA was determined by <t>RNA-RNA</t> gel-shift assays. Biotin-labeled
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1) Product Images from "The Staphylococcus aureus Protein-Coding Gene gdpS Modulates sarS Expression via mRNA-mRNA Interaction"

Article Title: The Staphylococcus aureus Protein-Coding Gene gdpS Modulates sarS Expression via mRNA-mRNA Interaction

Journal:

doi: 10.1128/IAI.00159-15

The gdpS mRNA binds to sarS mRNA in vitro . (A) Predicted base pairings between gdpS mRNA and sarS mRNA. The minimum free energy value is given. (B) The ability of gdpS mRNA to bind to sarS mRNA was determined by RNA-RNA gel-shift assays. Biotin-labeled
Figure Legend Snippet: The gdpS mRNA binds to sarS mRNA in vitro . (A) Predicted base pairings between gdpS mRNA and sarS mRNA. The minimum free energy value is given. (B) The ability of gdpS mRNA to bind to sarS mRNA was determined by RNA-RNA gel-shift assays. Biotin-labeled

Techniques Used: In Vitro, Electrophoretic Mobility Shift Assay, Labeling

Analysis of mRNA half-life in the wild-type, the gdpS mutant, and C/ATG-M strains. The WT, the gdpS mutant, and C/ATG-M strains were treated with rifampin to block de novo RNA synthesis. Samples were taken for RNA isolation at 0, 3, 5, and 10 min after
Figure Legend Snippet: Analysis of mRNA half-life in the wild-type, the gdpS mutant, and C/ATG-M strains. The WT, the gdpS mutant, and C/ATG-M strains were treated with rifampin to block de novo RNA synthesis. Samples were taken for RNA isolation at 0, 3, 5, and 10 min after

Techniques Used: Mutagenesis, Blocking Assay, Isolation

Northern blot assay of gdpS in S. aureus NCTC8325. (A) Two biotin-labeled probes complementary to the 3′ or 5′ end of gdpS were selected. (B) The probes were hybridized to the whole RNA of S. aureus NCTC8325 by Northern blotting, and the
Figure Legend Snippet: Northern blot assay of gdpS in S. aureus NCTC8325. (A) Two biotin-labeled probes complementary to the 3′ or 5′ end of gdpS were selected. (B) The probes were hybridized to the whole RNA of S. aureus NCTC8325 by Northern blotting, and the

Techniques Used: Northern Blot, Labeling

RNA-RNA gel-shift assays of gdpS mRNA and spa mRNA. Biotin-labeled gdpS mRNA probes were used in all reactions, and the antisense RNA was used as a positive control. Increasing amounts of spa mRNA were incubated with the biotin-labeled gdpS mRNA probes.
Figure Legend Snippet: RNA-RNA gel-shift assays of gdpS mRNA and spa mRNA. Biotin-labeled gdpS mRNA probes were used in all reactions, and the antisense RNA was used as a positive control. Increasing amounts of spa mRNA were incubated with the biotin-labeled gdpS mRNA probes.

Techniques Used: Electrophoretic Mobility Shift Assay, Labeling, Positive Control, Incubation

2) 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

3) Product Images from "Targeting the polyadenylation factor EhCFIm25 with RNA aptamers controls survival in Entamoeba histolytica"

Article Title: Targeting the polyadenylation factor EhCFIm25 with RNA aptamers controls survival in Entamoeba histolytica

Journal: Scientific Reports

doi: 10.1038/s41598-018-23997-w

Identification of aptamers against EhCFIm25. ( A ) Schematic representation of ssDNA oligonucleotides used to generate the ssRNA library for the SELEX protocol. ( B ) The predicted secondary structures of the C4 and C5 aptamers. ( C – G ) The RNA-electrophoretic mobility shift assay (REMSA). Proteins were incubated with a biotin-labelled RNA probe, and the RNA-protein complexes were resolved via PAGE and chemiluminescence assays. ( C ) A REMSA of the R7 aptamer population with wild-type EhCFIm25 and mutant EhCFIm25*L135T proteins. Proteinase K (1 µg), unspecific competitor (tRNA) and RNA molecules from the first round of SELEX (R0) were used as controls. The image results from the grouping of gels cropped from different parts of the same gel and a different gel. ( D ) A REMSA of C4 and C5 aptamers with wild-type EhCFIm25 and mutant EhCFIm25*L135T proteins. The image results from the grouping of gels cropped from two different gels. ( E ) A REMSA of C4 and C5 aptamers with E . histolytica protein extracts. Anti-EhCFIm25 antibodies were used as controls. ( F and G ) A REMSA of C4 and C5 aptamers with protein extracts from HeLa cells ( F ) and Trypanosoma cruzi parasites ( G ). Eh: E . histolytica ; Tc: T . cruzi ; single arrowhead: the RNA-protein complex; double arrowhead: the RNA-protein-antibody complex; asterisk: the free probe.
Figure Legend Snippet: Identification of aptamers against EhCFIm25. ( A ) Schematic representation of ssDNA oligonucleotides used to generate the ssRNA library for the SELEX protocol. ( B ) The predicted secondary structures of the C4 and C5 aptamers. ( C – G ) The RNA-electrophoretic mobility shift assay (REMSA). Proteins were incubated with a biotin-labelled RNA probe, and the RNA-protein complexes were resolved via PAGE and chemiluminescence assays. ( C ) A REMSA of the R7 aptamer population with wild-type EhCFIm25 and mutant EhCFIm25*L135T proteins. Proteinase K (1 µg), unspecific competitor (tRNA) and RNA molecules from the first round of SELEX (R0) were used as controls. The image results from the grouping of gels cropped from different parts of the same gel and a different gel. ( D ) A REMSA of C4 and C5 aptamers with wild-type EhCFIm25 and mutant EhCFIm25*L135T proteins. The image results from the grouping of gels cropped from two different gels. ( E ) A REMSA of C4 and C5 aptamers with E . histolytica protein extracts. Anti-EhCFIm25 antibodies were used as controls. ( F and G ) A REMSA of C4 and C5 aptamers with protein extracts from HeLa cells ( F ) and Trypanosoma cruzi parasites ( G ). Eh: E . histolytica ; Tc: T . cruzi ; single arrowhead: the RNA-protein complex; double arrowhead: the RNA-protein-antibody complex; asterisk: the free probe.

Techniques Used: Electrophoretic Mobility Shift Assay, Incubation, Polyacrylamide Gel Electrophoresis, Mutagenesis

4) Product Images from "Mammalian mitochondrial RNAs are degraded in the mitochondrial intermembrane space by RNASET2"

Article Title: Mammalian mitochondrial RNAs are degraded in the mitochondrial intermembrane space by RNASET2

Journal: Protein & Cell

doi: 10.1007/s13238-017-0448-9

Characterization of a ribonuclease activity in the mitochondrial IMS . (A) Immunoblots of total mitochondria and fractions. Mitochondria were separated into four fractions: total soluble (including IMS and Matrix), total membrane, IMS and matrix. Immunoblotting was performed using antibodies for Mortalin (Matrix), TIM23 (Inner membrane), DDP2 (IMS), and TOM40 (Outer membrane). (B) Four mitochondrial fractions were examined for ribonuclease activity using biotinylated UCP2 mRNA as a substrate. (C) No ribonuclease activity localizes at the outer surface of mitochondrial outer membrane. Isolated mitochondria were resuspended in mitoprep buffer (M buffer) or hypotonic buffer (H buffer) that ruptures the mitochondrial outer membrane. Biotinylated UCP2 RNA was added to the mixture and incubated at 37°C for 1 min or 5 min before the reaction was terminated. (D) IMS ribonuclease activity was tested for its sensitivity to EDTA (2 mmol/L) and Cu 2+ (0.5 mmol/L) using biotinylated UCP2 mRNA as a substrate. (E) IMS ribonuclease activity was tested for its sensitivity to different concentrations of Cu 2+ (0.5 mmol/L and 5 mmol/L), and Mg 2+ (0.5 mmol/L and 10 mmol/L) using RNAs purified from isolated mitochondria as substrates. (F) IMS ribonuclease activity and RNaseI were tested for sensitivity to different concentrations of ATP (0 mmol/L, 5 mmol/L, and 10 mmol/L). (G) IMS ribonuclease activity was tested for sensitivity to Proteinase K (ProK), EDTA (2 mmol/L), Cu 2+ (0.5 mmol/L), and heat (90°C 10 min)
Figure Legend Snippet: Characterization of a ribonuclease activity in the mitochondrial IMS . (A) Immunoblots of total mitochondria and fractions. Mitochondria were separated into four fractions: total soluble (including IMS and Matrix), total membrane, IMS and matrix. Immunoblotting was performed using antibodies for Mortalin (Matrix), TIM23 (Inner membrane), DDP2 (IMS), and TOM40 (Outer membrane). (B) Four mitochondrial fractions were examined for ribonuclease activity using biotinylated UCP2 mRNA as a substrate. (C) No ribonuclease activity localizes at the outer surface of mitochondrial outer membrane. Isolated mitochondria were resuspended in mitoprep buffer (M buffer) or hypotonic buffer (H buffer) that ruptures the mitochondrial outer membrane. Biotinylated UCP2 RNA was added to the mixture and incubated at 37°C for 1 min or 5 min before the reaction was terminated. (D) IMS ribonuclease activity was tested for its sensitivity to EDTA (2 mmol/L) and Cu 2+ (0.5 mmol/L) using biotinylated UCP2 mRNA as a substrate. (E) IMS ribonuclease activity was tested for its sensitivity to different concentrations of Cu 2+ (0.5 mmol/L and 5 mmol/L), and Mg 2+ (0.5 mmol/L and 10 mmol/L) using RNAs purified from isolated mitochondria as substrates. (F) IMS ribonuclease activity and RNaseI were tested for sensitivity to different concentrations of ATP (0 mmol/L, 5 mmol/L, and 10 mmol/L). (G) IMS ribonuclease activity was tested for sensitivity to Proteinase K (ProK), EDTA (2 mmol/L), Cu 2+ (0.5 mmol/L), and heat (90°C 10 min)

Techniques Used: Activity Assay, Western Blot, Isolation, Incubation, Purification

Characterization of mtRNA degradation using an in organello system . (A) Coomassie staining of human PNPASE samples purified from HEK293 mitochondria (HEK mito) or E . coli . For PNPASE purified from HEK mitochondria, two concentrations of samples were loaded. Con: same volume of eluate from cells harboring the empty vector; PNP: eluate from PNPASE-HisPC expressing cells. (B) PNPASE purified from E . coli or HEK mitochondria was incubated with Biotin-labeled UCP2 RNA. The lower panel shows immunoblotting of PNPASE. (C) PNPASE purified from E . coli or HEK mitochondria was incubated with total cytosolic RNA and the samples were resolved on an agarose gel. The lower panel shows immunoblotting of PNPASE. (D) Mitochondria were separated into total soluble and total membrane and the two fractions were examined for ribonuclease activity using biotinylated UCP2 mRNA as a substrate. Lower panels show Immunoblotting of PNPASE (Membrane) and DDP2 (Soluble). (E) Effect of temperature on in organello mtRNA degradation. Degradation was performed at 37°C (the temperature used for the other experiments if not specified) or 25°C. The three numbers (1, 2, and 3) represent three time points (5 min, 25 min, and 45 min). Top panel on the left shows the remaining labeled mtRNAs. Bottom panel is an immunoblot of mitochondrial protein Mortalin showing the amount of mitochondria taken out at each time point. Right panel shows the quantification of labeled mtRNAs ( n = 3). (F) Effect of pH on in organello mtRNA degradation. Degradation was performed at pH 7.4 (the pH used for the other experiments if not specified) or pH 6.5. (G) Effect of Cu 2+ on in organello mtRNA degradation. Two concentrations (0 mmol/L and 0.5 mmol/L) of Cu 2+ were used. (H) Effect of Mg 2+ on in organello mtRNA degradation. Three concentrations (0 mmol/L, 0.5 mmol/L, and 20 mmol/L) of Mg 2+ were used. (I) Effect of ATP on in organello mtRNA degradation. Three concentrations (0 mmol/L, 0.5 mmol/L, and 8 mmol/L) of ATP were used. Statistical comparisons are performed using unpaired t -tests ( n = 3 if not specified); * P
Figure Legend Snippet: Characterization of mtRNA degradation using an in organello system . (A) Coomassie staining of human PNPASE samples purified from HEK293 mitochondria (HEK mito) or E . coli . For PNPASE purified from HEK mitochondria, two concentrations of samples were loaded. Con: same volume of eluate from cells harboring the empty vector; PNP: eluate from PNPASE-HisPC expressing cells. (B) PNPASE purified from E . coli or HEK mitochondria was incubated with Biotin-labeled UCP2 RNA. The lower panel shows immunoblotting of PNPASE. (C) PNPASE purified from E . coli or HEK mitochondria was incubated with total cytosolic RNA and the samples were resolved on an agarose gel. The lower panel shows immunoblotting of PNPASE. (D) Mitochondria were separated into total soluble and total membrane and the two fractions were examined for ribonuclease activity using biotinylated UCP2 mRNA as a substrate. Lower panels show Immunoblotting of PNPASE (Membrane) and DDP2 (Soluble). (E) Effect of temperature on in organello mtRNA degradation. Degradation was performed at 37°C (the temperature used for the other experiments if not specified) or 25°C. The three numbers (1, 2, and 3) represent three time points (5 min, 25 min, and 45 min). Top panel on the left shows the remaining labeled mtRNAs. Bottom panel is an immunoblot of mitochondrial protein Mortalin showing the amount of mitochondria taken out at each time point. Right panel shows the quantification of labeled mtRNAs ( n = 3). (F) Effect of pH on in organello mtRNA degradation. Degradation was performed at pH 7.4 (the pH used for the other experiments if not specified) or pH 6.5. (G) Effect of Cu 2+ on in organello mtRNA degradation. Two concentrations (0 mmol/L and 0.5 mmol/L) of Cu 2+ were used. (H) Effect of Mg 2+ on in organello mtRNA degradation. Three concentrations (0 mmol/L, 0.5 mmol/L, and 20 mmol/L) of Mg 2+ were used. (I) Effect of ATP on in organello mtRNA degradation. Three concentrations (0 mmol/L, 0.5 mmol/L, and 8 mmol/L) of ATP were used. Statistical comparisons are performed using unpaired t -tests ( n = 3 if not specified); * P

Techniques Used: Staining, Purification, Plasmid Preparation, Expressing, Incubation, Labeling, Agarose Gel Electrophoresis, Activity Assay

Characterization of RNASET2 purified from HEK mitochondria . (A) Dual-tag purification of RNASET2 (His and HA). Purification was performed using IMS from control HEK cells (C) or RNASET2-overexpressing cells (T2) under native condition. (B) Ribonuclease activity were examined in IMS samples and the purification samples (Eluate) from control HEK cells (C) or RNASET2-overexpressing cells (T2) using biotinylated UCP2 mRNA as a substrate. The sensitivity of these activities to Cu 2+ (0.5 mmol/L) and proteinase K (ProK) was also tested. (C) RNASET2 was purified from RNASET2-overexpressing mitochondria under denaturing condition and checked for ribonuclease activity using RNA purified from isolated mitochondria as substrates; C (control pulldown from HEK mitochondria) and T2 (RNASET2). (D) RNASET2s purified under native conditions and denaturing conditions had the same responses to proteinase K (ProK) and Cu 2+ treatment. Lower panel is a coomassie staining gel of mitochondrial lysates as a positive control for proteinase K treatment. (E) Effect of temperature on RNASET2 purified from HEK mitochondria, RNaseI, and RNaseA (50 ng). Degradation was performed at 37°C (the temperature used for the other experiments if not specified) or 25°C. RNAs purified from isolated mitochondria were used as substrates. Con (control pulldown from HEK mitochondria). (F) Effect of pH on RNASET2 purified from HEK mitochondria, RNaseI, and RNaseA. Degradation was performed at pH 7.4 (the pH used for the other experiments if not specified), pH 6.5 or pH 5.5. (G) Effect of ATP on RNASET2 purified from HEK mitochondria, RNaseI and RNaseA. (H) Effect of Mg 2+ and Cu 2+ on RNASET2 purified from HEK mitochondria, RNaseI and RNaseA
Figure Legend Snippet: Characterization of RNASET2 purified from HEK mitochondria . (A) Dual-tag purification of RNASET2 (His and HA). Purification was performed using IMS from control HEK cells (C) or RNASET2-overexpressing cells (T2) under native condition. (B) Ribonuclease activity were examined in IMS samples and the purification samples (Eluate) from control HEK cells (C) or RNASET2-overexpressing cells (T2) using biotinylated UCP2 mRNA as a substrate. The sensitivity of these activities to Cu 2+ (0.5 mmol/L) and proteinase K (ProK) was also tested. (C) RNASET2 was purified from RNASET2-overexpressing mitochondria under denaturing condition and checked for ribonuclease activity using RNA purified from isolated mitochondria as substrates; C (control pulldown from HEK mitochondria) and T2 (RNASET2). (D) RNASET2s purified under native conditions and denaturing conditions had the same responses to proteinase K (ProK) and Cu 2+ treatment. Lower panel is a coomassie staining gel of mitochondrial lysates as a positive control for proteinase K treatment. (E) Effect of temperature on RNASET2 purified from HEK mitochondria, RNaseI, and RNaseA (50 ng). Degradation was performed at 37°C (the temperature used for the other experiments if not specified) or 25°C. RNAs purified from isolated mitochondria were used as substrates. Con (control pulldown from HEK mitochondria). (F) Effect of pH on RNASET2 purified from HEK mitochondria, RNaseI, and RNaseA. Degradation was performed at pH 7.4 (the pH used for the other experiments if not specified), pH 6.5 or pH 5.5. (G) Effect of ATP on RNASET2 purified from HEK mitochondria, RNaseI and RNaseA. (H) Effect of Mg 2+ and Cu 2+ on RNASET2 purified from HEK mitochondria, RNaseI and RNaseA

Techniques Used: Purification, Hemagglutination Assay, Activity Assay, Isolation, Staining, Positive Control

5) Product Images from "Ets-1 promoter-associated noncoding RNA regulates the NONO/ERG/Ets-1 axis to drive gastric cancer progression"

Article Title: Ets-1 promoter-associated noncoding RNA regulates the NONO/ERG/Ets-1 axis to drive gastric cancer progression

Journal: Oncogene

doi: 10.1038/s41388-018-0302-4

pancEts-1 is a lncRNA associated with poor survival of gastric cancer. a Scheme indicating the existence of pancEts-1 transcribed upstream the Ets-1 promoter region. b RNA fluorescence in situ hybridization images showing the nuclear and cytoplasmic localization of pancEts-1 in MKN-45 cells using a 138-bp antisense probe (red), with the nuclei staining by DAPI (blue). Sense probe and antisense probe with RNase A (20 μg) treatment were used as negative controls. Scale bars: 10 μm. c Real-time qRT-PCR assay revealing the pancEts-1 transcript levels (normalized to β-actin) in normal gastric mucosa ( n = 30) and cultured gastric cancer cell lines (mean ± SD, n = 5). d Real-time qRT-PCR assay indicating the differential expression of pancEts-1 transcript (normalized to β-actin) in normal gastric mucosa ( n = 30) and gastric cancer tissues ( n = 81). e , f Real-time qRT-PCR assay showing the pancEts-1 transcript levels (normalized to β-actin) in gastric cancer tissues with differential status of metastasis ( e ) or Ets-1 immunostaining ( f ). g The positive correlation between pancEts-1 and Ets-1 transcript levels in gastric cancer tissues ( n = 81). h Kaplan–Meier curves indicating overall survival of 81 gastric cancer patients (cutoff value = 2.833) and overall (OS) and first progression (FP) survival of those derived from Kaplan-Meier plotter with low or high pancEts-1 expression (cutoff values = 14.0 and 14.0). Student’s t test compared gene expression levels in c – f . Pearson’s correlation coefficient analysis in g . Log-rank test for survival comparison in h
Figure Legend Snippet: pancEts-1 is a lncRNA associated with poor survival of gastric cancer. a Scheme indicating the existence of pancEts-1 transcribed upstream the Ets-1 promoter region. b RNA fluorescence in situ hybridization images showing the nuclear and cytoplasmic localization of pancEts-1 in MKN-45 cells using a 138-bp antisense probe (red), with the nuclei staining by DAPI (blue). Sense probe and antisense probe with RNase A (20 μg) treatment were used as negative controls. Scale bars: 10 μm. c Real-time qRT-PCR assay revealing the pancEts-1 transcript levels (normalized to β-actin) in normal gastric mucosa ( n = 30) and cultured gastric cancer cell lines (mean ± SD, n = 5). d Real-time qRT-PCR assay indicating the differential expression of pancEts-1 transcript (normalized to β-actin) in normal gastric mucosa ( n = 30) and gastric cancer tissues ( n = 81). e , f Real-time qRT-PCR assay showing the pancEts-1 transcript levels (normalized to β-actin) in gastric cancer tissues with differential status of metastasis ( e ) or Ets-1 immunostaining ( f ). g The positive correlation between pancEts-1 and Ets-1 transcript levels in gastric cancer tissues ( n = 81). h Kaplan–Meier curves indicating overall survival of 81 gastric cancer patients (cutoff value = 2.833) and overall (OS) and first progression (FP) survival of those derived from Kaplan-Meier plotter with low or high pancEts-1 expression (cutoff values = 14.0 and 14.0). Student’s t test compared gene expression levels in c – f . Pearson’s correlation coefficient analysis in g . Log-rank test for survival comparison in h

Techniques Used: Fluorescence, In Situ Hybridization, Staining, Quantitative RT-PCR, Cell Culture, Expressing, Immunostaining, Derivative Assay

6) Product Images from "LncBRM initiates YAP1 signalling activation to drive self-renewal of liver cancer stem cells"

Article Title: LncBRM initiates YAP1 signalling activation to drive self-renewal of liver cancer stem cells

Journal: Nature Communications

doi: 10.1038/ncomms13608

LncBRM associates with BRM to initiate the BRG1/BRM switch. ( a ) LncBRM intron sequence (Ctrl), lncBRM antisense ( Lnc-AS ) and lncBRM transcripts were labelled with biotin and incubated with oncosphere lysates, followed by silver staining and mass spectrometry. Black arrow denotes BRM. ( b ) RNA pulldown was conducted using lncBRM transcript, followed by immunoblotting. ( c ) Domain mapping of lncBRM transcript. ( d ) LncBRM was incubated with increased doses of BRM, followed by electrophoretic mobility shift assay (EMSA). The 3 segment of lncBRM was labelled with biotin for probing. ( e ) Non-spheres and spheres were visualized by fluorescence in situ hybridization (FISH). Scale bar, 10 μm. ( f ) Antibodies against BRM or BRG1 were used for RNA immunoprecipitation, followed by RT–qPCR. ACTB served as a negative control. ( g ) Spheres (S) and non-sphere cells (N) were lysed and followed by immunoprecipitation with BAF170 and ARID1A antibodies. BRG1 and BRM enrichment was analysed with western blotting. ( h ) Different doses of lncBRM transcripts were incubated with oncosphere lysates and followed by co-immunoprecipitation (co-IP). ( i ) LncBRM -depleted HCC primary spheres were lysed for co-IP as in h . ( j , k ) The indicated oncosphere lysates were fractionated and followed by size fractionation with glycerol gradient ultracentrifugation. Elute gradients were used for western blotting. Data are shown as means±s.d. Two tailed Student's t -test was used for statistical analysis, *** P
Figure Legend Snippet: LncBRM associates with BRM to initiate the BRG1/BRM switch. ( a ) LncBRM intron sequence (Ctrl), lncBRM antisense ( Lnc-AS ) and lncBRM transcripts were labelled with biotin and incubated with oncosphere lysates, followed by silver staining and mass spectrometry. Black arrow denotes BRM. ( b ) RNA pulldown was conducted using lncBRM transcript, followed by immunoblotting. ( c ) Domain mapping of lncBRM transcript. ( d ) LncBRM was incubated with increased doses of BRM, followed by electrophoretic mobility shift assay (EMSA). The 3 segment of lncBRM was labelled with biotin for probing. ( e ) Non-spheres and spheres were visualized by fluorescence in situ hybridization (FISH). Scale bar, 10 μm. ( f ) Antibodies against BRM or BRG1 were used for RNA immunoprecipitation, followed by RT–qPCR. ACTB served as a negative control. ( g ) Spheres (S) and non-sphere cells (N) were lysed and followed by immunoprecipitation with BAF170 and ARID1A antibodies. BRG1 and BRM enrichment was analysed with western blotting. ( h ) Different doses of lncBRM transcripts were incubated with oncosphere lysates and followed by co-immunoprecipitation (co-IP). ( i ) LncBRM -depleted HCC primary spheres were lysed for co-IP as in h . ( j , k ) The indicated oncosphere lysates were fractionated and followed by size fractionation with glycerol gradient ultracentrifugation. Elute gradients were used for western blotting. Data are shown as means±s.d. Two tailed Student's t -test was used for statistical analysis, *** P

Techniques Used: Sequencing, Incubation, Silver Staining, Mass Spectrometry, Electrophoretic Mobility Shift Assay, Fluorescence, In Situ Hybridization, Fluorescence In Situ Hybridization, Immunoprecipitation, Quantitative RT-PCR, Negative Control, Western Blot, Co-Immunoprecipitation Assay, Fractionation, Two Tailed Test

KLF4 binds YAP1 promoter and recruits the BRG1-embedded BAF complex to initiate YAP1 expression. ( a ) HCC primary spheres were collected for co-IP assays with KLF4 antibody. ( b ) ChIP assay was performed using Klf4 antibody. ( c ) The interaction of KLF4 with YAP1 promoter was verified by EMSA assay. ( d ) HCC primary sphere cells were crosslinked with formaldehyde for ChIP assay with KLF4 antibody, followed by glycerol gradient ultracentrifugation. Elution gradients were concentrated for western blotting (upper panels) and PCR (lower panels) analyses. ( e , f ) KLF4 KO cells were established using CRISPR/Cas9 technology and allowed for sphere formation, followed by ChIP assay using BRG1 ( e ) and H3K4me3 ( f ) antibodies. ( g ) BRG1 was overexpressed in YAP1 promoter mutant ( YAP1p Mut) and wild-type (WT) cells, followed by sphere formation. Oncosphere cells were used for ChIP assays with H3K4me3 and H3K27ac antibodies, followed by examination for YAP1 promoter enrichment with real-time PCR. ( h ) LncBRM or BRG1 was overexpressed in YAP1 promoter mutant and WT cells and collected for RNA extraction. YAP1 mRNA expression was detected with northern blotting. ACTB served as a loading control. Data are shown as means±s.d. Two-tailed Student's t -test was used for statistical analysis; * P
Figure Legend Snippet: KLF4 binds YAP1 promoter and recruits the BRG1-embedded BAF complex to initiate YAP1 expression. ( a ) HCC primary spheres were collected for co-IP assays with KLF4 antibody. ( b ) ChIP assay was performed using Klf4 antibody. ( c ) The interaction of KLF4 with YAP1 promoter was verified by EMSA assay. ( d ) HCC primary sphere cells were crosslinked with formaldehyde for ChIP assay with KLF4 antibody, followed by glycerol gradient ultracentrifugation. Elution gradients were concentrated for western blotting (upper panels) and PCR (lower panels) analyses. ( e , f ) KLF4 KO cells were established using CRISPR/Cas9 technology and allowed for sphere formation, followed by ChIP assay using BRG1 ( e ) and H3K4me3 ( f ) antibodies. ( g ) BRG1 was overexpressed in YAP1 promoter mutant ( YAP1p Mut) and wild-type (WT) cells, followed by sphere formation. Oncosphere cells were used for ChIP assays with H3K4me3 and H3K27ac antibodies, followed by examination for YAP1 promoter enrichment with real-time PCR. ( h ) LncBRM or BRG1 was overexpressed in YAP1 promoter mutant and WT cells and collected for RNA extraction. YAP1 mRNA expression was detected with northern blotting. ACTB served as a loading control. Data are shown as means±s.d. Two-tailed Student's t -test was used for statistical analysis; * P

Techniques Used: Expressing, Co-Immunoprecipitation Assay, Chromatin Immunoprecipitation, Western Blot, Polymerase Chain Reaction, Gene Knockout, CRISPR, Mutagenesis, Real-time Polymerase Chain Reaction, RNA Extraction, Northern Blot, Two Tailed Test

LncBRM is highly expressed in HCC tumours and liver CSCs. ( a ) The indicated lncRNAs were silenced using pSiCoR lentivirus, followed by sphere formation assays. *, **, for Hep3B cells, Hep3B shlncBRM versus Hep3B shCtrl; #, ##, for Huh7 cells, Huh7 shlncBRM versus Huh7 shCtrl. ( b ) Total RNAs were extracted from peri-tumour (P) and tumour (T) tissues, followed by northern blotting. ACTB served as a loading control. ( c ) Primary HCC samples were prepared for examination of lncBRM expression using RT–qPCR. aHCC, advanced HCC; eHCC, early HCC. ( d ) LncBRM was detected by in situ hybridization. LncBRM highly expressed cells (middle panel) and lncBRM photon intensity (right panel) were calculated by Image-Pro Plus 6 and shown as scatter plot (means±s.e.m.). Scale bars, 100 μm. ( e ) Liver CSCs (CD13 + CD133 + ) and non-CSCs (CD13 − CD133 − ) were sorted from HCC samples, followed by detection of lncBRM using RT–qPCR (left panel). Oncospheres and non-spheres derived from HCC primary tumour cells were analysed similarly. Expression levels of lncBRM were normalized to that of non-tumour sample 17 as a baseline level. ( f ) lncBRM was examined in oncospheres and non-spheres with northern blotting. N, non-sphere; S, sphere. ( g ) Non-spheres and spheres were stained with lncBRM probes and CD13 antibody for confocal microscopy. Scale bars, 20 μm. ( h ) Nucleocytoplasmic fractionation of oncosphere cells was performed and followed by immunoblotting (upper panel) and RT–qPCR (lower panel). U1 RNA served as a nuclear location control and NKILA was used as a cytoplasmic location control. Data are shown as means±s.d. Two tailed Student's t -test was used for statistical analysis; * P
Figure Legend Snippet: LncBRM is highly expressed in HCC tumours and liver CSCs. ( a ) The indicated lncRNAs were silenced using pSiCoR lentivirus, followed by sphere formation assays. *, **, for Hep3B cells, Hep3B shlncBRM versus Hep3B shCtrl; #, ##, for Huh7 cells, Huh7 shlncBRM versus Huh7 shCtrl. ( b ) Total RNAs were extracted from peri-tumour (P) and tumour (T) tissues, followed by northern blotting. ACTB served as a loading control. ( c ) Primary HCC samples were prepared for examination of lncBRM expression using RT–qPCR. aHCC, advanced HCC; eHCC, early HCC. ( d ) LncBRM was detected by in situ hybridization. LncBRM highly expressed cells (middle panel) and lncBRM photon intensity (right panel) were calculated by Image-Pro Plus 6 and shown as scatter plot (means±s.e.m.). Scale bars, 100 μm. ( e ) Liver CSCs (CD13 + CD133 + ) and non-CSCs (CD13 − CD133 − ) were sorted from HCC samples, followed by detection of lncBRM using RT–qPCR (left panel). Oncospheres and non-spheres derived from HCC primary tumour cells were analysed similarly. Expression levels of lncBRM were normalized to that of non-tumour sample 17 as a baseline level. ( f ) lncBRM was examined in oncospheres and non-spheres with northern blotting. N, non-sphere; S, sphere. ( g ) Non-spheres and spheres were stained with lncBRM probes and CD13 antibody for confocal microscopy. Scale bars, 20 μm. ( h ) Nucleocytoplasmic fractionation of oncosphere cells was performed and followed by immunoblotting (upper panel) and RT–qPCR (lower panel). U1 RNA served as a nuclear location control and NKILA was used as a cytoplasmic location control. Data are shown as means±s.d. Two tailed Student's t -test was used for statistical analysis; * P

Techniques Used: Northern Blot, Expressing, Quantitative RT-PCR, In Situ Hybridization, Derivative Assay, Staining, Confocal Microscopy, Fractionation, Two Tailed Test

7) Product Images from "Long Noncoding RNA HEIH Promotes Colorectal Cancer Tumorigenesis via Counteracting miR-939‒Mediated Transcriptional Repression of Bcl-xL"

Article Title: Long Noncoding RNA HEIH Promotes Colorectal Cancer Tumorigenesis via Counteracting miR-939‒Mediated Transcriptional Repression of Bcl-xL

Journal: Cancer Research and Treatment : Official Journal of Korean Cancer Association

doi: 10.4143/crt.2017.226

Bcl-xL mRNA level is positively correlated with long noncoding RNA HEIH (lncRNA-HEIH) expression level in colorectal cancer (CRC) tissues. (A) Bcl-xL mRNA levels in 84 paired CRC and adjacent normal mucosa were detected by quantitative real-time polymerase chain reaction and normalized to glyceraldehyde 3-phosphate dehydrogenase. ***p
Figure Legend Snippet: Bcl-xL mRNA level is positively correlated with long noncoding RNA HEIH (lncRNA-HEIH) expression level in colorectal cancer (CRC) tissues. (A) Bcl-xL mRNA levels in 84 paired CRC and adjacent normal mucosa were detected by quantitative real-time polymerase chain reaction and normalized to glyceraldehyde 3-phosphate dehydrogenase. ***p

Techniques Used: Expressing, Real-time Polymerase Chain Reaction

Long noncoding RNA HEIH (lncRNA-HEIH) counteracts miR-939‒mediated transcriptional repression of Bcl-xL. (A) After transient transfection of lncRNA-HEIH or lncRNA-HEIH-mut into HT-29 cells, chromatin immunoprecipitation (ChIP) assays with nuclear factor κB (NF-κB) specific antibody were performed, and the retrieved DNA was detected by quantitative real-time polymerase chain reaction (qRT-PCR) and normalized to input. (B) After co-transfection of lncRNA-HEIH knockdown plasmid and miR-939 inhibitors into LoVo cells, ChIP assays with NF-κB specific antibody were performed, and the retrieved DNA was detected by qRT-PCR and normalized to input. (C) After co-transfection of lncRNA-HEIH or lncRNA-HEIH-mut and luciferase reporter containing Bcl-xL promoterinto HT-29 cells, the luciferase activities were measured. Results are shown as the relative ratio of firefly luciferase activity to Renilla luciferase activity. (D) After co-transfection of lncRNA-HEIH knockdown plasmid, miR-939 inhibitors, and luciferase reporter containing Bcl-xL promoter into LoVo cells, the luciferase activities were measured. Results are shown as in panel C. (E) After transient transfection of lncRNA-HEIH or lncRNA-HEIH-mut into HT-29 cells, Bcl-xL mRNA levels were detected by qRT-PCR and normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH). (F) After co-transfection of lncRNA-HEIH knockdown plasmid and miR-939 inhibitors into LoVo cells, Bcl-xL mRNA levels were detected by qRT-PCR and normalized to GAPDH. (G) After transient transfection of lncRNA-HEIH or lncRNA-HEIH-mut into HT-29 cells, Bcl-xL protein levels were detected by western blot and normalized to GAPDH. (H) After co-transfection of lncRNA-HEIH knockdown plasmid and miR-939 inhibitors into LoVo cells, Bcl-xL protein levels were detected by western blot and normalized to GAPDH. Results are shown as mean±standard deviation from three independent experiments. **p
Figure Legend Snippet: Long noncoding RNA HEIH (lncRNA-HEIH) counteracts miR-939‒mediated transcriptional repression of Bcl-xL. (A) After transient transfection of lncRNA-HEIH or lncRNA-HEIH-mut into HT-29 cells, chromatin immunoprecipitation (ChIP) assays with nuclear factor κB (NF-κB) specific antibody were performed, and the retrieved DNA was detected by quantitative real-time polymerase chain reaction (qRT-PCR) and normalized to input. (B) After co-transfection of lncRNA-HEIH knockdown plasmid and miR-939 inhibitors into LoVo cells, ChIP assays with NF-κB specific antibody were performed, and the retrieved DNA was detected by qRT-PCR and normalized to input. (C) After co-transfection of lncRNA-HEIH or lncRNA-HEIH-mut and luciferase reporter containing Bcl-xL promoterinto HT-29 cells, the luciferase activities were measured. Results are shown as the relative ratio of firefly luciferase activity to Renilla luciferase activity. (D) After co-transfection of lncRNA-HEIH knockdown plasmid, miR-939 inhibitors, and luciferase reporter containing Bcl-xL promoter into LoVo cells, the luciferase activities were measured. Results are shown as in panel C. (E) After transient transfection of lncRNA-HEIH or lncRNA-HEIH-mut into HT-29 cells, Bcl-xL mRNA levels were detected by qRT-PCR and normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH). (F) After co-transfection of lncRNA-HEIH knockdown plasmid and miR-939 inhibitors into LoVo cells, Bcl-xL mRNA levels were detected by qRT-PCR and normalized to GAPDH. (G) After transient transfection of lncRNA-HEIH or lncRNA-HEIH-mut into HT-29 cells, Bcl-xL protein levels were detected by western blot and normalized to GAPDH. (H) After co-transfection of lncRNA-HEIH knockdown plasmid and miR-939 inhibitors into LoVo cells, Bcl-xL protein levels were detected by western blot and normalized to GAPDH. Results are shown as mean±standard deviation from three independent experiments. **p

Techniques Used: Transfection, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Cotransfection, Plasmid Preparation, Luciferase, Activity Assay, Western Blot, Standard Deviation

Long noncoding RNA HEIH (lncRNA-HEIH) physically binds to miR-939 and counteracts the binding between miR-939 and nuclear factor κB (NF-κB). (A) Schematic outlining the predicted miR-939 binding sites on lncRNA-HEIH. The red nucleotides indict the seed sequences of miR-939. (B) HT-29 cell lysates were incubated with biotinylated lncRNA-HEIH or the miR-939 binding sites mutated lncRNA-HEIH (lncRNA-HEIH-mut); after pull-down, RNAs were retrieved and detected by quantitative real-time polymerase chain reaction (qRT-PCR), and normalized to input. (C) After transient transfection of lncRNA-HEIH or lncRNA-HEIH-mut into HT-29 cells, RNA immunoprecipitation assay (RIP) assays with NF-κB specific antibody were performed, and the retrieved RNA was detected by qRT-PCR and normalized to input. (D) After co-transfection of lncRNA-HEIH knockdown plasmid and miR-939 inhibitors into LoVo cells, RIP assays with NF-κB specific antibody were performed, and the retrieved RNA was detected by qRT-PCR and normalized to input. Results are shown as mean±standard deviation from three independent experiments. **p
Figure Legend Snippet: Long noncoding RNA HEIH (lncRNA-HEIH) physically binds to miR-939 and counteracts the binding between miR-939 and nuclear factor κB (NF-κB). (A) Schematic outlining the predicted miR-939 binding sites on lncRNA-HEIH. The red nucleotides indict the seed sequences of miR-939. (B) HT-29 cell lysates were incubated with biotinylated lncRNA-HEIH or the miR-939 binding sites mutated lncRNA-HEIH (lncRNA-HEIH-mut); after pull-down, RNAs were retrieved and detected by quantitative real-time polymerase chain reaction (qRT-PCR), and normalized to input. (C) After transient transfection of lncRNA-HEIH or lncRNA-HEIH-mut into HT-29 cells, RNA immunoprecipitation assay (RIP) assays with NF-κB specific antibody were performed, and the retrieved RNA was detected by qRT-PCR and normalized to input. (D) After co-transfection of lncRNA-HEIH knockdown plasmid and miR-939 inhibitors into LoVo cells, RIP assays with NF-κB specific antibody were performed, and the retrieved RNA was detected by qRT-PCR and normalized to input. Results are shown as mean±standard deviation from three independent experiments. **p

Techniques Used: Binding Assay, Incubation, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Transfection, Immunoprecipitation, Cotransfection, Plasmid Preparation, Standard Deviation

Long noncoding RNA HEIH (lncRNA-HEIH) is up-regulated in colorectal cancer (CRC) and indicts poor prognosis of CRC patients. (A) The expression of lncRNA-HEIH in 84 paired CRC and adjacent normal mucosa was detected by quantitative real-time polymerase chain reaction (qRT-PCR) and normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH). ***p
Figure Legend Snippet: Long noncoding RNA HEIH (lncRNA-HEIH) is up-regulated in colorectal cancer (CRC) and indicts poor prognosis of CRC patients. (A) The expression of lncRNA-HEIH in 84 paired CRC and adjacent normal mucosa was detected by quantitative real-time polymerase chain reaction (qRT-PCR) and normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH). ***p

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

The mutation of miR-939 binding sites on long noncoding RNA HEIH (lncRNA-HEIH) abolished the effects of lncRNA-HEIH on colorectal cancer tumorigenesis. (A) The expression of lncRNA-HEIH in lncRNA-HEIH or lncRNA-HEIH-mut stably overexpressed and control HT-29 cells was detected by quantitative real-time polymerase chain reaction and normalized to glyceraldehyde 3-phosphate dehydrogenase. (B) Cell proliferation rate of lncRNA-HEIH or lncRNA-HEIH-mut stably overexpressed and control HT-29 cells were detected by the Cell Counting Kit-8 assays. (C) Proliferative cells of lncRNA-HEIH or lncRNA-HEIH-mut stably overexpressed and control HT-29 were labeled with ethynyl deoxyuridine (EdU). Red color indicts EdU-positive cells. Scale bars=100 μm. (D) The level of apoptosis in lncRNA-HEIH or lncRNA-HEIH-mut stably overexpressed and control HT-29 cells was detected by TdT-mediated dUTP nick end labeling (TUNEL) staining. Blue color indicts TUNEL-positive cells. Scale bars=100 μm. For A-D, results are shown as mean±standard deviation (SD) from three independent experiments. **p
Figure Legend Snippet: The mutation of miR-939 binding sites on long noncoding RNA HEIH (lncRNA-HEIH) abolished the effects of lncRNA-HEIH on colorectal cancer tumorigenesis. (A) The expression of lncRNA-HEIH in lncRNA-HEIH or lncRNA-HEIH-mut stably overexpressed and control HT-29 cells was detected by quantitative real-time polymerase chain reaction and normalized to glyceraldehyde 3-phosphate dehydrogenase. (B) Cell proliferation rate of lncRNA-HEIH or lncRNA-HEIH-mut stably overexpressed and control HT-29 cells were detected by the Cell Counting Kit-8 assays. (C) Proliferative cells of lncRNA-HEIH or lncRNA-HEIH-mut stably overexpressed and control HT-29 were labeled with ethynyl deoxyuridine (EdU). Red color indicts EdU-positive cells. Scale bars=100 μm. (D) The level of apoptosis in lncRNA-HEIH or lncRNA-HEIH-mut stably overexpressed and control HT-29 cells was detected by TdT-mediated dUTP nick end labeling (TUNEL) staining. Blue color indicts TUNEL-positive cells. Scale bars=100 μm. For A-D, results are shown as mean±standard deviation (SD) from three independent experiments. **p

Techniques Used: Mutagenesis, Binding Assay, Expressing, Stable Transfection, Real-time Polymerase Chain Reaction, Cell Counting, Labeling, End Labeling, TUNEL Assay, Staining, Standard Deviation

Knockdown of long noncoding RNA HEIH (lncRNA-HEIH) inhibits colorectal cancer tumorigenesis. (A) The expression of lncRNA-HEIH in lncRNA-HEIH stably knocked-down and control LoVo cells was detected by quantitative real-time polymerase chain reaction and normalized to glyceraldehyde 3-phosphate dehydrogenase. (B) Cell proliferation rate of lncRNA-HEIH stably knocked-down and control LoVo cells were detected by the Cell Counting Kit-8 assays. (C) Proliferative cells of lncRNA-HEIH stably knocked-down and control LoVo were labeled with ethynyl deoxyuridine (EdU). Red color indicts EdU-positive cells. Scale bars=100 μm. (D) The level of apoptosis in lncRNA-HEIH stably knocked-down and control LoVo cells was detected by TdT-mediated dUTP nick end labeling (TUNEL) staining. Blue color indicts TUNEL-positive cells. Scale bars=100 μm. For A-D, results are shown as mean±standard deviation (SD) from three independent experiments. *p
Figure Legend Snippet: Knockdown of long noncoding RNA HEIH (lncRNA-HEIH) inhibits colorectal cancer tumorigenesis. (A) The expression of lncRNA-HEIH in lncRNA-HEIH stably knocked-down and control LoVo cells was detected by quantitative real-time polymerase chain reaction and normalized to glyceraldehyde 3-phosphate dehydrogenase. (B) Cell proliferation rate of lncRNA-HEIH stably knocked-down and control LoVo cells were detected by the Cell Counting Kit-8 assays. (C) Proliferative cells of lncRNA-HEIH stably knocked-down and control LoVo were labeled with ethynyl deoxyuridine (EdU). Red color indicts EdU-positive cells. Scale bars=100 μm. (D) The level of apoptosis in lncRNA-HEIH stably knocked-down and control LoVo cells was detected by TdT-mediated dUTP nick end labeling (TUNEL) staining. Blue color indicts TUNEL-positive cells. Scale bars=100 μm. For A-D, results are shown as mean±standard deviation (SD) from three independent experiments. *p

Techniques Used: Expressing, Stable Transfection, Real-time Polymerase Chain Reaction, Cell Counting, Labeling, End Labeling, TUNEL Assay, Staining, Standard Deviation

Enhanced expression of long noncoding RNA HEIH (lncRNA-HEIH) promotes colorectal cancer tumorigenesis. (A) The expression of lncRNA-HEIH in lncRNA-HEIH stably overexpressed and control HT-29 cells was detected by quantitative real-time polymerase chain reaction and normalized to glyceraldehyde 3-phosphat e dehydrogenase. (B) Cell proliferation rate of lncRNA-HEIH stably overexpressed and control HT-29 cells were detected by the Cell Counting Kit-8 assays. (C) Proliferative cells of lncRNA-HEIH stably overexpressed and control HT-29 were labeled with ethynyl deoxyuridine (EdU). Red color indicts EdU-positive cells. Scale bars=100 μm. (D) The level of apoptosis in lncRNA-HEIH stably overexpressed and control HT-29 cells was detected by TdT-mediated dUTP nick end labeling (TUNEL) staining. Blue color indicts TUNEL-positive cells. Scale bars=100 μm. For A-D, results are shown as mean±standard deviation (SD). from three independent experiments. **p
Figure Legend Snippet: Enhanced expression of long noncoding RNA HEIH (lncRNA-HEIH) promotes colorectal cancer tumorigenesis. (A) The expression of lncRNA-HEIH in lncRNA-HEIH stably overexpressed and control HT-29 cells was detected by quantitative real-time polymerase chain reaction and normalized to glyceraldehyde 3-phosphat e dehydrogenase. (B) Cell proliferation rate of lncRNA-HEIH stably overexpressed and control HT-29 cells were detected by the Cell Counting Kit-8 assays. (C) Proliferative cells of lncRNA-HEIH stably overexpressed and control HT-29 were labeled with ethynyl deoxyuridine (EdU). Red color indicts EdU-positive cells. Scale bars=100 μm. (D) The level of apoptosis in lncRNA-HEIH stably overexpressed and control HT-29 cells was detected by TdT-mediated dUTP nick end labeling (TUNEL) staining. Blue color indicts TUNEL-positive cells. Scale bars=100 μm. For A-D, results are shown as mean±standard deviation (SD). from three independent experiments. **p

Techniques Used: Expressing, Stable Transfection, Real-time Polymerase Chain Reaction, Cell Counting, Labeling, End Labeling, TUNEL Assay, Staining, Standard Deviation

8) Product Images from "LncRNA ZFAS1 promotes growth and metastasis by regulating BMI1 and ZEB2 in osteosarcoma"

Article Title: LncRNA ZFAS1 promotes growth and metastasis by regulating BMI1 and ZEB2 in osteosarcoma

Journal:

doi:

ZFAS1 regulates ZEB2 stability. A. Biotin-RNA pull-downs were performed with extracts of MG-63 cells using full-length ZFAS1 transcript (ZFAS1) and antisense ZFAS1 (ZFAS1-AS). This was followed by mass spectrometry. B. The interaction of ZFAS1 with ZEB2
Figure Legend Snippet: ZFAS1 regulates ZEB2 stability. A. Biotin-RNA pull-downs were performed with extracts of MG-63 cells using full-length ZFAS1 transcript (ZFAS1) and antisense ZFAS1 (ZFAS1-AS). This was followed by mass spectrometry. B. The interaction of ZFAS1 with ZEB2

Techniques Used: Mass Spectrometry

9) Product Images from "Upregulation of LncRNA‐HIT promotes migration and invasion of non‐small cell lung cancer cells by association with ZEB1"

Article Title: Upregulation of LncRNA‐HIT promotes migration and invasion of non‐small cell lung cancer cells by association with ZEB1

Journal: Cancer Medicine

doi: 10.1002/cam4.948

lnc RNA ‐ HIT promotes migration and invasion via regulation of ZEB 1 expression. (A) ZEB 1 protein expression in A549 control and lnc RNA ‐ HIT knockdown cells expressing control and ZEB 1. (B) Migration of A549 cells expressing control and lnc RNA ‐ HIT sh RNA s with and without ZEB 1. (C) Invasion of A549 cells expressing control and lnc RNA ‐ HIT sh RNA s with and without ZEB 1. Data are shown as mean ± SD. * P
Figure Legend Snippet: lnc RNA ‐ HIT promotes migration and invasion via regulation of ZEB 1 expression. (A) ZEB 1 protein expression in A549 control and lnc RNA ‐ HIT knockdown cells expressing control and ZEB 1. (B) Migration of A549 cells expressing control and lnc RNA ‐ HIT sh RNA s with and without ZEB 1. (C) Invasion of A549 cells expressing control and lnc RNA ‐ HIT sh RNA s with and without ZEB 1. Data are shown as mean ± SD. * P

Techniques Used: Migration, Expressing

lnc RNA ‐ HIT is upregulated in NSCLC tissues and cell lines and correlates with prognosis.(A) The relative expression of lnc RNA ‐ HIT in different NSCLC cell lines and normal lung epithelial cells ( NLEC ). (B) The relative expression of lnc RNA ‐ HIT in NSCLC tumor tissues (T) compared to matched adjacent nontumor tissues ( NT ) from 60 NSCLC patients. (C) Kaplan–Meier analyses of the correlations between lnc RNA ‐ HIT expression level and overall survival of 60 patients with NSCLC . The cut point of high and low Lnc RNA ‐ HIT expressers is the median. Data are shown as mean ± SD. * P
Figure Legend Snippet: lnc RNA ‐ HIT is upregulated in NSCLC tissues and cell lines and correlates with prognosis.(A) The relative expression of lnc RNA ‐ HIT in different NSCLC cell lines and normal lung epithelial cells ( NLEC ). (B) The relative expression of lnc RNA ‐ HIT in NSCLC tumor tissues (T) compared to matched adjacent nontumor tissues ( NT ) from 60 NSCLC patients. (C) Kaplan–Meier analyses of the correlations between lnc RNA ‐ HIT expression level and overall survival of 60 patients with NSCLC . The cut point of high and low Lnc RNA ‐ HIT expressers is the median. Data are shown as mean ± SD. * P

Techniques Used: Expressing

Silence of lnc RNA ‐ HIT inhibits the migration and invasion of NSCLC cells. (A) The relative expression of lnc RNA ‐ HIT in control and lnc RNA ‐ HIT knockdown cells. (B) The EMT markers detected by western blot in control and lnc RNA ‐ HIT knockdown cells. (C) Knockdown of lnc RNA ‐ HIT suppressed migration in NSCLC cells. (D) Knockdown of lnc RNA ‐ HIT suppressed invasion in NSCLC cells. Data are shown as mean ± SD . * P
Figure Legend Snippet: Silence of lnc RNA ‐ HIT inhibits the migration and invasion of NSCLC cells. (A) The relative expression of lnc RNA ‐ HIT in control and lnc RNA ‐ HIT knockdown cells. (B) The EMT markers detected by western blot in control and lnc RNA ‐ HIT knockdown cells. (C) Knockdown of lnc RNA ‐ HIT suppressed migration in NSCLC cells. (D) Knockdown of lnc RNA ‐ HIT suppressed invasion in NSCLC cells. Data are shown as mean ± SD . * P

Techniques Used: Migration, Expressing, Western Blot

lnc RNA ‐ HIT associates with ZEB 1. (A) RIP assays for lnc RNA ‐ HIT were performed using indicated antibodies. (B) Deletion mapping of ZEB 1‐binding domain in lnc RNA ‐ HIT (Up). Western blot for ZEB 1 in samples pulled down by different lnc RNA ‐ HIT fragments (Down). Antisense lnc RNA ‐ HIT was used as a negative control. (C) The relative ZEB 1 mRNA expression in control and lnc RNA ‐ HIT knockdown cells. (D) The ZEB 1 protein level in control and lnc RNA ‐ HIT knockdown cells. (E) The relative ZEB 1 mRNA expression in control and lnc RNA ‐ HIT overexpressed cells. (F) The ZEB 1 protein level in control and lnc RNA ‐ HIT overexpressed cells. (G) The stability of ZEB 1 protein over time was measured by western blot relative to time 0 after blocking new protein synthesis with 100 mg/ml CHX in control and lnc RNA ‐ HIT knockdown A549 cells. (H) ZEB 1 protein expression in control and lnc RNA ‐ HIT knockdown A549 cells treated with vehicle control ( DMSO ) or 10 μ mol/L MG 132 for 12 h. (I) The occupancy of ZEB 1 in the promoter of CDH 1 was measured by ZEB 1 Ch IP assay followed by qRT ‐ PCR in control and lnc RNA ‐ HIT knockdown A549 cells. (J) The occupancy of ZEB 1 in the promoter of CDH 1 was measured by ZEB 1 Ch IP assay followed by qRT ‐ PCR in control and lnc RNA ‐ HIT overexpressed A549 cells. Data are shown as mean ± SD. * P
Figure Legend Snippet: lnc RNA ‐ HIT associates with ZEB 1. (A) RIP assays for lnc RNA ‐ HIT were performed using indicated antibodies. (B) Deletion mapping of ZEB 1‐binding domain in lnc RNA ‐ HIT (Up). Western blot for ZEB 1 in samples pulled down by different lnc RNA ‐ HIT fragments (Down). Antisense lnc RNA ‐ HIT was used as a negative control. (C) The relative ZEB 1 mRNA expression in control and lnc RNA ‐ HIT knockdown cells. (D) The ZEB 1 protein level in control and lnc RNA ‐ HIT knockdown cells. (E) The relative ZEB 1 mRNA expression in control and lnc RNA ‐ HIT overexpressed cells. (F) The ZEB 1 protein level in control and lnc RNA ‐ HIT overexpressed cells. (G) The stability of ZEB 1 protein over time was measured by western blot relative to time 0 after blocking new protein synthesis with 100 mg/ml CHX in control and lnc RNA ‐ HIT knockdown A549 cells. (H) ZEB 1 protein expression in control and lnc RNA ‐ HIT knockdown A549 cells treated with vehicle control ( DMSO ) or 10 μ mol/L MG 132 for 12 h. (I) The occupancy of ZEB 1 in the promoter of CDH 1 was measured by ZEB 1 Ch IP assay followed by qRT ‐ PCR in control and lnc RNA ‐ HIT knockdown A549 cells. (J) The occupancy of ZEB 1 in the promoter of CDH 1 was measured by ZEB 1 Ch IP assay followed by qRT ‐ PCR in control and lnc RNA ‐ HIT overexpressed A549 cells. Data are shown as mean ± SD. * P

Techniques Used: Binding Assay, Western Blot, Negative Control, Expressing, Blocking Assay, Quantitative RT-PCR

Overexpression of lnc RNA ‐ HIT promotes the migration and invasion of NSCLC cells. (A) The relative expression of lnc RNA ‐ HIT in control and lnc RNA ‐ HIT overexpressed cells. (B) The EMT markers detected by western blot in control and lnc RNA ‐ HIT overexpressed cells. (C) Overexpression of lnc RNA ‐ HIT promoted migration in NSCLC cells. (D) Overexpression of lnc RNA ‐ HIT promoted invasion in NSCLC cells. Data are shown as mean ± SD. * P
Figure Legend Snippet: Overexpression of lnc RNA ‐ HIT promotes the migration and invasion of NSCLC cells. (A) The relative expression of lnc RNA ‐ HIT in control and lnc RNA ‐ HIT overexpressed cells. (B) The EMT markers detected by western blot in control and lnc RNA ‐ HIT overexpressed cells. (C) Overexpression of lnc RNA ‐ HIT promoted migration in NSCLC cells. (D) Overexpression of lnc RNA ‐ HIT promoted invasion in NSCLC cells. Data are shown as mean ± SD. * P

Techniques Used: Over Expression, Migration, Expressing, Western Blot

10) Product Images from "A positive feedback loop between ZNF205‐AS1 and EGR4 promotes non‐small cell lung cancer growth. A positive feedback loop between ZNF205‐AS1 and EGR4 promotes non‐small cell lung cancer growth"

Article Title: A positive feedback loop between ZNF205‐AS1 and EGR4 promotes non‐small cell lung cancer growth. A positive feedback loop between ZNF205‐AS1 and EGR4 promotes non‐small cell lung cancer growth

Journal: Journal of Cellular and Molecular Medicine

doi: 10.1111/jcmm.14056

ZNF205‐AS1 stabilized EGR4 mRNA via RNA‐RNA interaction. A, The expression of ZNF205‐AS1 in ZNF205‐AS1 stably overexpressed and control PC‐9 cells was detected by qPCR. B, The expression of ZNF205‐AS1 in ZNF205‐AS1 stably depleted and control SPC‐A1 cells was detected by qPCR. C, The mRNA levels of EGR4 in ZNF205‐AS1 stably overexpressed and control PC‐9 cells were detected by qPCR. D, The mRNA levels of EGR4 in ZNF205‐AS1 stably depleted and control SPC‐A1 cells was detected by qPCR. E, The protein levels of EGR4 in ZNF205‐AS1 stably overexpressed and control PC‐9 cells were detected by western blot. F, The protein levels of EGR4 in ZNF205‐AS1 stably depleted and control SPC‐A1 cells was detected by western blot. G, The subcellular distribution of ZNF205‐AS1 in SPC‐A1 cells was detected by qPCR. U6 and β‐actin were used as nuclear and cytoplasmic control, respectively. H, Schematic diagram of the predicted RNA‐RNA interaction between EGR4 mRNA and ZNF205‐AS1 transcript. I, The RNA‐RNA interaction between EGR4 mRNA and ZNF205‐AS1 transcript was detected by RNA pulldown assay using in vitro transcribed biotin‐labelled ZNF205‐AS1. The retrieved RNA was quantified by qPCR and displayed as percentage of input RNA. J, After transiently transfecting ZNF205‐AS1 overexpression plasmids into PC‐9 cells, the stability of EGR4 mRNA over time was detected by qPCR relative to time 0 after blocking new RNA synthesis with α‐amanitin (50 µM) and normalized to 18S rRNA (transcribed by RNA polymerase I and not influenced by α‐amanitin). K, After transiently transfecting ZNF205‐AS1 specific shRNAs into SPC‐A1 cells, the stability of EGR4 mRNA over time was detected by qPCR relative to time 0 after blocking new RNA synthesis with α‐amanitin (50 µM) and normalized to 18S rRNA. L, After transiently transfecting EGR4 overexpression plasmids into PC‐9 cells, the stability of ZNF205‐AS1 transcript over time was detected by qPCR relative to time 0 after blocking new RNA synthesis with α‐amanitin (50 µM) and normalized to 18S rRNA. M, After transiently transfecting EGR4 specific shRNAs into SPC‐A1 cells, the stability of ZNF205‐AS1 transcript over time was detected by qPCR relative to time 0 after blocking new RNA synthesis with α‐amanitin (50 µM) and normalized to 18S rRNA. Results are displayed as mean ± SD of three independent experiments. ** P
Figure Legend Snippet: ZNF205‐AS1 stabilized EGR4 mRNA via RNA‐RNA interaction. A, The expression of ZNF205‐AS1 in ZNF205‐AS1 stably overexpressed and control PC‐9 cells was detected by qPCR. B, The expression of ZNF205‐AS1 in ZNF205‐AS1 stably depleted and control SPC‐A1 cells was detected by qPCR. C, The mRNA levels of EGR4 in ZNF205‐AS1 stably overexpressed and control PC‐9 cells were detected by qPCR. D, The mRNA levels of EGR4 in ZNF205‐AS1 stably depleted and control SPC‐A1 cells was detected by qPCR. E, The protein levels of EGR4 in ZNF205‐AS1 stably overexpressed and control PC‐9 cells were detected by western blot. F, The protein levels of EGR4 in ZNF205‐AS1 stably depleted and control SPC‐A1 cells was detected by western blot. G, The subcellular distribution of ZNF205‐AS1 in SPC‐A1 cells was detected by qPCR. U6 and β‐actin were used as nuclear and cytoplasmic control, respectively. H, Schematic diagram of the predicted RNA‐RNA interaction between EGR4 mRNA and ZNF205‐AS1 transcript. I, The RNA‐RNA interaction between EGR4 mRNA and ZNF205‐AS1 transcript was detected by RNA pulldown assay using in vitro transcribed biotin‐labelled ZNF205‐AS1. The retrieved RNA was quantified by qPCR and displayed as percentage of input RNA. J, After transiently transfecting ZNF205‐AS1 overexpression plasmids into PC‐9 cells, the stability of EGR4 mRNA over time was detected by qPCR relative to time 0 after blocking new RNA synthesis with α‐amanitin (50 µM) and normalized to 18S rRNA (transcribed by RNA polymerase I and not influenced by α‐amanitin). K, After transiently transfecting ZNF205‐AS1 specific shRNAs into SPC‐A1 cells, the stability of EGR4 mRNA over time was detected by qPCR relative to time 0 after blocking new RNA synthesis with α‐amanitin (50 µM) and normalized to 18S rRNA. L, After transiently transfecting EGR4 overexpression plasmids into PC‐9 cells, the stability of ZNF205‐AS1 transcript over time was detected by qPCR relative to time 0 after blocking new RNA synthesis with α‐amanitin (50 µM) and normalized to 18S rRNA. M, After transiently transfecting EGR4 specific shRNAs into SPC‐A1 cells, the stability of ZNF205‐AS1 transcript over time was detected by qPCR relative to time 0 after blocking new RNA synthesis with α‐amanitin (50 µM) and normalized to 18S rRNA. Results are displayed as mean ± SD of three independent experiments. ** P

Techniques Used: Expressing, Stable Transfection, Real-time Polymerase Chain Reaction, Western Blot, In Vitro, Over Expression, Blocking Assay

11) Product Images from "LncRNA-SARCC suppresses renal cell carcinoma (RCC) progression via altering the androgen receptor(AR)/miRNA-143-3p signals"

Article Title: LncRNA-SARCC suppresses renal cell carcinoma (RCC) progression via altering the androgen receptor(AR)/miRNA-143-3p signals

Journal:

doi: 10.1038/cdd.2017.74

LncRNA-SARCC is physically associated with and negatively correlated with AR. ( a ) Immunoblot of AR expression in a series of RCC cell lines or immortalized proximal tubule epithelial cell line from normal adult human kidney (HK2, A498, SW839, 769-P, ACHN, 786-O, OSRC-2, Caki-1 and Caki-2), with prostate cancer cell line C4-2 as positive control. ( b ) A schematic illustration of the procedure used to discover and define LncRNAs binding to AR in RCC tissues. ( c ) RIP assays for the potential LncRNA candidates endogenously associated with AR in SW839 cells. Total RNA was subjected to qRT-PCR assays. ( d ) Primary RCC and adjacent non-cancerous renal tissues were subjected to RNA FISH and analyzed by ultraviolet light excitation using a fluorescence microscope. ( e ) qRT-PCR for LncRNA-SARCC, GAPDH and U1 from RNA extracted from cytoplasmic and nuclear fractions. ( f ) SW839 and OSRC-2 cells were cross-linked with/without 4% paraformaldehyde before RIP assays were carried out. ( g ) RIP assay in 10 nM DHT-treated SW839 cells at the indicated time points. ( h ) RNAs corresponding to different fragments of LncRNA-SARCC were biotinylated and incubated with SW839 cell extracts, targeted with streptavidin beads and washed. Associated AR protein was detected by WB and compared between full length LncRNA-SARCC and its antisense RNA. ( i and j ) qRT-PCR assays (left panels) for the shRNA-SARCC mRNA level in stable SW839 ( i ) and OSRC-2 ( j ) cell clones. AR protein and luciferase level were measured by WB (middle panels) and luciferase reporter assay (right panels). ( k and l ) qRT-PCR assays for the oe-SARCC mRNA levels in stable SW839 ( k ) and OSRC-2 ( l ) cell clones. AR protein and luciferase level were measured by WB (middle panels) and luciferase reporter assay (right panels). ( m ) Immunofluorescence staining of AR. SW839-control and SW839-LncRNA-SARCC cells were hormone-starved for 3 days and the SW839-control cells treated with DMSO or 1 nM R1881, whereas the SW839-LncRNA-SARCC cells were treated with R1881 for 24 h before subjected to immunostaining using an anti-AR antibody. ( n ) SW839 cells expressing control shRNA or LncRNA-SARCC shRNA were treated with 20 mg/ml cycloheximide (CHX) for the indicated time periods and cell lysates analyzed by WB. ( o ) SW839 cells expressing mock or oe-LncRNA-SARCC were treated as in ( n ) and analyzed by WB. ( p ) SW839 cells expressing shRNA-control (−) or shRNA-SARCC (+) were cultured with/without 5 mM MG132 for 10 h and cell lysates analyzed by WB. ( q ) CoIP showing AR-Hsp90 protein interaction with the absence or presence of LncRNA-SARCC. AR, Hsp90 and LncRNA-SARCC were expressed in 293T cells through transient transfection followed by AR immunoprecipitation. AR and associated Hsp90 protein was detected by immunoblot analysis. Data shown are mean±S.D. ( n =3). * P < 0.05, ** P < 0.01
Figure Legend Snippet: LncRNA-SARCC is physically associated with and negatively correlated with AR. ( a ) Immunoblot of AR expression in a series of RCC cell lines or immortalized proximal tubule epithelial cell line from normal adult human kidney (HK2, A498, SW839, 769-P, ACHN, 786-O, OSRC-2, Caki-1 and Caki-2), with prostate cancer cell line C4-2 as positive control. ( b ) A schematic illustration of the procedure used to discover and define LncRNAs binding to AR in RCC tissues. ( c ) RIP assays for the potential LncRNA candidates endogenously associated with AR in SW839 cells. Total RNA was subjected to qRT-PCR assays. ( d ) Primary RCC and adjacent non-cancerous renal tissues were subjected to RNA FISH and analyzed by ultraviolet light excitation using a fluorescence microscope. ( e ) qRT-PCR for LncRNA-SARCC, GAPDH and U1 from RNA extracted from cytoplasmic and nuclear fractions. ( f ) SW839 and OSRC-2 cells were cross-linked with/without 4% paraformaldehyde before RIP assays were carried out. ( g ) RIP assay in 10 nM DHT-treated SW839 cells at the indicated time points. ( h ) RNAs corresponding to different fragments of LncRNA-SARCC were biotinylated and incubated with SW839 cell extracts, targeted with streptavidin beads and washed. Associated AR protein was detected by WB and compared between full length LncRNA-SARCC and its antisense RNA. ( i and j ) qRT-PCR assays (left panels) for the shRNA-SARCC mRNA level in stable SW839 ( i ) and OSRC-2 ( j ) cell clones. AR protein and luciferase level were measured by WB (middle panels) and luciferase reporter assay (right panels). ( k and l ) qRT-PCR assays for the oe-SARCC mRNA levels in stable SW839 ( k ) and OSRC-2 ( l ) cell clones. AR protein and luciferase level were measured by WB (middle panels) and luciferase reporter assay (right panels). ( m ) Immunofluorescence staining of AR. SW839-control and SW839-LncRNA-SARCC cells were hormone-starved for 3 days and the SW839-control cells treated with DMSO or 1 nM R1881, whereas the SW839-LncRNA-SARCC cells were treated with R1881 for 24 h before subjected to immunostaining using an anti-AR antibody. ( n ) SW839 cells expressing control shRNA or LncRNA-SARCC shRNA were treated with 20 mg/ml cycloheximide (CHX) for the indicated time periods and cell lysates analyzed by WB. ( o ) SW839 cells expressing mock or oe-LncRNA-SARCC were treated as in ( n ) and analyzed by WB. ( p ) SW839 cells expressing shRNA-control (−) or shRNA-SARCC (+) were cultured with/without 5 mM MG132 for 10 h and cell lysates analyzed by WB. ( q ) CoIP showing AR-Hsp90 protein interaction with the absence or presence of LncRNA-SARCC. AR, Hsp90 and LncRNA-SARCC were expressed in 293T cells through transient transfection followed by AR immunoprecipitation. AR and associated Hsp90 protein was detected by immunoblot analysis. Data shown are mean±S.D. ( n =3). * P < 0.05, ** P < 0.01

Techniques Used: Expressing, Positive Control, Binding Assay, Quantitative RT-PCR, Fluorescence In Situ Hybridization, Fluorescence, Microscopy, Incubation, Western Blot, shRNA, Clone Assay, Luciferase, Reporter Assay, Immunofluorescence, Staining, Immunostaining, Cell Culture, Co-Immunoprecipitation Assay, Transfection, Immunoprecipitation

12) Product Images from "FBXL19-AS1 exerts oncogenic function by sponging miR-431-5p to regulate RAF1 expression in lung cancer"

Article Title: FBXL19-AS1 exerts oncogenic function by sponging miR-431-5p to regulate RAF1 expression in lung cancer

Journal: Bioscience Reports

doi: 10.1042/BSR20181804

FBXL19-AS1 acts as a sponge of miR-431-5p in lung cancer ( A ) The predicted binding sites of FBXL19-AS1 and miR-431-5p, and the mutant sites in mutant-type FBXL19-AS1 reporter. ( B ) FBXL19-AS1 (Wt) or FBXL19-AS1 (Mut) reporter were co-transfected with miR-NC, miR-431-5p mimic, or miR-431-5p inhibitor into A549 and H1299 cells. After 48 h, relative luciferase activity was detected by using luciferase reporter assay. ( C ) RIP and RT-qPCR assays were performed to determine the enrichment degrees of FBXL19-AS1 and miR-431-5p in IgG or Ago2 immunoprecipitate. The whole sample was divided in four, with 10% as input, 30% as positive control, 30% as negative control, and 30% as IP sample. ( D ) RNA pull-down assay was performed to further confirm the binding ability between FBXL19-AS1 and miR-431-5p in A549 and H1299 cells. ( E ) RT-qPCR results showed that overexpression of miR-431-5p reduced the expression of FBXL19-AS1. ( F ) RT-qPCR results showed that overexpression of FBXL19-AS1 reduced the expression of miR-431-5p. ( G ) RT-qPCR results showed that expression of miR-431-5p was lower in tumor tissues than in adjacent non-tumor tissues. ( H ) Spearman’s correlation analysis showed the negative correlation between expression of FBXL19-AS1 and miR-431-5p. Error bars represent the mean ± SD of at least three independent experiments. * P
Figure Legend Snippet: FBXL19-AS1 acts as a sponge of miR-431-5p in lung cancer ( A ) The predicted binding sites of FBXL19-AS1 and miR-431-5p, and the mutant sites in mutant-type FBXL19-AS1 reporter. ( B ) FBXL19-AS1 (Wt) or FBXL19-AS1 (Mut) reporter were co-transfected with miR-NC, miR-431-5p mimic, or miR-431-5p inhibitor into A549 and H1299 cells. After 48 h, relative luciferase activity was detected by using luciferase reporter assay. ( C ) RIP and RT-qPCR assays were performed to determine the enrichment degrees of FBXL19-AS1 and miR-431-5p in IgG or Ago2 immunoprecipitate. The whole sample was divided in four, with 10% as input, 30% as positive control, 30% as negative control, and 30% as IP sample. ( D ) RNA pull-down assay was performed to further confirm the binding ability between FBXL19-AS1 and miR-431-5p in A549 and H1299 cells. ( E ) RT-qPCR results showed that overexpression of miR-431-5p reduced the expression of FBXL19-AS1. ( F ) RT-qPCR results showed that overexpression of FBXL19-AS1 reduced the expression of miR-431-5p. ( G ) RT-qPCR results showed that expression of miR-431-5p was lower in tumor tissues than in adjacent non-tumor tissues. ( H ) Spearman’s correlation analysis showed the negative correlation between expression of FBXL19-AS1 and miR-431-5p. Error bars represent the mean ± SD of at least three independent experiments. * P

Techniques Used: Binding Assay, Mutagenesis, Transfection, Luciferase, Activity Assay, Reporter Assay, Quantitative RT-PCR, Positive Control, Negative Control, Pull Down Assay, Over Expression, Expressing

MiR-431-5p directly targets RAF1 and is negatively correlated to RAF1 expression ( A ) The predicted binding sites of miR-431-5p and RAF1 3′-UTR, and mutant sites in mutant-type RAF1 reporter. ( B ) RAF1 (Wt) or RAF1 (Mut) reporter were co-transfected with miR-NC, miR-431-5p mimic or miR-431-5p mimic +pcDNA3.1/FBXL19-AS1 into A549 and H1299 cells. After 48 h, relative luciferase activity was detected by using luciferase reporter assay. ( C ) RNA pull-down assay was performed to further confirm the binding ability between RAF1 and miR-431-5p in A549 and H1299 cells. ( D ) After 48 h of transfection, the protein expression of RAF1 was measured by western blot assay. ( E ) RT-qPCR results showed that overexpression of RAF1 reduced the expression of miR-431-5p. ( F ) RT-qPCR results showed that expression of RAF1 was higher in tumor tissues than in adjacent non-tumor tissues. ( G ) Spearman’s correlation analysis demonstrated the negative correlation between expression of RAF1 and miR-431-5p. ( H ) Spearman’s correlation analysis showed the positive correlation between expression of FBXL19-AS1 and RAF1. Error bars represent the mean ± SD of at least three independent experiments. * P
Figure Legend Snippet: MiR-431-5p directly targets RAF1 and is negatively correlated to RAF1 expression ( A ) The predicted binding sites of miR-431-5p and RAF1 3′-UTR, and mutant sites in mutant-type RAF1 reporter. ( B ) RAF1 (Wt) or RAF1 (Mut) reporter were co-transfected with miR-NC, miR-431-5p mimic or miR-431-5p mimic +pcDNA3.1/FBXL19-AS1 into A549 and H1299 cells. After 48 h, relative luciferase activity was detected by using luciferase reporter assay. ( C ) RNA pull-down assay was performed to further confirm the binding ability between RAF1 and miR-431-5p in A549 and H1299 cells. ( D ) After 48 h of transfection, the protein expression of RAF1 was measured by western blot assay. ( E ) RT-qPCR results showed that overexpression of RAF1 reduced the expression of miR-431-5p. ( F ) RT-qPCR results showed that expression of RAF1 was higher in tumor tissues than in adjacent non-tumor tissues. ( G ) Spearman’s correlation analysis demonstrated the negative correlation between expression of RAF1 and miR-431-5p. ( H ) Spearman’s correlation analysis showed the positive correlation between expression of FBXL19-AS1 and RAF1. Error bars represent the mean ± SD of at least three independent experiments. * P

Techniques Used: Expressing, Binding Assay, Mutagenesis, Transfection, Luciferase, Activity Assay, Reporter Assay, Pull Down Assay, Western Blot, Quantitative RT-PCR, Over Expression

13) Product Images from "Circular RNA hsa-circ-0012129 Promotes Cell Proliferation and Invasion in 30 Cases of Human Glioma and Human Glioma Cell Lines U373, A172, and SHG44, by Targeting MicroRNA-661 (miR-661)"

Article Title: Circular RNA hsa-circ-0012129 Promotes Cell Proliferation and Invasion in 30 Cases of Human Glioma and Human Glioma Cell Lines U373, A172, and SHG44, by Targeting MicroRNA-661 (miR-661)

Journal: Medical Science Monitor : International Medical Journal of Experimental and Clinical Research

doi: 10.12659/MSM.909229

Silence of circular RNA (circRNA), hsa-circ-0012129 by transfection of the U373 and SHG44 human glioma cells with small interfering RNA (siRNA) suppresses glioma cell proliferation. ( A, B ) Circular RNA (circRNA), hsa-circ-0012129 expression was assessed by quantitative real-time polymerase chain reaction (qRT-PCR) in the transfected U373 and SHG44 human glioma cells, respectively (*P
Figure Legend Snippet: Silence of circular RNA (circRNA), hsa-circ-0012129 by transfection of the U373 and SHG44 human glioma cells with small interfering RNA (siRNA) suppresses glioma cell proliferation. ( A, B ) Circular RNA (circRNA), hsa-circ-0012129 expression was assessed by quantitative real-time polymerase chain reaction (qRT-PCR) in the transfected U373 and SHG44 human glioma cells, respectively (*P

Techniques Used: Transfection, Small Interfering RNA, Expressing, Real-time Polymerase Chain Reaction, Quantitative RT-PCR

Circular RNA (circRNA), hsa-circ-0012129 knockdown inhibits glioma cell proliferation and invasion by targeting miR-661. ( A ) Quantitative real-time polymerase chain reaction (qRT-PCR) was used to detect the expression level of the microRNA, miR-661 in glioma tissues and normal tissues (* P
Figure Legend Snippet: Circular RNA (circRNA), hsa-circ-0012129 knockdown inhibits glioma cell proliferation and invasion by targeting miR-661. ( A ) Quantitative real-time polymerase chain reaction (qRT-PCR) was used to detect the expression level of the microRNA, miR-661 in glioma tissues and normal tissues (* P

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

Circular RNA (circRNA), hsa-circ-0012129 is highly expressed in glioma tumor tissues and the U373 and SHG44 human glioma cells. ( A ) Quantitative real-time polymerase chain react ion (qRT-PCR) was used to detect the expression of hsa-circ-0012129 and miR-661 in glioma tissues from 31 patients, compared with adjacent normal tissue, and showed a significant increase in expression in human glioma tissue (* P
Figure Legend Snippet: Circular RNA (circRNA), hsa-circ-0012129 is highly expressed in glioma tumor tissues and the U373 and SHG44 human glioma cells. ( A ) Quantitative real-time polymerase chain react ion (qRT-PCR) was used to detect the expression of hsa-circ-0012129 and miR-661 in glioma tissues from 31 patients, compared with adjacent normal tissue, and showed a significant increase in expression in human glioma tissue (* P

Techniques Used: Quantitative RT-PCR, Expressing

14) Product Images from "LincK contributes to breast tumorigenesis by promoting proliferation and epithelial-to-mesenchymal transition"

Article Title: LincK contributes to breast tumorigenesis by promoting proliferation and epithelial-to-mesenchymal transition

Journal: Journal of Hematology & Oncology

doi: 10.1186/s13045-019-0707-8

LincK was physically associated with miR-200 s. a Schematic outlining the predicted binding sites of miR-200 s on LincK. b Dual luciferase reporter assay in 293 T cells co-transfected with microRNAs (NC or miR-200b mimics) and psiCHECK2 (containing wild-type or mutant transcripts). Data were presented as the relative ratio of Renilla luciferase activity to Firefly luciferase activity. c Schematic outline of MS2-Flag RIP assay. 293 T cells were co-transfected by MS2bp-FLAG and MS2bs-LincK or MS2bs. Cell lysates were immunoprecipitated by FLAG M2 beads, and miR-200b endogenously associated with MS2bs-LincK were measured by qRT-PCR. Data were shown as means ± S.D. ( n = 3). d RNA pull down assay of the binding of LincK and mir-200b. MCF-7 cell lysates were incubated with biotin-labeled LincK or LacZ, and the mir-200b expression level was detected by qRT-PCR after pull down. RNAs associated with biotin-LincK were compared to that of biotin-LacZ, and U6 was used as non-specific control. Data were shown as means ± S.D. ( n = 3). e RIP assay of AGO2 enriched LincK in MCF-7 cells. IgG was used as negative controls. All relative abundances were compared to 1% input. Data were shown as means ± S.D. ( n = 3). * p
Figure Legend Snippet: LincK was physically associated with miR-200 s. a Schematic outlining the predicted binding sites of miR-200 s on LincK. b Dual luciferase reporter assay in 293 T cells co-transfected with microRNAs (NC or miR-200b mimics) and psiCHECK2 (containing wild-type or mutant transcripts). Data were presented as the relative ratio of Renilla luciferase activity to Firefly luciferase activity. c Schematic outline of MS2-Flag RIP assay. 293 T cells were co-transfected by MS2bp-FLAG and MS2bs-LincK or MS2bs. Cell lysates were immunoprecipitated by FLAG M2 beads, and miR-200b endogenously associated with MS2bs-LincK were measured by qRT-PCR. Data were shown as means ± S.D. ( n = 3). d RNA pull down assay of the binding of LincK and mir-200b. MCF-7 cell lysates were incubated with biotin-labeled LincK or LacZ, and the mir-200b expression level was detected by qRT-PCR after pull down. RNAs associated with biotin-LincK were compared to that of biotin-LacZ, and U6 was used as non-specific control. Data were shown as means ± S.D. ( n = 3). e RIP assay of AGO2 enriched LincK in MCF-7 cells. IgG was used as negative controls. All relative abundances were compared to 1% input. Data were shown as means ± S.D. ( n = 3). * p

Techniques Used: Binding Assay, Luciferase, Reporter Assay, Transfection, Mutagenesis, Activity Assay, Immunoprecipitation, Quantitative RT-PCR, Pull Down Assay, Incubation, Labeling, Expressing

15) Product Images from "LncSox4 promotes the self-renewal of liver tumour-initiating cells through Stat3-mediated Sox4 expression"

Article Title: LncSox4 promotes the self-renewal of liver tumour-initiating cells through Stat3-mediated Sox4 expression

Journal: Nature Communications

doi: 10.1038/ncomms12598

LncSox4 drives liver TIC self-renewal through Sox4. ( a , b ) LncSox4 is required for Sox4 expression. mRNA expression of the indicated genes in LncSox4 -silenced (sh LncSox4 ) and control cells (shCtrl) were examined using real-time PCR ( a ). Impaired Sox4 expression in LncSox4 -silenced cells was confirmed by western blot ( b ). ( c , d ) LncSox4 drives liver TIC self-renewal through Sox4 expression. Sox4 expression was rescued using PBPLV lentivirus in LncSox4 -silenced cells, and then sphere formation ( c ) and tumour initiation ( d ) assays were performed. Five thousand indicated primary HCC cells were used for sphere formation, and 10, 1 × 10 2 , 1 × 10 3 , 1 × 10 4 and 1 × 10 5 cells were subcutaneously injected into BALB/c nude mice for tumour initiation. ( e ) Primary HCC cells were treated with CRISPR/Cas9 lentivirus for Sox4 deficiency, followed by LncSox4 overexpression (oeLnc). The indicated cells were used for in vivo tumour initiation, and the ratios of tumour-free mice were calculated 3 months later. ( f ) LncSox4 binds to Sox4 promoter. RNA ChIP assay was performed and fold enrichment was examined using real-time PCR. ( g , h ) The interaction of LncSox4 and Sox4 promoter was required for LncSox4 function. The Sox4 promoter region for LncSox4 binding (−3,647∼−3,537) was deleted using CRISPR/Cas9 approach ( Sox4PKO ), followed by LncSox4 overexpression. The established cells were examined for Sox4 expression and sphere formation capacities. Six samples were examined and similar results were found. Scale bars, C, H 500 μm. Data were shown as means±s.d. Two-tailed Student's t -test was used for statistical analysis. * P
Figure Legend Snippet: LncSox4 drives liver TIC self-renewal through Sox4. ( a , b ) LncSox4 is required for Sox4 expression. mRNA expression of the indicated genes in LncSox4 -silenced (sh LncSox4 ) and control cells (shCtrl) were examined using real-time PCR ( a ). Impaired Sox4 expression in LncSox4 -silenced cells was confirmed by western blot ( b ). ( c , d ) LncSox4 drives liver TIC self-renewal through Sox4 expression. Sox4 expression was rescued using PBPLV lentivirus in LncSox4 -silenced cells, and then sphere formation ( c ) and tumour initiation ( d ) assays were performed. Five thousand indicated primary HCC cells were used for sphere formation, and 10, 1 × 10 2 , 1 × 10 3 , 1 × 10 4 and 1 × 10 5 cells were subcutaneously injected into BALB/c nude mice for tumour initiation. ( e ) Primary HCC cells were treated with CRISPR/Cas9 lentivirus for Sox4 deficiency, followed by LncSox4 overexpression (oeLnc). The indicated cells were used for in vivo tumour initiation, and the ratios of tumour-free mice were calculated 3 months later. ( f ) LncSox4 binds to Sox4 promoter. RNA ChIP assay was performed and fold enrichment was examined using real-time PCR. ( g , h ) The interaction of LncSox4 and Sox4 promoter was required for LncSox4 function. The Sox4 promoter region for LncSox4 binding (−3,647∼−3,537) was deleted using CRISPR/Cas9 approach ( Sox4PKO ), followed by LncSox4 overexpression. The established cells were examined for Sox4 expression and sphere formation capacities. Six samples were examined and similar results were found. Scale bars, C, H 500 μm. Data were shown as means±s.d. Two-tailed Student's t -test was used for statistical analysis. * P

Techniques Used: Expressing, Real-time Polymerase Chain Reaction, Western Blot, Injection, Mouse Assay, CRISPR, Over Expression, In Vivo, Chromatin Immunoprecipitation, Binding Assay, Two Tailed Test

LncSox4 interacts with Stat3. ( a ) RNA pull-down assay was performed using LncSox4 , Lnc-AS ( LncSox4 antisense RNA) and control RNA, and the samples were separated by Coomassie staining, followed by identification using mass spectrum. The black arrow indicates Stat3. KD, kiloDalton. ( b ) The interaction between LncSox4 and Stat3 was confirmed by RNA pull-down and western blot. Lnc-AS, LncSox4 anti-sense. β-Actin served as loading controls. ( c ) RIP was performed using anti-Stat3 and control IgG antibodies, followed by real-time PCR to examine the enrichment of LncSox4 and GAPDH. GAPDH served as a negative control. ( d ) LncSox4 is co-localized with Stat3. LncSox4 probes and anti-Stat3 antibody were used to examine the subcellular location using flow cytometer sorted CD133 - (Non-TIC), CD133 + (TIC) cells and stem-like oncospheres (Sphere). Scale bars, 10 μm. ( e ) Three regions of LncSox4 were examined for interaction with Stat3. The indicated truncates of LncSox4 were constructed and RNA pull-down assays were performed. The samples were examined by western blot with anti-Stat3 antibody. ( f ) LncSox4 interacts with Stat3. Biotin-labelled LncSox4 was obtained by in vitro transcription assays and incubated with recombination Stat3 protein, followed by RNA EMSA. 100 × unlabelled probes were used for competitive EMSA. ( g ) Bead-based proximity assay of LncSox4 , LncSox4 truncate (-#3 region) and LncSox4 -antisense (Lnc-AS ) (50 nnol) were incubated with various concentrations (horizontal axis) of Stat3 (left) or Stat3 (50 ng μl −1 ) mixed with various concentrations (horizontal axis) of biotin-labelled RNA (right); and units generated by the assay system were shown. ( h ) LncSox4 (FL), LncSox4 truncate (-#3 region) overexpressing primary HCC cells were established, followed by sphere-formation assay. Typical spheres were shown in left panels, and sphere formation ratios in right panels. Scale bars, 500 μm. Data were shown as means±s.d. Two-tailed Student's t -test was used for statistical analysis. * P
Figure Legend Snippet: LncSox4 interacts with Stat3. ( a ) RNA pull-down assay was performed using LncSox4 , Lnc-AS ( LncSox4 antisense RNA) and control RNA, and the samples were separated by Coomassie staining, followed by identification using mass spectrum. The black arrow indicates Stat3. KD, kiloDalton. ( b ) The interaction between LncSox4 and Stat3 was confirmed by RNA pull-down and western blot. Lnc-AS, LncSox4 anti-sense. β-Actin served as loading controls. ( c ) RIP was performed using anti-Stat3 and control IgG antibodies, followed by real-time PCR to examine the enrichment of LncSox4 and GAPDH. GAPDH served as a negative control. ( d ) LncSox4 is co-localized with Stat3. LncSox4 probes and anti-Stat3 antibody were used to examine the subcellular location using flow cytometer sorted CD133 - (Non-TIC), CD133 + (TIC) cells and stem-like oncospheres (Sphere). Scale bars, 10 μm. ( e ) Three regions of LncSox4 were examined for interaction with Stat3. The indicated truncates of LncSox4 were constructed and RNA pull-down assays were performed. The samples were examined by western blot with anti-Stat3 antibody. ( f ) LncSox4 interacts with Stat3. Biotin-labelled LncSox4 was obtained by in vitro transcription assays and incubated with recombination Stat3 protein, followed by RNA EMSA. 100 × unlabelled probes were used for competitive EMSA. ( g ) Bead-based proximity assay of LncSox4 , LncSox4 truncate (-#3 region) and LncSox4 -antisense (Lnc-AS ) (50 nnol) were incubated with various concentrations (horizontal axis) of Stat3 (left) or Stat3 (50 ng μl −1 ) mixed with various concentrations (horizontal axis) of biotin-labelled RNA (right); and units generated by the assay system were shown. ( h ) LncSox4 (FL), LncSox4 truncate (-#3 region) overexpressing primary HCC cells were established, followed by sphere-formation assay. Typical spheres were shown in left panels, and sphere formation ratios in right panels. Scale bars, 500 μm. Data were shown as means±s.d. Two-tailed Student's t -test was used for statistical analysis. * P

Techniques Used: Pull Down Assay, Staining, Western Blot, Real-time Polymerase Chain Reaction, Negative Control, Flow Cytometry, Cytometry, Construct, In Vitro, Incubation, Proximity Assay, Generated, Tube Formation Assay, Two Tailed Test

16) Product Images from "LncHOXA10 drives liver TICs self-renewal and tumorigenesis via HOXA10 transcription activation"

Article Title: LncHOXA10 drives liver TICs self-renewal and tumorigenesis via HOXA10 transcription activation

Journal: Molecular Cancer

doi: 10.1186/s12943-018-0921-y

LncHOXA10 interacted with SNF2L. a RNA pulldown was performed and the specific band in lncHOXA10 sample was identified as SNF2L by mass spectrum. b The combination between lncHOXA10 and SNF2L was examined by RNA-pulldown and Western blot. c Diagram showing the procedure of RAP (RNA antisense purification). DNA probes targeting endogenous lncHOXA10 were grouped into Probeset #2. d RAP assay was performed and a specific band in Probeset #2 sample was identified as SNF2L. e Eluate samples of RAP assay were utilized for SDS-PAGE, followed by Western blot for SNF2L detection. f LncHOXA10 truncates were generated (upper panels) and incubated with sphere lysates. The interaction between lncHOXA10 truncates and SNF2L was examined by Western blot (lower panels). g RNA electrophoretic mobility shift assay (RNA EMSA) was performed to validate the combination of lncHOXA10 and SNF2L. h RNA immunoprecipitation (RIP) assay was performed using oncospheres and enrichment of lncHOXA10 and GAPDH mRNA were examined through realtime PCR. IgG was an antibody control. i Double FISH assay showed the co-localization of lncHOXA10 and SNF2L in oncospheres. Scale bars, 10 μm. j LncHOXA10 depleted cells were used for SNF2L, H3K4me3 and RNA polymerase II (RNA PolII) ChIP assays, and HOXA10 promoter enrichment was detected with realtime PCR. (K, L) HOXA10 promoter was cloned into PGL3 luciferase reporter plasmid, and luciferase activity was measured in lncHOXA10 silenced ( k ) and overexpressing cells ( l ). m SNF2L knockout cells were generated through CRISPR/Cas9 approach, and the knockout efficiency was confirmed by Western blot. The expression of HOXA10 was also detected by Western blot. n , o Full length (FL) lncHOXA10 or truncated (△#1) lncHOXA10 was overexpressed in WT and SNF2L KO cells, followed by sphere formation assay (N) and transwell invasion assay ( o ). Data are representative of three independent experiments
Figure Legend Snippet: LncHOXA10 interacted with SNF2L. a RNA pulldown was performed and the specific band in lncHOXA10 sample was identified as SNF2L by mass spectrum. b The combination between lncHOXA10 and SNF2L was examined by RNA-pulldown and Western blot. c Diagram showing the procedure of RAP (RNA antisense purification). DNA probes targeting endogenous lncHOXA10 were grouped into Probeset #2. d RAP assay was performed and a specific band in Probeset #2 sample was identified as SNF2L. e Eluate samples of RAP assay were utilized for SDS-PAGE, followed by Western blot for SNF2L detection. f LncHOXA10 truncates were generated (upper panels) and incubated with sphere lysates. The interaction between lncHOXA10 truncates and SNF2L was examined by Western blot (lower panels). g RNA electrophoretic mobility shift assay (RNA EMSA) was performed to validate the combination of lncHOXA10 and SNF2L. h RNA immunoprecipitation (RIP) assay was performed using oncospheres and enrichment of lncHOXA10 and GAPDH mRNA were examined through realtime PCR. IgG was an antibody control. i Double FISH assay showed the co-localization of lncHOXA10 and SNF2L in oncospheres. Scale bars, 10 μm. j LncHOXA10 depleted cells were used for SNF2L, H3K4me3 and RNA polymerase II (RNA PolII) ChIP assays, and HOXA10 promoter enrichment was detected with realtime PCR. (K, L) HOXA10 promoter was cloned into PGL3 luciferase reporter plasmid, and luciferase activity was measured in lncHOXA10 silenced ( k ) and overexpressing cells ( l ). m SNF2L knockout cells were generated through CRISPR/Cas9 approach, and the knockout efficiency was confirmed by Western blot. The expression of HOXA10 was also detected by Western blot. n , o Full length (FL) lncHOXA10 or truncated (△#1) lncHOXA10 was overexpressed in WT and SNF2L KO cells, followed by sphere formation assay (N) and transwell invasion assay ( o ). Data are representative of three independent experiments

Techniques Used: Western Blot, Purification, SDS Page, Generated, Incubation, Electrophoretic Mobility Shift Assay, Immunoprecipitation, Polymerase Chain Reaction, Fluorescence In Situ Hybridization, Chromatin Immunoprecipitation, Clone Assay, Luciferase, Plasmid Preparation, Activity Assay, Knock-Out, CRISPR, Expressing, Tube Formation Assay, Transwell Invasion Assay

17) Product Images from "Long Stress Induced Non-Coding Transcripts 5 (LSINCT5) Promotes Hepatocellular Carcinoma Progression Through Interaction with High-Mobility Group AT-hook 2 and MiR-4516"

Article Title: Long Stress Induced Non-Coding Transcripts 5 (LSINCT5) Promotes Hepatocellular Carcinoma Progression Through Interaction with High-Mobility Group AT-hook 2 and MiR-4516

Journal: Medical Science Monitor : International Medical Journal of Experimental and Clinical Research

doi: 10.12659/MSM.911179

LSINCT5 acts as a ceRNA of miR-4516 to promote HCC progression. ( A ) RIP assays followed by qPCR to identify putative miRNAs associated with LSINCT5. ( B ) The miRNAs were verified by RNA pulldown a with biotin-labeled sense or antisense LSINCT5. ( C ) Luciferase reporter assays identified miR-4516 could interact with LSINCT5 (top). RNA pulldown assay with anti-AGO2 was performed in HepG2 cells with miR-4516 overexpression followed by qPCR to enrich LSINCT5 (bottom). ( D ) The predicted base-pairing between LSINCT5 and miR-4516 at miRDB. Three hits were detected. ( E ) RNA-FISH assay revealed the co-localization of LSINCT5 (Alexa Fluor 488, green dots) and miR-4516 (Cy3, red dots) in HepG2 cells. Scale bar, 5 μm. ( F ) CCK-8 viability assay for Huh7 cells and HepG2 cells transfected with plasmids as indicated. ( G ) Tumor sphere formation assay for HepG2 cells transfected with negative control (NC), miR-4516 mimics + oe Vec or miR-4516 mimics + LSINCT5. Quantification results were shown on the right panel. ( H ) Western blot assays for BclxL, STAT3, and pSTAT3 (Tyr705) expression in HepG2 cells by knocking down or overexpressing LSINCT5. CCK-8– cell counting Kit-8; ceRNA – competing endogenous RNA; FISH – fluorescent in situ hybridization; HCC – hepatocellular carcinoma; LSINCT5 – long stress induced non-coding transcripts 5; miRNAs – micro RNAs; oe – overexpression; RIP – RNA immunoprecipitation.
Figure Legend Snippet: LSINCT5 acts as a ceRNA of miR-4516 to promote HCC progression. ( A ) RIP assays followed by qPCR to identify putative miRNAs associated with LSINCT5. ( B ) The miRNAs were verified by RNA pulldown a with biotin-labeled sense or antisense LSINCT5. ( C ) Luciferase reporter assays identified miR-4516 could interact with LSINCT5 (top). RNA pulldown assay with anti-AGO2 was performed in HepG2 cells with miR-4516 overexpression followed by qPCR to enrich LSINCT5 (bottom). ( D ) The predicted base-pairing between LSINCT5 and miR-4516 at miRDB. Three hits were detected. ( E ) RNA-FISH assay revealed the co-localization of LSINCT5 (Alexa Fluor 488, green dots) and miR-4516 (Cy3, red dots) in HepG2 cells. Scale bar, 5 μm. ( F ) CCK-8 viability assay for Huh7 cells and HepG2 cells transfected with plasmids as indicated. ( G ) Tumor sphere formation assay for HepG2 cells transfected with negative control (NC), miR-4516 mimics + oe Vec or miR-4516 mimics + LSINCT5. Quantification results were shown on the right panel. ( H ) Western blot assays for BclxL, STAT3, and pSTAT3 (Tyr705) expression in HepG2 cells by knocking down or overexpressing LSINCT5. CCK-8– cell counting Kit-8; ceRNA – competing endogenous RNA; FISH – fluorescent in situ hybridization; HCC – hepatocellular carcinoma; LSINCT5 – long stress induced non-coding transcripts 5; miRNAs – micro RNAs; oe – overexpression; RIP – RNA immunoprecipitation.

Techniques Used: Real-time Polymerase Chain Reaction, Labeling, Luciferase, Over Expression, Fluorescence In Situ Hybridization, CCK-8 Assay, Viability Assay, Transfection, Tube Formation Assay, Negative Control, Western Blot, Expressing, Cell Counting, In Situ Hybridization, Immunoprecipitation

18) Product Images from "LncFZD6 initiates Wnt/β-catenin and liver TIC self-renewal through BRG1-mediated FZD6 transcriptional activation"

Article Title: LncFZD6 initiates Wnt/β-catenin and liver TIC self-renewal through BRG1-mediated FZD6 transcriptional activation

Journal: Oncogene

doi: 10.1038/s41388-018-0203-6

LncFZD6 recruits BRG1 to FZD6 promoter. a RNA pulldown as performed and the specific band of lncFZD6 was identified as BRG1 with mass spectrum. b The interaction between lncFZD6 and BRG1 was confirmed by western blot. β-Actin served as a loading control. c LncFZD6 truncates were constructed (left panels), followed by RNA pulldown and western blot (right panels). d RNA immunoprecipitation (RIP) assay were performed with oncospheres derived from clinical samples, and enrichment of lncFZD6 and GAPDH were examined with real-time PCR. IgG is an isotype antibody control. Data were shown as means ± s.d. e Double FISH assays showed the colocalization of lncFZD6 and BRG1. Scale bars, 10 μm. f ChIP and ChIRP assays were performed with BRG1 antibody and lncFZD6 probes, respectively, followed by FZD6 promoter detection with real-time PCR. BRG1 and lncFZD6 bind to the same region of FZD6 promoter. g LncFZD6 silenced and control cells were used for BRG1 ChIP assay and impaired combination between BRG1 and FZD6 promoter was found in lncFZD6 silenced cells. h LncFZD6 and BRG1 silenced cells were used for ChIP assays with H3K4me3, H3K27Ac and RNA polymerase II. FZD6 promoter enrichment was analyzed by real-time PCR. i DNase sensibility assays were performed using lncFZD6 and BRG1 silenced cells. j LncFZD6 and BRG1 silenced cells were lyzed for FZD6 western blot. β-Actin served as a loading control. k The lncFZD6 -BRG1 binding region on FZD6 promoter was deleted using CRISPR/Cas9 approach (upper panel), and confirmed by DNA sequencing (lower panel). l Sphere formation assays were performed using FZD6 promoter deleted cells (promoter KO) and FZD6 rescued cells (promoter KO/oeFZD6) cells, and typical images were shown. Data are representative of three independent experiments
Figure Legend Snippet: LncFZD6 recruits BRG1 to FZD6 promoter. a RNA pulldown as performed and the specific band of lncFZD6 was identified as BRG1 with mass spectrum. b The interaction between lncFZD6 and BRG1 was confirmed by western blot. β-Actin served as a loading control. c LncFZD6 truncates were constructed (left panels), followed by RNA pulldown and western blot (right panels). d RNA immunoprecipitation (RIP) assay were performed with oncospheres derived from clinical samples, and enrichment of lncFZD6 and GAPDH were examined with real-time PCR. IgG is an isotype antibody control. Data were shown as means ± s.d. e Double FISH assays showed the colocalization of lncFZD6 and BRG1. Scale bars, 10 μm. f ChIP and ChIRP assays were performed with BRG1 antibody and lncFZD6 probes, respectively, followed by FZD6 promoter detection with real-time PCR. BRG1 and lncFZD6 bind to the same region of FZD6 promoter. g LncFZD6 silenced and control cells were used for BRG1 ChIP assay and impaired combination between BRG1 and FZD6 promoter was found in lncFZD6 silenced cells. h LncFZD6 and BRG1 silenced cells were used for ChIP assays with H3K4me3, H3K27Ac and RNA polymerase II. FZD6 promoter enrichment was analyzed by real-time PCR. i DNase sensibility assays were performed using lncFZD6 and BRG1 silenced cells. j LncFZD6 and BRG1 silenced cells were lyzed for FZD6 western blot. β-Actin served as a loading control. k The lncFZD6 -BRG1 binding region on FZD6 promoter was deleted using CRISPR/Cas9 approach (upper panel), and confirmed by DNA sequencing (lower panel). l Sphere formation assays were performed using FZD6 promoter deleted cells (promoter KO) and FZD6 rescued cells (promoter KO/oeFZD6) cells, and typical images were shown. Data are representative of three independent experiments

Techniques Used: Western Blot, Construct, Immunoprecipitation, Derivative Assay, Real-time Polymerase Chain Reaction, Fluorescence In Situ Hybridization, Chromatin Immunoprecipitation, Binding Assay, CRISPR, DNA Sequencing, Gene Knockout

LncFZD6 was required for liver TIC self-renewal. a LncFZD6 silenced cells were established (left panels) with CRISPRi and sphere formation assays were performed. The efficiency of lncFZD6 knockdown was detected by northern blot (left panels). Typical pictures were shown in middle panels and calculated ratios were shown in right panels. b In all, 1 × 10 6 lncFZD6 silenced or control TICs were injected into BALB/c nude mice. One month later, mice were sacrificed and the weight of tumors were detected and shown as scatter diagram. Six mice were used for each assay. c The indicated tumors were obtained and stained with CD133 antibody, followed by FACS examination. The ratios of CD133 + cells were shown. n = 6 for each group. d Total RNA were extracted from the indicated tumors, and expression levels of the indicated transcripts were examined by real-time PCR. e CD133 + TICs and CD133 - non-TICs were enriched and tumor invasion capacity was examined. Typical images and calculated ratios were shown. f lncFZD6 silenced or control cells were used for transwell assay, and typical images were shown. g , h In all, 10, 1 × 10 2 , 1 × 10 3 , 1 × 10 4 and 1 × 10 5 lncFZD6 silenced cells and control cells were injected into BALB/c nude mice for 3 months’ tumor formation. Three months later, tumor formation was observed and the ratios of tumor-free mice were calculated g . TIC ratios were analyzed using extreme limiting dilution analysis h . 95% CI 95% confidence interval of the estimation, vs versus. i FZD6 knockout cells were generated with CRISPR/Cas9 approach, and knockout efficiency was determined with western blot. β-Actin served as a loading control. j , k In all, 10, 1 × 10 2 , 1 × 10 3 , 1 × 10 4 and 1 × 10 5 FZD6 knockout cells and control cells were injected into BALB/c nude mice and tumor formation was analyzed as g , h . l LncFZD6 was silenced in FZD6 knockout cells or control cells, followed by sphere formation assays. Typical pictures were shown in left panels and calculated sphere-initiating ratios were shown in right panels. m FZD6 expression was rescued in lncFZD6 depleted TICs, followed by oncosphere formation. Typical pictures were shown in left panels and TIC ratios were shown in right panels. Scale bars, 500 μm. Data were shown as means ± s.d. * P
Figure Legend Snippet: LncFZD6 was required for liver TIC self-renewal. a LncFZD6 silenced cells were established (left panels) with CRISPRi and sphere formation assays were performed. The efficiency of lncFZD6 knockdown was detected by northern blot (left panels). Typical pictures were shown in middle panels and calculated ratios were shown in right panels. b In all, 1 × 10 6 lncFZD6 silenced or control TICs were injected into BALB/c nude mice. One month later, mice were sacrificed and the weight of tumors were detected and shown as scatter diagram. Six mice were used for each assay. c The indicated tumors were obtained and stained with CD133 antibody, followed by FACS examination. The ratios of CD133 + cells were shown. n = 6 for each group. d Total RNA were extracted from the indicated tumors, and expression levels of the indicated transcripts were examined by real-time PCR. e CD133 + TICs and CD133 - non-TICs were enriched and tumor invasion capacity was examined. Typical images and calculated ratios were shown. f lncFZD6 silenced or control cells were used for transwell assay, and typical images were shown. g , h In all, 10, 1 × 10 2 , 1 × 10 3 , 1 × 10 4 and 1 × 10 5 lncFZD6 silenced cells and control cells were injected into BALB/c nude mice for 3 months’ tumor formation. Three months later, tumor formation was observed and the ratios of tumor-free mice were calculated g . TIC ratios were analyzed using extreme limiting dilution analysis h . 95% CI 95% confidence interval of the estimation, vs versus. i FZD6 knockout cells were generated with CRISPR/Cas9 approach, and knockout efficiency was determined with western blot. β-Actin served as a loading control. j , k In all, 10, 1 × 10 2 , 1 × 10 3 , 1 × 10 4 and 1 × 10 5 FZD6 knockout cells and control cells were injected into BALB/c nude mice and tumor formation was analyzed as g , h . l LncFZD6 was silenced in FZD6 knockout cells or control cells, followed by sphere formation assays. Typical pictures were shown in left panels and calculated sphere-initiating ratios were shown in right panels. m FZD6 expression was rescued in lncFZD6 depleted TICs, followed by oncosphere formation. Typical pictures were shown in left panels and TIC ratios were shown in right panels. Scale bars, 500 μm. Data were shown as means ± s.d. * P

Techniques Used: Northern Blot, Injection, Mouse Assay, Staining, FACS, Expressing, Real-time Polymerase Chain Reaction, Transwell Assay, Knock-Out, Generated, CRISPR, Western Blot

19) Product Images from "The LINC01138 drives malignancies via activating arginine methyltransferase 5 in hepatocellular carcinoma"

Article Title: The LINC01138 drives malignancies via activating arginine methyltransferase 5 in hepatocellular carcinoma

Journal: Nature Communications

doi: 10.1038/s41467-018-04006-0

The association between LINC01138 and PRMT5 is a candidate therapeutic target for HCC. a RNA pull-down assays for the specific association of PRMT5 and LINC01138, in the cells exposed to PRMT5 inhibitors at the indicated concentration for 48 h. b SMMC-7721 cells were transfected with plasmids containing HA-tagged PRMT5, and RIP assays were performed using HA antibodies in the cells treated with PRMT5 inhibitors at the indicated concentration for 48 h. c CCK-8 assays for pWPXL-LINC01138 or vector cells, exposed to PRMT5 inhibitors at the indicated concentration for 3 days. d CCK-8 assays in cells transfected with si-NC or si-LINC01138 mixture, exposed to PRMT5 inhibitors at the indicated concentration for 3 days. e IC 50 were analysed in cells transfected with si-NC or si-LINC01138 mixture, exposed to PRMT5 inhibitors for 3 days. Values are expressed as the mean ± SEM, n = 3 b – e . ** P
Figure Legend Snippet: The association between LINC01138 and PRMT5 is a candidate therapeutic target for HCC. a RNA pull-down assays for the specific association of PRMT5 and LINC01138, in the cells exposed to PRMT5 inhibitors at the indicated concentration for 48 h. b SMMC-7721 cells were transfected with plasmids containing HA-tagged PRMT5, and RIP assays were performed using HA antibodies in the cells treated with PRMT5 inhibitors at the indicated concentration for 48 h. c CCK-8 assays for pWPXL-LINC01138 or vector cells, exposed to PRMT5 inhibitors at the indicated concentration for 3 days. d CCK-8 assays in cells transfected with si-NC or si-LINC01138 mixture, exposed to PRMT5 inhibitors at the indicated concentration for 3 days. e IC 50 were analysed in cells transfected with si-NC or si-LINC01138 mixture, exposed to PRMT5 inhibitors for 3 days. Values are expressed as the mean ± SEM, n = 3 b – e . ** P

Techniques Used: Concentration Assay, Transfection, Hemagglutination Assay, CCK-8 Assay, Plasmid Preparation

The oncogenic IGF2BP1/IGF2BP3-LINC01138-PRMT5 axis in HCC cells. a Relative RNA levels of LINC01138 in SMMC-7721 cells with IGF2BP1/IGF2BP3 knockdown or overexpression, using qPCR. b The half-life of LINC01138 after treatment with 2.5 μM actinomycin D for indicated times, with IGF2BP1/IGF2BP3 knockdown or overexpression in SMMC-7721 cells. c CCK-8 rescue assays were performed after LINC01138 knockdown in pWPXL-IGF2BP1 or pWPXL-IGF2BP3 cells. d Transwell invasion rescue assays were performed after LINC01138 knockdown in pWPXL-IGF2BP1 or pWPXL-IGF2BP3 cells. e Immunoblotting to detect the protein levels of PRMT5 after knockdown of IGF2BP1/IGF2BP3 or LINC01138 in SMMC-7721 cells. f Integrated model depicting lncRNA LINC01138 as an oncogene in liver cancer. The working model shows that LINC01138 loci is amplified, and its transcript is stabilized by IGF2BP1 or IGF2BP3 in HCC; LINC01138 exerts its oncogenic activity through interacting with and stabilizing PRMT5, which can be disrupted by small molecule inhibitors in HCC. Values are expressed as the mean ± SEM, n = 3 in a – d . *** P
Figure Legend Snippet: The oncogenic IGF2BP1/IGF2BP3-LINC01138-PRMT5 axis in HCC cells. a Relative RNA levels of LINC01138 in SMMC-7721 cells with IGF2BP1/IGF2BP3 knockdown or overexpression, using qPCR. b The half-life of LINC01138 after treatment with 2.5 μM actinomycin D for indicated times, with IGF2BP1/IGF2BP3 knockdown or overexpression in SMMC-7721 cells. c CCK-8 rescue assays were performed after LINC01138 knockdown in pWPXL-IGF2BP1 or pWPXL-IGF2BP3 cells. d Transwell invasion rescue assays were performed after LINC01138 knockdown in pWPXL-IGF2BP1 or pWPXL-IGF2BP3 cells. e Immunoblotting to detect the protein levels of PRMT5 after knockdown of IGF2BP1/IGF2BP3 or LINC01138 in SMMC-7721 cells. f Integrated model depicting lncRNA LINC01138 as an oncogene in liver cancer. The working model shows that LINC01138 loci is amplified, and its transcript is stabilized by IGF2BP1 or IGF2BP3 in HCC; LINC01138 exerts its oncogenic activity through interacting with and stabilizing PRMT5, which can be disrupted by small molecule inhibitors in HCC. Values are expressed as the mean ± SEM, n = 3 in a – d . *** P

Techniques Used: Over Expression, Real-time Polymerase Chain Reaction, CCK-8 Assay, Amplification, Activity Assay

LINC01138 is associated with clinical outcomes in patients with HCC. a The flow chart for selecting candidate lincRNAs in HCC; four lincRNAs were identified for further study. b The copy numbers of LINC01138 were determined in 72 pairs of HCC tissues and adjacent normal tissues using qPCR. c Fold-change of LINC01138 copy number variations in 72 paired tissues (deletion, blue; no-change, yellow; amplification, red.). d The RNA levels of LINC01138 were quantified in 120 pairs of HCC tissues and adjacent normal tissues using qPCR. e Fold-changes of expression of LINC01138 in 120 paired tissues (downexpression, blue; no-change, yellow; upexpression, red.). f Clinical significance of LINC01138 in patients with HCC; high LINC01138 expression positively correlated with tumour size (≥5 cm), AFP ( > 200 ng/ml) and HBsAg-positive patients. g Kaplan–Meier analyses of the correlation between LINC01138 RNA levels and the overall survival in 120 patients with HCC. Patients were stratified for the analysis by the median value. Values are expressed as the median with interquartile range in b , d , f
Figure Legend Snippet: LINC01138 is associated with clinical outcomes in patients with HCC. a The flow chart for selecting candidate lincRNAs in HCC; four lincRNAs were identified for further study. b The copy numbers of LINC01138 were determined in 72 pairs of HCC tissues and adjacent normal tissues using qPCR. c Fold-change of LINC01138 copy number variations in 72 paired tissues (deletion, blue; no-change, yellow; amplification, red.). d The RNA levels of LINC01138 were quantified in 120 pairs of HCC tissues and adjacent normal tissues using qPCR. e Fold-changes of expression of LINC01138 in 120 paired tissues (downexpression, blue; no-change, yellow; upexpression, red.). f Clinical significance of LINC01138 in patients with HCC; high LINC01138 expression positively correlated with tumour size (≥5 cm), AFP ( > 200 ng/ml) and HBsAg-positive patients. g Kaplan–Meier analyses of the correlation between LINC01138 RNA levels and the overall survival in 120 patients with HCC. Patients were stratified for the analysis by the median value. Values are expressed as the median with interquartile range in b , d , f

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

LINC01138 physically interacts with IGF2BP1, IGF2BP3 and PRMT5 in HCC cells. a Immunoblotting for the specific associations of IGF2BP1, IGF2BP3 or PRMT5 with biotinylated-LINC01138 from three independent streptavidin RNA pull-down assays. b RIP assays were performed using the indicated antibodies. Real-time PCR was used to detect LINC01138 enrichment, using GAPDH antibody as the antibody control and 2036-nt PVT1 as the LincRNA control. c Immunoblotting of IGF2BP1, IGF2BP3 or PRMT5 in pull-down samples by full-length biotinylated-LINC01138 (#1) or truncated biotinylated-LINC01138 RNA motifs (#2: 1–219 nt; #3: 1–630 nt; #4: 1–1560 nt; #5: 630–2075 nt; #6: 1560–2075 nt; #7: 220–2075 nt), with GAPDH as the negative control. d – f Deletion mapping for the domains of IGF2BP1 ( d ), IGF2BP3 ( e ) or PRMT5 ( f ) that bind to LINC01138. RIP analysis for LINC01138 enrichment in cells transiently transfected with plasmids containing the indicated FLAG-tagged or HA-tagged full-length or truncated constructs. Values are expressed as the mean ± SEM, n = 3 in b , d – f
Figure Legend Snippet: LINC01138 physically interacts with IGF2BP1, IGF2BP3 and PRMT5 in HCC cells. a Immunoblotting for the specific associations of IGF2BP1, IGF2BP3 or PRMT5 with biotinylated-LINC01138 from three independent streptavidin RNA pull-down assays. b RIP assays were performed using the indicated antibodies. Real-time PCR was used to detect LINC01138 enrichment, using GAPDH antibody as the antibody control and 2036-nt PVT1 as the LincRNA control. c Immunoblotting of IGF2BP1, IGF2BP3 or PRMT5 in pull-down samples by full-length biotinylated-LINC01138 (#1) or truncated biotinylated-LINC01138 RNA motifs (#2: 1–219 nt; #3: 1–630 nt; #4: 1–1560 nt; #5: 630–2075 nt; #6: 1560–2075 nt; #7: 220–2075 nt), with GAPDH as the negative control. d – f Deletion mapping for the domains of IGF2BP1 ( d ), IGF2BP3 ( e ) or PRMT5 ( f ) that bind to LINC01138. RIP analysis for LINC01138 enrichment in cells transiently transfected with plasmids containing the indicated FLAG-tagged or HA-tagged full-length or truncated constructs. Values are expressed as the mean ± SEM, n = 3 in b , d – f

Techniques Used: Real-time Polymerase Chain Reaction, Negative Control, Transfection, Hemagglutination Assay, Construct

20) Product Images from "Ets-1 promoter-associated noncoding RNA regulates the NONO/ERG/Ets-1 axis to drive gastric cancer progression"

Article Title: Ets-1 promoter-associated noncoding RNA regulates the NONO/ERG/Ets-1 axis to drive gastric cancer progression

Journal: Oncogene

doi: 10.1038/s41388-018-0302-4

pancEts-1 is a lncRNA associated with poor survival of gastric cancer. a Scheme indicating the existence of pancEts-1 transcribed upstream the Ets-1 promoter region. b RNA fluorescence in situ hybridization images showing the nuclear and cytoplasmic localization of pancEts-1 in MKN-45 cells using a 138-bp antisense probe (red), with the nuclei staining by DAPI (blue). Sense probe and antisense probe with RNase A (20 μg) treatment were used as negative controls. Scale bars: 10 μm. c Real-time qRT-PCR assay revealing the pancEts-1 transcript levels (normalized to β-actin) in normal gastric mucosa ( n = 30) and cultured gastric cancer cell lines (mean ± SD, n = 5). d Real-time qRT-PCR assay indicating the differential expression of pancEts-1 transcript (normalized to β-actin) in normal gastric mucosa ( n = 30) and gastric cancer tissues ( n = 81). e , f Real-time qRT-PCR assay showing the pancEts-1 transcript levels (normalized to β-actin) in gastric cancer tissues with differential status of metastasis ( e ) or Ets-1 immunostaining ( f ). g The positive correlation between pancEts-1 and Ets-1 transcript levels in gastric cancer tissues ( n = 81). h Kaplan–Meier curves indicating overall survival of 81 gastric cancer patients (cutoff value = 2.833) and overall (OS) and first progression (FP) survival of those derived from Kaplan-Meier plotter with low or high pancEts-1 expression (cutoff values = 14.0 and 14.0). Student’s t test compared gene expression levels in c – f . Pearson’s correlation coefficient analysis in g . Log-rank test for survival comparison in h
Figure Legend Snippet: pancEts-1 is a lncRNA associated with poor survival of gastric cancer. a Scheme indicating the existence of pancEts-1 transcribed upstream the Ets-1 promoter region. b RNA fluorescence in situ hybridization images showing the nuclear and cytoplasmic localization of pancEts-1 in MKN-45 cells using a 138-bp antisense probe (red), with the nuclei staining by DAPI (blue). Sense probe and antisense probe with RNase A (20 μg) treatment were used as negative controls. Scale bars: 10 μm. c Real-time qRT-PCR assay revealing the pancEts-1 transcript levels (normalized to β-actin) in normal gastric mucosa ( n = 30) and cultured gastric cancer cell lines (mean ± SD, n = 5). d Real-time qRT-PCR assay indicating the differential expression of pancEts-1 transcript (normalized to β-actin) in normal gastric mucosa ( n = 30) and gastric cancer tissues ( n = 81). e , f Real-time qRT-PCR assay showing the pancEts-1 transcript levels (normalized to β-actin) in gastric cancer tissues with differential status of metastasis ( e ) or Ets-1 immunostaining ( f ). g The positive correlation between pancEts-1 and Ets-1 transcript levels in gastric cancer tissues ( n = 81). h Kaplan–Meier curves indicating overall survival of 81 gastric cancer patients (cutoff value = 2.833) and overall (OS) and first progression (FP) survival of those derived from Kaplan-Meier plotter with low or high pancEts-1 expression (cutoff values = 14.0 and 14.0). Student’s t test compared gene expression levels in c – f . Pearson’s correlation coefficient analysis in g . Log-rank test for survival comparison in h

Techniques Used: Fluorescence, In Situ Hybridization, Staining, Quantitative RT-PCR, Cell Culture, Expressing, Immunostaining, Derivative Assay

pancEts-1 regulates Ets-1 expression by facilitating NONO-mediated ERG transactivation. a IP, Commassie blue staining (left) and mass spectrometry (MS) assay (right) showing the changes in NONO interacting proteins in NCI-N87 cells stably transfected with empty vector (mock) or pancEts-1 . b IP and western blot revealing the endogenous interaction between NONO and ERG in the NCI-N87 and AGS cells, c , d IP and western blot indicating the interaction between NONO and ERG in NCI-N87 cells transfected with HA-tagged ERG or FLAG-tagged NONO truncates. e IP and western blot showing the interaction between NONO and ERG in NCI-N87 and MKN-45 cells stably transfected with mock, pancEts-1 , scramble shRNA (sh-RNA), or sh-pancEts-1 #2. f ChIP and qPCR assays indicating the binding of ERG to Ets-1 promoter in gastric cancer cells, and its changes in those stably transfected with mock, NONO , pancEts-1 , sh-Scb, sh-NONO #2, or sh-pancEts-1 #2 (mean ± SD, n = 4). g , h Dual-luciferase ( g ) and western blot ( h ) assays showing the promoter activity and expression of Ets-1 in gastric cancer cells, and their changes in those stably transfected with mock, ERG , pancEts-1 , sh-Scb, sh-ERG #1, sh-ERG #2, or sh-pancEts-1 #2 (mean ± SD, n = 4). Student’s t test analyzed the difference in f and g . * P
Figure Legend Snippet: pancEts-1 regulates Ets-1 expression by facilitating NONO-mediated ERG transactivation. a IP, Commassie blue staining (left) and mass spectrometry (MS) assay (right) showing the changes in NONO interacting proteins in NCI-N87 cells stably transfected with empty vector (mock) or pancEts-1 . b IP and western blot revealing the endogenous interaction between NONO and ERG in the NCI-N87 and AGS cells, c , d IP and western blot indicating the interaction between NONO and ERG in NCI-N87 cells transfected with HA-tagged ERG or FLAG-tagged NONO truncates. e IP and western blot showing the interaction between NONO and ERG in NCI-N87 and MKN-45 cells stably transfected with mock, pancEts-1 , scramble shRNA (sh-RNA), or sh-pancEts-1 #2. f ChIP and qPCR assays indicating the binding of ERG to Ets-1 promoter in gastric cancer cells, and its changes in those stably transfected with mock, NONO , pancEts-1 , sh-Scb, sh-NONO #2, or sh-pancEts-1 #2 (mean ± SD, n = 4). g , h Dual-luciferase ( g ) and western blot ( h ) assays showing the promoter activity and expression of Ets-1 in gastric cancer cells, and their changes in those stably transfected with mock, ERG , pancEts-1 , sh-Scb, sh-ERG #1, sh-ERG #2, or sh-pancEts-1 #2 (mean ± SD, n = 4). Student’s t test analyzed the difference in f and g . * P

Techniques Used: Expressing, Staining, Mass Spectrometry, Stable Transfection, Transfection, Plasmid Preparation, Western Blot, Hemagglutination Assay, shRNA, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Binding Assay, Luciferase, Activity Assay

pancEts-1 interacts with NONO protein in gastric cancer cells. a Biotin-labeled RNA pull-down (left) and mass spectrometry (MS) assay (right) showing the interaction between pancEts-1 and NONO protein in MKN-45 cells. The pancEts-1 antisense (AS)- and bead-bound protein served as negative controls. b RIP assay using NONO antibody indicating the interaction between pancEts-1 and NONO protein in NCI-N87 cells transfected with a series of pancEts-1 truncates. The IgG-bound RNA was taken as negative control. c , d , Western blot assay depicting the recovered NONO levels of cellular nuclear extracts pulled down by biotin-labeled pancEts-1 truncates. e In vitro binding assay showing the recovered pancEts-1 levels by RIP (lower) after incubation with full-length (1–471 amino acids), ΔN (75–471 amino acids), ΔC (1–374 amino acids), Drosophila behavior/human splicing (DBHS, 75–374 amino acids), ΔRRM1 (149–374 amino acids), ΔRRM1 + 2 (231–374 amino acids), or coiled-coil (269–374 amino acids) of GST-tagged recombinant NONO protein validated by western blot (upper). f RNA EMSA determining the interaction between recombinant or endogenous NONO protein and biotin-labeled RNA probes for pancEts-1 (arrowhead), with or without competition using an excess of unlabeled homologous RNA probe or treatment using NONO antibody.
Figure Legend Snippet: pancEts-1 interacts with NONO protein in gastric cancer cells. a Biotin-labeled RNA pull-down (left) and mass spectrometry (MS) assay (right) showing the interaction between pancEts-1 and NONO protein in MKN-45 cells. The pancEts-1 antisense (AS)- and bead-bound protein served as negative controls. b RIP assay using NONO antibody indicating the interaction between pancEts-1 and NONO protein in NCI-N87 cells transfected with a series of pancEts-1 truncates. The IgG-bound RNA was taken as negative control. c , d , Western blot assay depicting the recovered NONO levels of cellular nuclear extracts pulled down by biotin-labeled pancEts-1 truncates. e In vitro binding assay showing the recovered pancEts-1 levels by RIP (lower) after incubation with full-length (1–471 amino acids), ΔN (75–471 amino acids), ΔC (1–374 amino acids), Drosophila behavior/human splicing (DBHS, 75–374 amino acids), ΔRRM1 (149–374 amino acids), ΔRRM1 + 2 (231–374 amino acids), or coiled-coil (269–374 amino acids) of GST-tagged recombinant NONO protein validated by western blot (upper). f RNA EMSA determining the interaction between recombinant or endogenous NONO protein and biotin-labeled RNA probes for pancEts-1 (arrowhead), with or without competition using an excess of unlabeled homologous RNA probe or treatment using NONO antibody.

Techniques Used: Labeling, Mass Spectrometry, Transfection, Negative Control, Western Blot, In Vitro, Binding Assay, Incubation, Recombinant

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Clone Assay:

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Centrifugation:

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Amplification:

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Synthesized:

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Quantitative RT-PCR:

Article Title: Long Noncoding RNA HEIH Promotes Colorectal Cancer Tumorigenesis via Counteracting miR-939‒Mediated Transcriptional Repression of Bcl-xL
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Electrophoresis:

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Incubation:

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Article Title: Upregulation of LncRNA‐HIT promotes migration and invasion of non‐small cell lung cancer cells by association with ZEB1
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Article Title: Mammalian mitochondrial RNAs are degraded in the mitochondrial intermembrane space by RNASET2
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Article Title: Mammalian mitochondrial RNAs are degraded in the mitochondrial intermembrane space by RNASET2
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Article Title: Defining potentially conserved RNA regulons of homologous zinc-finger RNA-binding proteins
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Mass Spectrometry:

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Western Blot:

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Over Expression:

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Hybridization:

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Transfection:

Article Title: LncBRM initiates YAP1 signalling activation to drive self-renewal of liver cancer stem cells
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Chromatography:

Article Title: Involvement of KSRP in the post-transcriptional regulation of human iNOS expression-complex interplay of KSRP with TTP and HuR
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Mobility Shift:

Article Title: LncBRM initiates YAP1 signalling activation to drive self-renewal of liver cancer stem cells
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Protease Inhibitor:

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

Article Title: LncBRM initiates YAP1 signalling activation to drive self-renewal of liver cancer stem cells
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Northern Blot:

Article Title: lncAKHE enhances cell growth and migration in hepatocellular carcinoma via activation of NOTCH2 signaling
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SDS Page:

Article Title: Long noncoding RNA OCC-1 suppresses cell growth through destabilizing HuR protein in colorectal cancer
Article Snippet: Briefly, the relative long 918-nucleotide OCC-1 3′UTR RNA was synthesized and labeled with Biotin RNA Labeling Mix (Roche) by in vitro transcription. .. After incubation, RNA-protein complexes were retrieved by streptavidin-coupled T1 beads (Dynabeads), washed five times in IP buffer and eluted in Laemmli buffer.

Article Title: LncSHRG promotes hepatocellular carcinoma progression by activating HES6
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Article Title: Mammalian mitochondrial RNAs are degraded in the mitochondrial intermembrane space by RNASET2
Article Snippet: For in organello degradation assays that examined their responses to mitoplasting, temperature, pH, ATP, and metal ions, 500 μg mitochondria first underwent mtRNA synthesis in 200 μL mitoprep buffer containing 4 mmol/L ATP pH 7.4, 20 mmol/L succinate, 1 mmol/L CaCl2 , and 1 μL Biotin RNA Labeling Mix (Roche) at 37°C for 45 min. Mitochondria were pelleted at 12,000 ×g for 4 min at 4°C, washed with 1 mL ice cold mitoprep buffer, resuspended in 150 μL mitoprep buffer, and incubated on ice for 15 min with one vortexing at 8 min. 50 μL ice cold cocktail containing 4 mmol/L UTP, 40 mmol/L Ca2+ , 1 μL Ribolock RNase inhibitor (Thermo), and 1 μg RNaseI (Thermo) (original buffer exchanged to mitoprep buffer) in mitoprep buffer was added to the sample (RNase inhibitor was used to eliminate cytosolic interference and RNaseI was used to quickly degrade the RNA from broken mitochondria). .. Samples were then cooled to room temperature and 0.5 μg of proteinase K was added for a 5 min incubation at 37°C.

Imaging:

Article Title: Monitoring the Transcriptional Activity of Human Endogenous Retroviral HERV-W Family Using PNA Strand Invasion into Double-Stranded DNA
Article Snippet: The reaction products of PNA strand invasion were loaded onto 6%, 1-mm-thick polyacrylamide gel (29:1 acrylamide/Bis-acrylamide ratio) and resolved for 2.5 h at 100 V. After electrophoresis, gels were stained for 10 min in the solution containing 1× concentrated SYBR Green I nucleic acid gel stain (Sigma-Aldrich Co, Poznań, Poland), then washed with high-pure water for 5 min, and digitalized using Gel Logic 100 Imaging System (Eastman Kodak Company, Rochester, NY, USA). .. An ERVWE1-specific, biotinylated RNA probe was synthetized using T3 RNA polymerase and Biotin RNA Labeling Mix (Roche Diagnostics, Warsaw, Poland); XbaI-linearized pSC-B-ERVWE1270 plasmid served as a template in transcription reaction.

Polymerase Chain Reaction:

Article Title: Involvement of KSRP in the post-transcriptional regulation of human iNOS expression-complex interplay of KSRP with TTP and HuR
Article Snippet: The immunoreactive proteins on the blots were visualized by the enhanced chemiluminescence detection system (ECL, Amersham). .. To generate biotinylated-RNA sense probes for the affinity purification 0.5–1 µg of the linearized plasmids pCR-iNOS 3′-UTR or pCR-iNOS 3′-UTR-non-AU ( ) were in vitro transcribed using the biotin RNA labeling mix (Roche Diagnostics, Mannheim, Germany) as described by the manufacturer. .. Protein extracts from CM-induced DLD-1 cells were first precleared by incubating 200 mg of cytoplasmic proteins with 3 ml of a 50% slurry pre-blocked streptavidin-coated agarose beads, for 3 h at 4°C in 50 ml binding buffer [10 mM HEPES (pH 7.6), 3 mM MgCl2 , 5 mM EDTA, 2 mM DTT, 5% Glycerol, 0.5% NP-40, 3 mg/ml Heparin and 0.5 mg/ml Yeast RNA] supplemented with 40 mM KCl and RNasin (0.3 U/μl, final).

Article Title: LncSHRG promotes hepatocellular carcinoma progression by activating HES6
Article Snippet: For RNA pulldown assays, Biotin-labeled lncSHRG and anti-sense control of lncSHRG were obtained by T7 transcription in vitro using Biotin RNA labeling Mix (Roche) and added into cell lysates containing His-SATB1. .. For lncSHRG domain mapping, truncated lncSHRGs were cloned into pCDNA3 plasmid.

Article Title: Circular RNA hsa-circ-0012129 Promotes Cell Proliferation and Invasion in 30 Cases of Human Glioma and Human Glioma Cell Lines U373, A172, and SHG44, by Targeting MicroRNA-661 (miR-661)
Article Snippet: PCR products were obtained with the specific primers for the back-splice region of hsa-circ-0012129. .. PCR products were labeled with biotin-labeled RNA probes by using biotin RNA labeling mix (Roche Applied Science, Mannheim, Germany) (Cat. No: 11685597910) and T7 RNA polymerase (Cat. No: M0251L) according to the manufacturer’s instructions. .. Cells were seeded in six-well plates and were hybridized in Ambion ULTRAhyb hybridization buffer (Cat. No: AM8670), with biotin-labeled RNA probes to hsa-circ-0012129, at 60°C overnight.

Article Title: Defining potentially conserved RNA regulons of homologous zinc-finger RNA-binding proteins
Article Snippet: DNA templates for biotin-RNA synthesis were either prepared by PCR from S. cerevisiae or human DNA with oligonucleotides bearing a T7 RNA polymerase promotor sequence at their 5' ends, or by direct annealing of complementary oligonucleotide pairs containing a 5' T7 RNA polymerase promotor (a list of oligonucleotide primers is given in Additional file ). .. Biotin-labeled RNAs were prepared with the Biotin RNA Labeling Mix (Roche) and T7 RNA polymerase (Promega).

Sonication:

Article Title: linc-HOXA1 is a noncoding RNA that represses Hoxa1 transcription in cis
Article Snippet: Biotin-labeled RNA of the three isoforms of linc-HOXA1 , along with deletions of these isoforms and antisense versions, were prepared using the Biotin RNA labeling mix (Roche) and T7 RNA polymerase (Roche). .. Biotin-labeled RNA of the three isoforms of linc-HOXA1 , along with deletions of these isoforms and antisense versions, were prepared using the Biotin RNA labeling mix (Roche) and T7 RNA polymerase (Roche).

Affinity Purification:

Article Title: Involvement of KSRP in the post-transcriptional regulation of human iNOS expression-complex interplay of KSRP with TTP and HuR
Article Snippet: The immunoreactive proteins on the blots were visualized by the enhanced chemiluminescence detection system (ECL, Amersham). .. To generate biotinylated-RNA sense probes for the affinity purification 0.5–1 µg of the linearized plasmids pCR-iNOS 3′-UTR or pCR-iNOS 3′-UTR-non-AU ( ) were in vitro transcribed using the biotin RNA labeling mix (Roche Diagnostics, Mannheim, Germany) as described by the manufacturer. .. Protein extracts from CM-induced DLD-1 cells were first precleared by incubating 200 mg of cytoplasmic proteins with 3 ml of a 50% slurry pre-blocked streptavidin-coated agarose beads, for 3 h at 4°C in 50 ml binding buffer [10 mM HEPES (pH 7.6), 3 mM MgCl2 , 5 mM EDTA, 2 mM DTT, 5% Glycerol, 0.5% NP-40, 3 mg/ml Heparin and 0.5 mg/ml Yeast RNA] supplemented with 40 mM KCl and RNasin (0.3 U/μl, final).

Binding Assay:

Article Title: Involvement of KSRP in the post-transcriptional regulation of human iNOS expression-complex interplay of KSRP with TTP and HuR
Article Snippet: To generate biotinylated-RNA sense probes for the affinity purification 0.5–1 µg of the linearized plasmids pCR-iNOS 3′-UTR or pCR-iNOS 3′-UTR-non-AU ( ) were in vitro transcribed using the biotin RNA labeling mix (Roche Diagnostics, Mannheim, Germany) as described by the manufacturer. .. Protein extracts from CM-induced DLD-1 cells were first precleared by incubating 200 mg of cytoplasmic proteins with 3 ml of a 50% slurry pre-blocked streptavidin-coated agarose beads, for 3 h at 4°C in 50 ml binding buffer [10 mM HEPES (pH 7.6), 3 mM MgCl2 , 5 mM EDTA, 2 mM DTT, 5% Glycerol, 0.5% NP-40, 3 mg/ml Heparin and 0.5 mg/ml Yeast RNA] supplemented with 40 mM KCl and RNasin (0.3 U/μl, final).

Article Title: Long Noncoding RNA HEIH Promotes Colorectal Cancer Tumorigenesis via Counteracting miR-939‒Mediated Transcriptional Repression of Bcl-xL
Article Snippet: Then TUNEL staining was performed using an In Situ Cell Death Detection Kit (Roche). .. lncRNA-HEIH and miR-939 binding sites mutated lncRNA-HEIH (lncRNA-HEIH-mut) were in vitro transcribed from pSPT19-HEIH, and pSPT19-HEIH-mut, respectively, using the SP6 RNA polymerase (Roche) and biotinlabeled with the Biotin RNA Labeling Mix (Roche) in accordance with the manufacturer’s protocols. .. After being treated with RNase-free DNase I (Roche), the in vitro transcribed biotinylated RNA was purified using an RNeasy Mini Kit (Qiagen, Valencia, CA).

Article Title: Upregulation of LncRNA‐HIT promotes migration and invasion of non‐small cell lung cancer cells by association with ZEB1
Article Snippet: Briefly, biotin‐labeled lncRNA‐HIT were in vitro transcribed with the Biotin RNA Labeling Mix (Roche, Cambridge, UK) and T7 RNA polymerase (Roche), treated with RNase‐free DNase I (Roche), and purified with the RNeasy Mini Kit (Qiagen, Dusseldolf, Germany). .. Briefly, biotin‐labeled lncRNA‐HIT were in vitro transcribed with the Biotin RNA Labeling Mix (Roche, Cambridge, UK) and T7 RNA polymerase (Roche), treated with RNase‐free DNase I (Roche), and purified with the RNeasy Mini Kit (Qiagen, Dusseldolf, Germany).

Article Title: Long noncoding RNA OCC-1 suppresses cell growth through destabilizing HuR protein in colorectal cancer
Article Snippet: Briefly, the relative long 918-nucleotide OCC-1 3′UTR RNA was synthesized and labeled with Biotin RNA Labeling Mix (Roche) by in vitro transcription. .. After incubation, RNA-protein complexes were retrieved by streptavidin-coupled T1 beads (Dynabeads), washed five times in IP buffer and eluted in Laemmli buffer.

Article Title: LncBRM initiates YAP1 signalling activation to drive self-renewal of liver cancer stem cells
Article Snippet: Probes and precipitated proteins were incubated in EMSA binding buffer and mobility shift assay was performed using gel electrophoresis. .. LncBRM-specific probes were labelled with Biotin using Biotin RNA Labeling Mix (Roche).

Nucleic Acid Electrophoresis:

Article Title: The LINC01138 drives malignancies via activating arginine methyltransferase 5 in hepatocellular carcinoma
Article Snippet: LINC01138 or antisense-LINC01138 RNAs were transcribed and labelled by the Biotin RNA Labeling Mix (Roche, USA), treated with RNase-free DNase I (Takara, Japan) and purified with an RNeasy Mini Kit (QIAGEN, USA). .. LINC01138 or antisense-LINC01138 RNAs were transcribed and labelled by the Biotin RNA Labeling Mix (Roche, USA), treated with RNase-free DNase I (Takara, Japan) and purified with an RNeasy Mini Kit (QIAGEN, USA).

Article Title: LncBRM initiates YAP1 signalling activation to drive self-renewal of liver cancer stem cells
Article Snippet: Probes and precipitated proteins were incubated in EMSA binding buffer and mobility shift assay was performed using gel electrophoresis. .. LncBRM-specific probes were labelled with Biotin using Biotin RNA Labeling Mix (Roche).

Article Title: Monitoring the Transcriptional Activity of Human Endogenous Retroviral HERV-W Family Using PNA Strand Invasion into Double-Stranded DNA
Article Snippet: Paragraph title: DNA Electrophoresis and Blotting ... An ERVWE1-specific, biotinylated RNA probe was synthetized using T3 RNA polymerase and Biotin RNA Labeling Mix (Roche Diagnostics, Warsaw, Poland); XbaI-linearized pSC-B-ERVWE1270 plasmid served as a template in transcription reaction.

Article Title: m6A-induced lncRNA RP11 triggers the dissemination of colorectal cancer cells via upregulation of Zeb1
Article Snippet: LncRNA-RP11 and its antisense RNA were transcribed in vitro from the pGEM-T-RP11 vector, biotin-labelled with the Biotin RNA Labeling Mix (Roche Diagnostics, Indianapolis, IN, USA) and T7/SP6 RNA polymerase (Roche), treated with RNase-free DNase I (Roche), and purified with an RNeasy Mini Kit (Qiagen, Valencia, CA, USA). .. One milligram of protein from the extracts of HCT-15 cells stably transfected with pcDNA3.1-RP11 was then mixed with 50 pmoles of biotinylated RNA, incubated with streptavidin agarose beads (Invitrogen, Carlsbad, CA, USA), and washed.

In Vivo:

Article Title: Regulation of mitochondrion-associated cytosolic ribosomes by mammalian mitochondrial ribonuclease T2 (RNASET2)
Article Snippet: In vivo , the level of an RNA is the result of a fine balance among synthesis, transport, and degradation. .. To examine whether manipulation of RNASET2 level has an effect on nuclear rRNA transcription, in vivo RNA synthesis was performed using live cells and biotin RNA labeling mix (Roche). .. Two major biotin-labeled rRNA bands were observed.

Pull Down Assay:

Article Title: Long Noncoding RNA HEIH Promotes Colorectal Cancer Tumorigenesis via Counteracting miR-939‒Mediated Transcriptional Repression of Bcl-xL
Article Snippet: Paragraph title: 11. RNA pull-down assay ... lncRNA-HEIH and miR-939 binding sites mutated lncRNA-HEIH (lncRNA-HEIH-mut) were in vitro transcribed from pSPT19-HEIH, and pSPT19-HEIH-mut, respectively, using the SP6 RNA polymerase (Roche) and biotinlabeled with the Biotin RNA Labeling Mix (Roche) in accordance with the manufacturer’s protocols.

Article Title: Upregulation of LncRNA‐HIT promotes migration and invasion of non‐small cell lung cancer cells by association with ZEB1
Article Snippet: Paragraph title: RNA pull‐down assay ... Briefly, biotin‐labeled lncRNA‐HIT were in vitro transcribed with the Biotin RNA Labeling Mix (Roche, Cambridge, UK) and T7 RNA polymerase (Roche), treated with RNase‐free DNase I (Roche), and purified with the RNeasy Mini Kit (Qiagen, Dusseldolf, Germany).

Article Title: Long noncoding RNA OCC-1 suppresses cell growth through destabilizing HuR protein in colorectal cancer
Article Snippet: Paragraph title: RNA pull-down assay ... Briefly, the relative long 918-nucleotide OCC-1 3′UTR RNA was synthesized and labeled with Biotin RNA Labeling Mix (Roche) by in vitro transcription.

Article Title: Human colorectal cancer-specific CCAT1-L lncRNA regulates long-range chromatin interactions at the MYC locus
Article Snippet: Paragraph title: Biotin-labeled RNA pull-down assay ... Biotin-labeled CCAT1-L truncation probes were in vitro transcribed with the Biotin RNA Labeling Mix (Roche) and AmpliScripe T7/SP6-flash Transcription Kit (Epicentre).

Fluorescence:

Article Title: Circular RNA hsa-circ-0012129 Promotes Cell Proliferation and Invasion in 30 Cases of Human Glioma and Human Glioma Cell Lines U373, A172, and SHG44, by Targeting MicroRNA-661 (miR-661)
Article Snippet: Paragraph title: Fluorescence in situ hybridization (FISH) assay ... PCR products were labeled with biotin-labeled RNA probes by using biotin RNA labeling mix (Roche Applied Science, Mannheim, Germany) (Cat. No: 11685597910) and T7 RNA polymerase (Cat. No: M0251L) according to the manufacturer’s instructions.

Isolation:

Article Title: Involvement of KSRP in the post-transcriptional regulation of human iNOS expression-complex interplay of KSRP with TTP and HuR
Article Snippet: To generate biotinylated-RNA sense probes for the affinity purification 0.5–1 µg of the linearized plasmids pCR-iNOS 3′-UTR or pCR-iNOS 3′-UTR-non-AU ( ) were in vitro transcribed using the biotin RNA labeling mix (Roche Diagnostics, Mannheim, Germany) as described by the manufacturer. .. Beads were pelleted and the supernatant was incubated with 600 µg biotinylated-RNA probe as described above for 2 h. The binding mixture was then incubated with 3 ml of the 50% slurry pre-blocked streptavidin-coated agarose beads for 2 h. Beads were pelleted and washed three times in binding buffer with 40 mM KCl and twice in binding buffer with 300 mM KCl.

Article Title: LncBRM initiates YAP1 signalling activation to drive self-renewal of liver cancer stem cells
Article Snippet: For RNA EMSA, human BRM plasmid was transfected into Huh7 cells, then cell nuclear extracts were isolated from BRM-overexpressed oncospheres. .. LncBRM-specific probes were labelled with Biotin using Biotin RNA Labeling Mix (Roche).

Article Title: lncAKHE enhances cell growth and migration in hepatocellular carcinoma via activation of NOTCH2 signaling
Article Snippet: Biotin-conjugated probes were purchased from Invitrogen. lncAKHE probe sequences as follows: #1: 5′-TTGGGTTAGGCAAACACTGT-3′; #2: 5′-ACAACAGATTGATAGTCCAT-3′; #3: 5′-ACAATAGCACCCCAATAAGA-3′. .. RNAs were isolated from HCC samples and cell lines using TRIZOL (Invitrogen). lncAKHE and 18S probes for Northern blot were achieved by Biotin RNA labeling mix (Roche). .. The RNA samples were separated by electrophoresis and transferred to NC membrane.

Labeling:

Article Title: Involvement of KSRP in the post-transcriptional regulation of human iNOS expression-complex interplay of KSRP with TTP and HuR
Article Snippet: The immunoreactive proteins on the blots were visualized by the enhanced chemiluminescence detection system (ECL, Amersham). .. To generate biotinylated-RNA sense probes for the affinity purification 0.5–1 µg of the linearized plasmids pCR-iNOS 3′-UTR or pCR-iNOS 3′-UTR-non-AU ( ) were in vitro transcribed using the biotin RNA labeling mix (Roche Diagnostics, Mannheim, Germany) as described by the manufacturer. .. Protein extracts from CM-induced DLD-1 cells were first precleared by incubating 200 mg of cytoplasmic proteins with 3 ml of a 50% slurry pre-blocked streptavidin-coated agarose beads, for 3 h at 4°C in 50 ml binding buffer [10 mM HEPES (pH 7.6), 3 mM MgCl2 , 5 mM EDTA, 2 mM DTT, 5% Glycerol, 0.5% NP-40, 3 mg/ml Heparin and 0.5 mg/ml Yeast RNA] supplemented with 40 mM KCl and RNasin (0.3 U/μl, final).

Article Title: Long Noncoding RNA HEIH Promotes Colorectal Cancer Tumorigenesis via Counteracting miR-939‒Mediated Transcriptional Repression of Bcl-xL
Article Snippet: Then TUNEL staining was performed using an In Situ Cell Death Detection Kit (Roche). .. lncRNA-HEIH and miR-939 binding sites mutated lncRNA-HEIH (lncRNA-HEIH-mut) were in vitro transcribed from pSPT19-HEIH, and pSPT19-HEIH-mut, respectively, using the SP6 RNA polymerase (Roche) and biotinlabeled with the Biotin RNA Labeling Mix (Roche) in accordance with the manufacturer’s protocols. .. After being treated with RNase-free DNase I (Roche), the in vitro transcribed biotinylated RNA was purified using an RNeasy Mini Kit (Qiagen, Valencia, CA).

Article Title: Upregulation of LncRNA‐HIT promotes migration and invasion of non‐small cell lung cancer cells by association with ZEB1
Article Snippet: RNA pull‐down and deletion mapping were performed as described previously . .. Briefly, biotin‐labeled lncRNA‐HIT were in vitro transcribed with the Biotin RNA Labeling Mix (Roche, Cambridge, UK) and T7 RNA polymerase (Roche), treated with RNase‐free DNase I (Roche), and purified with the RNeasy Mini Kit (Qiagen, Dusseldolf, Germany). .. Cell nuclear proteins were extracted using the Cytoplasmic and Nuclear Protein Extraction Kit (Tiangen, Beijing China).

Article Title: Long noncoding RNA OCC-1 suppresses cell growth through destabilizing HuR protein in colorectal cancer
Article Snippet: RNA pull-down assay was carried out as previously described ( ). .. Briefly, the relative long 918-nucleotide OCC-1 3′UTR RNA was synthesized and labeled with Biotin RNA Labeling Mix (Roche) by in vitro transcription. .. The biotin-labeled RNA (1 μg) was first folded in RNA structure buffer (20 mM Tris–Cl [pH 7.0], 0.2 M KCl and 20 mM MgCl2 ) and then incubated with Caco-2 whole-cell lysate at 4°C for 1 h with rotation.

Article Title: The LINC01138 drives malignancies via activating arginine methyltransferase 5 in hepatocellular carcinoma
Article Snippet: All experiments were performed in accordance with relevant institutional and national guidelines and regulations of Shanghai Medical Experimental Animal Care Commission. .. LINC01138 or antisense-LINC01138 RNAs were transcribed and labelled by the Biotin RNA Labeling Mix (Roche, USA), treated with RNase-free DNase I (Takara, Japan) and purified with an RNeasy Mini Kit (QIAGEN, USA). .. Next, 1 pmol biotinylated RNA was pretreated with RNA structure buffer (Beyotime Biotechnology, Shanghai, China) to obtain an appropriate secondary structure formation.

Article Title: Human colorectal cancer-specific CCAT1-L lncRNA regulates long-range chromatin interactions at the MYC locus
Article Snippet: Biotin-labeled RNAs pull-down assay was performed as described , , with minor modifications. .. Biotin-labeled CCAT1-L truncation probes were in vitro transcribed with the Biotin RNA Labeling Mix (Roche) and AmpliScripe T7/SP6-flash Transcription Kit (Epicentre). .. 4 μg biotinylated RNA was denatured for 5 min at 65 °C in PA buffer (10 mM Tris-HCl, pH 7.5, 10 mM MgCl2 , 100 mM NH4 Cl) and slowly cooled down to RT.

Article Title: Ets-1 promoter-associated noncoding RNA regulates the NONO/ERG/Ets-1 axis to drive gastric cancer progression
Article Snippet: PCR amplification was undertaken using SMARTer RACE cDNA Amplification Kit (Clontech, Mountain View, CA), gene specific primers (Supplementary Table ), and universal primer mix. .. To prepare biotin-labeled sense or antisense RNA probe of pancEts-1 , the biotin RNA Labeling Mix (Roche) and T7 RNA polymerase were used. .. Fixed cells were hybridized with sense or antisense probe for 16 h at 37 °C, with or without treatment of RNase A (20 μg).

Article Title: The Staphylococcus aureus Protein-Coding Gene gdpS Modulates sarS Expression via mRNA-mRNA Interaction
Article Snippet: For the gel-shift assays, the sarS and spa promoters were identified as previously described ( ). .. In vitro transcription was carried out with RiboMAX large-scale RNA production systems (Promega). gdpS mRNA was amplified and biotin labeled with a Roche biotin RNA-labeling mix (Roche). .. Labeled probes were incubated with increasing amounts of sarS mRNA or spa mRNA at 85°C for 2 min and 37°C for 30 min. Then, the mixture was processed using a 4% native polyacrylamide gel and transferred to a charged nylon membrane.

Article Title: A Prader-Willi locus lncRNA cloud modulates diurnal genes and energy expenditure
Article Snippet: A 1 cm square piece of gel from 80–140 kDa was excised and submitted to the UC Davis Proteomics core for mass-spec identification. .. Template DNA was linearized and 250 ng of DNA was IVT using Biotin RNA labeling Mix (Roche), DNAse I (NEB) treated and nucleotides removed using Micro-Biospin columns (Biorad). .. IVT RNA was denatured at 65°C for 5 min and then slow-cooled to 4°C.

Article Title: Regulation of mitochondrion-associated cytosolic ribosomes by mammalian mitochondrial ribonuclease T2 (RNASET2)
Article Snippet: In vivo , the level of an RNA is the result of a fine balance among synthesis, transport, and degradation. .. To examine whether manipulation of RNASET2 level has an effect on nuclear rRNA transcription, in vivo RNA synthesis was performed using live cells and biotin RNA labeling mix (Roche). .. Two major biotin-labeled rRNA bands were observed.

Article Title: LncSHRG promotes hepatocellular carcinoma progression by activating HES6
Article Snippet: After transferred on a nylon membrane (Amersham Biosciences), the labeled DNA was cross-linked by UV, probed with streptavidin-HRP conjugate and then incubated with the detection substrate. .. For RNA pulldown assays, Biotin-labeled lncSHRG and anti-sense control of lncSHRG were obtained by T7 transcription in vitro using Biotin RNA labeling Mix (Roche) and added into cell lysates containing His-SATB1. .. After incubation overnight at 4°C, Streptavidin-magnetic C1 beads was added and incubated for another 4 h at 4°C.

Article Title: Circular RNA hsa-circ-0012129 Promotes Cell Proliferation and Invasion in 30 Cases of Human Glioma and Human Glioma Cell Lines U373, A172, and SHG44, by Targeting MicroRNA-661 (miR-661)
Article Snippet: PCR products were obtained with the specific primers for the back-splice region of hsa-circ-0012129. .. PCR products were labeled with biotin-labeled RNA probes by using biotin RNA labeling mix (Roche Applied Science, Mannheim, Germany) (Cat. No: 11685597910) and T7 RNA polymerase (Cat. No: M0251L) according to the manufacturer’s instructions. .. Cells were seeded in six-well plates and were hybridized in Ambion ULTRAhyb hybridization buffer (Cat. No: AM8670), with biotin-labeled RNA probes to hsa-circ-0012129, at 60°C overnight.

Article Title: LncBRM initiates YAP1 signalling activation to drive self-renewal of liver cancer stem cells
Article Snippet: For RNA EMSA, human BRM plasmid was transfected into Huh7 cells, then cell nuclear extracts were isolated from BRM-overexpressed oncospheres. .. LncBRM-specific probes were labelled with Biotin using Biotin RNA Labeling Mix (Roche). .. BRM nuclear extracts and biotin-labelled probe were incubated in 1 × REMSA binding buffer supplementing with Glycerol, transfer RNA and dithiothreitol for 30 min according to LightShift Chemiluminescent RNA EMSA Kit (Thermo Scientific).

Article Title: Monitoring the Transcriptional Activity of Human Endogenous Retroviral HERV-W Family Using PNA Strand Invasion into Double-Stranded DNA
Article Snippet: DNA was fixed on the membrane by baking it at 80 °C for 1 h in a sterilization oven. .. An ERVWE1-specific, biotinylated RNA probe was synthetized using T3 RNA polymerase and Biotin RNA Labeling Mix (Roche Diagnostics, Warsaw, Poland); XbaI-linearized pSC-B-ERVWE1270 plasmid served as a template in transcription reaction. .. Hybridization was performed in a roller bottle overnight at 55 °C using 2.5 μl of RNA probe diluted in 4 ml of hybridization buffer (Minidizer Hybridizer, UVP, LLC, Upland, CA, USA).

Article Title: m6A-induced lncRNA RP11 triggers the dissemination of colorectal cancer cells via upregulation of Zeb1
Article Snippet: RNA expression was normalized to the total amount of RNA used for reverse transcription. .. LncRNA-RP11 and its antisense RNA were transcribed in vitro from the pGEM-T-RP11 vector, biotin-labelled with the Biotin RNA Labeling Mix (Roche Diagnostics, Indianapolis, IN, USA) and T7/SP6 RNA polymerase (Roche), treated with RNase-free DNase I (Roche), and purified with an RNeasy Mini Kit (Qiagen, Valencia, CA, USA). .. One milligram of protein from the extracts of HCT-15 cells stably transfected with pcDNA3.1-RP11 was then mixed with 50 pmoles of biotinylated RNA, incubated with streptavidin agarose beads (Invitrogen, Carlsbad, CA, USA), and washed.

Article Title: Mammalian mitochondrial RNAs are degraded in the mitochondrial intermembrane space by RNASET2
Article Snippet: Biotinylated samples were run on SDS-PAGE, transferred to a nylon membrane, and detected with nucleic acid detection kit (Thermo). mtRNA samples were run either on SDS-PAGE or agarose gels and stained with EtBr. .. In organello RNA synthesis was performed in 200 μL mitoprep buffer containing 4 mmol/L ATP pH 7.4, 20 mmol/L succinate, 1 mmol/L CaCl2 and 1 μL Biotin RNA Labeling Mix (Roche) with 500 μg mitochondria at 37°C. .. For each time point (0 min, 30 min, and 60 min), 60 μL reaction mix was taken out and mitochondria were pelleted at 18,000 ×g for 2 min. Pellets were stored at −80°C for at least 15 min before next preparation step.

Article Title: Mammalian mitochondrial RNAs are degraded in the mitochondrial intermembrane space by RNASET2
Article Snippet: Therefore, in organello mtRNA synthesis were carried out in buffer containing 4 mmol/L ATP pH 7.4, 20 mmol/L succinate, 0.5 mmol/L CaCl2 , 10 mmol/L MgCl2 , and 1 mg/mL HEK cytosol. .. For in organello degradation assays that examined their responses to mitoplasting, temperature, pH, ATP, and metal ions, 500 μg mitochondria first underwent mtRNA synthesis in 200 μL mitoprep buffer containing 4 mmol/L ATP pH 7.4, 20 mmol/L succinate, 1 mmol/L CaCl2 , and 1 μL Biotin RNA Labeling Mix (Roche) at 37°C for 45 min. Mitochondria were pelleted at 12,000 ×g for 4 min at 4°C, washed with 1 mL ice cold mitoprep buffer, resuspended in 150 μL mitoprep buffer, and incubated on ice for 15 min with one vortexing at 8 min. 50 μL ice cold cocktail containing 4 mmol/L UTP, 40 mmol/L Ca2+ , 1 μL Ribolock RNase inhibitor (Thermo), and 1 μg RNaseI (Thermo) (original buffer exchanged to mitoprep buffer) in mitoprep buffer was added to the sample (RNase inhibitor was used to eliminate cytosolic interference and RNaseI was used to quickly degrade the RNA from broken mitochondria). .. For each time point (0 min, 30 min, and 60 min), 60 μL reaction mix was taken out and mitochondria were pelleted at 18,000 ×g for 2 min. Pellets were stored at −80°C for at least 15 min before next preparation step.

Article Title: lncAKHE enhances cell growth and migration in hepatocellular carcinoma via activation of NOTCH2 signaling
Article Snippet: Biotin-conjugated probes were purchased from Invitrogen. lncAKHE probe sequences as follows: #1: 5′-TTGGGTTAGGCAAACACTGT-3′; #2: 5′-ACAACAGATTGATAGTCCAT-3′; #3: 5′-ACAATAGCACCCCAATAAGA-3′. .. RNAs were isolated from HCC samples and cell lines using TRIZOL (Invitrogen). lncAKHE and 18S probes for Northern blot were achieved by Biotin RNA labeling mix (Roche). .. The RNA samples were separated by electrophoresis and transferred to NC membrane.

Article Title: linc-HOXA1 is a noncoding RNA that represses Hoxa1 transcription in cis
Article Snippet: A database of all possible peptide masses encoded by the linc-HOXA1 isoforms (Supplemental Table 3) was included along with the UniProt Human database in the analysis of the raw data. .. Biotin-labeled RNA of the three isoforms of linc-HOXA1 , along with deletions of these isoforms and antisense versions, were prepared using the Biotin RNA labeling mix (Roche) and T7 RNA polymerase (Roche). .. Biotinylated RNAs were treated with RNase-free DNase I (Roche) and purified on prepacked spin columns with Bio-Gel P-30 in 1× saline sodium citrate (Bio-Rad).

Article Title: Defining potentially conserved RNA regulons of homologous zinc-finger RNA-binding proteins
Article Snippet: For the annealing of oligos, a mixture of 1 μM of forward and reverse complementary oligonucleotides was incubated for 1 minute at 95°C in 20 μl of water and cooled down slowly to room temperature. .. Biotin-labeled RNAs were prepared with the Biotin RNA Labeling Mix (Roche) and T7 RNA polymerase (Promega). .. Biotinylated RNAs were treated with RNase-free DNase I and purified with the PureLink Micro-to-Midi Kit (Invitrogen).

Purification:

Article Title: Long Noncoding RNA HEIH Promotes Colorectal Cancer Tumorigenesis via Counteracting miR-939‒Mediated Transcriptional Repression of Bcl-xL
Article Snippet: lncRNA-HEIH and miR-939 binding sites mutated lncRNA-HEIH (lncRNA-HEIH-mut) were in vitro transcribed from pSPT19-HEIH, and pSPT19-HEIH-mut, respectively, using the SP6 RNA polymerase (Roche) and biotinlabeled with the Biotin RNA Labeling Mix (Roche) in accordance with the manufacturer’s protocols. .. After being treated with RNase-free DNase I (Roche), the in vitro transcribed biotinylated RNA was purified using an RNeasy Mini Kit (Qiagen, Valencia, CA).

Article Title: Upregulation of LncRNA‐HIT promotes migration and invasion of non‐small cell lung cancer cells by association with ZEB1
Article Snippet: RNA pull‐down and deletion mapping were performed as described previously . .. Briefly, biotin‐labeled lncRNA‐HIT were in vitro transcribed with the Biotin RNA Labeling Mix (Roche, Cambridge, UK) and T7 RNA polymerase (Roche), treated with RNase‐free DNase I (Roche), and purified with the RNeasy Mini Kit (Qiagen, Dusseldolf, Germany). .. Cell nuclear proteins were extracted using the Cytoplasmic and Nuclear Protein Extraction Kit (Tiangen, Beijing China).

Article Title: The LINC01138 drives malignancies via activating arginine methyltransferase 5 in hepatocellular carcinoma
Article Snippet: All experiments were performed in accordance with relevant institutional and national guidelines and regulations of Shanghai Medical Experimental Animal Care Commission. .. LINC01138 or antisense-LINC01138 RNAs were transcribed and labelled by the Biotin RNA Labeling Mix (Roche, USA), treated with RNase-free DNase I (Takara, Japan) and purified with an RNeasy Mini Kit (QIAGEN, USA). .. Next, 1 pmol biotinylated RNA was pretreated with RNA structure buffer (Beyotime Biotechnology, Shanghai, China) to obtain an appropriate secondary structure formation.

Article Title: m6A-induced lncRNA RP11 triggers the dissemination of colorectal cancer cells via upregulation of Zeb1
Article Snippet: RNA expression was normalized to the total amount of RNA used for reverse transcription. .. LncRNA-RP11 and its antisense RNA were transcribed in vitro from the pGEM-T-RP11 vector, biotin-labelled with the Biotin RNA Labeling Mix (Roche Diagnostics, Indianapolis, IN, USA) and T7/SP6 RNA polymerase (Roche), treated with RNase-free DNase I (Roche), and purified with an RNeasy Mini Kit (Qiagen, Valencia, CA, USA). .. One milligram of protein from the extracts of HCT-15 cells stably transfected with pcDNA3.1-RP11 was then mixed with 50 pmoles of biotinylated RNA, incubated with streptavidin agarose beads (Invitrogen, Carlsbad, CA, USA), and washed.

Article Title: Defining potentially conserved RNA regulons of homologous zinc-finger RNA-binding proteins
Article Snippet: Biotin-labeled RNAs were prepared with the Biotin RNA Labeling Mix (Roche) and T7 RNA polymerase (Promega). .. Biotinylated RNAs were treated with RNase-free DNase I and purified with the PureLink Micro-to-Midi Kit (Invitrogen).

Sequencing:

Article Title: Defining potentially conserved RNA regulons of homologous zinc-finger RNA-binding proteins
Article Snippet: DNA templates for biotin-RNA synthesis were either prepared by PCR from S. cerevisiae or human DNA with oligonucleotides bearing a T7 RNA polymerase promotor sequence at their 5' ends, or by direct annealing of complementary oligonucleotide pairs containing a 5' T7 RNA polymerase promotor (a list of oligonucleotide primers is given in Additional file ). .. Biotin-labeled RNAs were prepared with the Biotin RNA Labeling Mix (Roche) and T7 RNA polymerase (Promega).

Electrophoretic Mobility Shift Assay:

Article Title: The Staphylococcus aureus Protein-Coding Gene gdpS Modulates sarS Expression via mRNA-mRNA Interaction
Article Snippet: Paragraph title: In vitro transcription and gel-shift assays. ... In vitro transcription was carried out with RiboMAX large-scale RNA production systems (Promega). gdpS mRNA was amplified and biotin labeled with a Roche biotin RNA-labeling mix (Roche).

Spectroscopy:

Article Title: m6A-induced lncRNA RP11 triggers the dissemination of colorectal cancer cells via upregulation of Zeb1
Article Snippet: Paragraph title: RNA pull-down/mass spectroscopy analysis ... LncRNA-RP11 and its antisense RNA were transcribed in vitro from the pGEM-T-RP11 vector, biotin-labelled with the Biotin RNA Labeling Mix (Roche Diagnostics, Indianapolis, IN, USA) and T7/SP6 RNA polymerase (Roche), treated with RNase-free DNase I (Roche), and purified with an RNeasy Mini Kit (Qiagen, Valencia, CA, USA).

Lysis:

Article Title: The LINC01138 drives malignancies via activating arginine methyltransferase 5 in hepatocellular carcinoma
Article Snippet: LINC01138 or antisense-LINC01138 RNAs were transcribed and labelled by the Biotin RNA Labeling Mix (Roche, USA), treated with RNase-free DNase I (Takara, Japan) and purified with an RNeasy Mini Kit (QIAGEN, USA). .. LINC01138 or antisense-LINC01138 RNAs were transcribed and labelled by the Biotin RNA Labeling Mix (Roche, USA), treated with RNase-free DNase I (Takara, Japan) and purified with an RNeasy Mini Kit (QIAGEN, USA).

Silver Staining:

Article Title: Long noncoding RNA OCC-1 suppresses cell growth through destabilizing HuR protein in colorectal cancer
Article Snippet: Briefly, the relative long 918-nucleotide OCC-1 3′UTR RNA was synthesized and labeled with Biotin RNA Labeling Mix (Roche) by in vitro transcription. .. After incubation, RNA-protein complexes were retrieved by streptavidin-coupled T1 beads (Dynabeads), washed five times in IP buffer and eluted in Laemmli buffer.

Article Title: The LINC01138 drives malignancies via activating arginine methyltransferase 5 in hepatocellular carcinoma
Article Snippet: LINC01138 or antisense-LINC01138 RNAs were transcribed and labelled by the Biotin RNA Labeling Mix (Roche, USA), treated with RNase-free DNase I (Takara, Japan) and purified with an RNeasy Mini Kit (QIAGEN, USA). .. LINC01138 or antisense-LINC01138 RNAs were transcribed and labelled by the Biotin RNA Labeling Mix (Roche, USA), treated with RNase-free DNase I (Takara, Japan) and purified with an RNeasy Mini Kit (QIAGEN, USA).

In Situ Hybridization:

Article Title: Circular RNA hsa-circ-0012129 Promotes Cell Proliferation and Invasion in 30 Cases of Human Glioma and Human Glioma Cell Lines U373, A172, and SHG44, by Targeting MicroRNA-661 (miR-661)
Article Snippet: Paragraph title: Fluorescence in situ hybridization (FISH) assay ... PCR products were labeled with biotin-labeled RNA probes by using biotin RNA labeling mix (Roche Applied Science, Mannheim, Germany) (Cat. No: 11685597910) and T7 RNA polymerase (Cat. No: M0251L) according to the manufacturer’s instructions.

Plasmid Preparation:

Article Title: LncSHRG promotes hepatocellular carcinoma progression by activating HES6
Article Snippet: For RNA pulldown assays, Biotin-labeled lncSHRG and anti-sense control of lncSHRG were obtained by T7 transcription in vitro using Biotin RNA labeling Mix (Roche) and added into cell lysates containing His-SATB1. .. Then biotin-enriched components were separated with SDS–PAGE and examined by WB.

Article Title: LncBRM initiates YAP1 signalling activation to drive self-renewal of liver cancer stem cells
Article Snippet: For RNA EMSA, human BRM plasmid was transfected into Huh7 cells, then cell nuclear extracts were isolated from BRM-overexpressed oncospheres. .. LncBRM-specific probes were labelled with Biotin using Biotin RNA Labeling Mix (Roche).

Article Title: Monitoring the Transcriptional Activity of Human Endogenous Retroviral HERV-W Family Using PNA Strand Invasion into Double-Stranded DNA
Article Snippet: DNA was fixed on the membrane by baking it at 80 °C for 1 h in a sterilization oven. .. An ERVWE1-specific, biotinylated RNA probe was synthetized using T3 RNA polymerase and Biotin RNA Labeling Mix (Roche Diagnostics, Warsaw, Poland); XbaI-linearized pSC-B-ERVWE1270 plasmid served as a template in transcription reaction. .. Hybridization was performed in a roller bottle overnight at 55 °C using 2.5 μl of RNA probe diluted in 4 ml of hybridization buffer (Minidizer Hybridizer, UVP, LLC, Upland, CA, USA).

Article Title: m6A-induced lncRNA RP11 triggers the dissemination of colorectal cancer cells via upregulation of Zeb1
Article Snippet: RNA expression was normalized to the total amount of RNA used for reverse transcription. .. LncRNA-RP11 and its antisense RNA were transcribed in vitro from the pGEM-T-RP11 vector, biotin-labelled with the Biotin RNA Labeling Mix (Roche Diagnostics, Indianapolis, IN, USA) and T7/SP6 RNA polymerase (Roche), treated with RNase-free DNase I (Roche), and purified with an RNeasy Mini Kit (Qiagen, Valencia, CA, USA). .. One milligram of protein from the extracts of HCT-15 cells stably transfected with pcDNA3.1-RP11 was then mixed with 50 pmoles of biotinylated RNA, incubated with streptavidin agarose beads (Invitrogen, Carlsbad, CA, USA), and washed.

Software:

Article Title: Monitoring the Transcriptional Activity of Human Endogenous Retroviral HERV-W Family Using PNA Strand Invasion into Double-Stranded DNA
Article Snippet: Integrated optical density (IOD) values for every DNA band were calculated using ImageJ, v. 1.41 software [ ]. .. An ERVWE1-specific, biotinylated RNA probe was synthetized using T3 RNA polymerase and Biotin RNA Labeling Mix (Roche Diagnostics, Warsaw, Poland); XbaI-linearized pSC-B-ERVWE1270 plasmid served as a template in transcription reaction.

Microscopy:

Article Title: Circular RNA hsa-circ-0012129 Promotes Cell Proliferation and Invasion in 30 Cases of Human Glioma and Human Glioma Cell Lines U373, A172, and SHG44, by Targeting MicroRNA-661 (miR-661)
Article Snippet: PCR products were labeled with biotin-labeled RNA probes by using biotin RNA labeling mix (Roche Applied Science, Mannheim, Germany) (Cat. No: 11685597910) and T7 RNA polymerase (Cat. No: M0251L) according to the manufacturer’s instructions. .. Cell nuclei were stained with 4,6-diamidino-2-phenylindole (DAPI) (Invitrogen).

SYBR Green Assay:

Article Title: Monitoring the Transcriptional Activity of Human Endogenous Retroviral HERV-W Family Using PNA Strand Invasion into Double-Stranded DNA
Article Snippet: The reaction products of PNA strand invasion were loaded onto 6%, 1-mm-thick polyacrylamide gel (29:1 acrylamide/Bis-acrylamide ratio) and resolved for 2.5 h at 100 V. After electrophoresis, gels were stained for 10 min in the solution containing 1× concentrated SYBR Green I nucleic acid gel stain (Sigma-Aldrich Co, Poznań, Poland), then washed with high-pure water for 5 min, and digitalized using Gel Logic 100 Imaging System (Eastman Kodak Company, Rochester, NY, USA). .. An ERVWE1-specific, biotinylated RNA probe was synthetized using T3 RNA polymerase and Biotin RNA Labeling Mix (Roche Diagnostics, Warsaw, Poland); XbaI-linearized pSC-B-ERVWE1270 plasmid served as a template in transcription reaction.

Agarose Gel Electrophoresis:

Article Title: Defining potentially conserved RNA regulons of homologous zinc-finger RNA-binding proteins
Article Snippet: Biotin-labeled RNAs were prepared with the Biotin RNA Labeling Mix (Roche) and T7 RNA polymerase (Promega). .. RNAs smaller than 60 nucleotides were purified with the mirVana PARIS Kit (Ambion, catalogue number AM1556).

In Vitro:

Article Title: Involvement of KSRP in the post-transcriptional regulation of human iNOS expression-complex interplay of KSRP with TTP and HuR
Article Snippet: The immunoreactive proteins on the blots were visualized by the enhanced chemiluminescence detection system (ECL, Amersham). .. To generate biotinylated-RNA sense probes for the affinity purification 0.5–1 µg of the linearized plasmids pCR-iNOS 3′-UTR or pCR-iNOS 3′-UTR-non-AU ( ) were in vitro transcribed using the biotin RNA labeling mix (Roche Diagnostics, Mannheim, Germany) as described by the manufacturer. .. Protein extracts from CM-induced DLD-1 cells were first precleared by incubating 200 mg of cytoplasmic proteins with 3 ml of a 50% slurry pre-blocked streptavidin-coated agarose beads, for 3 h at 4°C in 50 ml binding buffer [10 mM HEPES (pH 7.6), 3 mM MgCl2 , 5 mM EDTA, 2 mM DTT, 5% Glycerol, 0.5% NP-40, 3 mg/ml Heparin and 0.5 mg/ml Yeast RNA] supplemented with 40 mM KCl and RNasin (0.3 U/μl, final).

Article Title: Long Noncoding RNA HEIH Promotes Colorectal Cancer Tumorigenesis via Counteracting miR-939‒Mediated Transcriptional Repression of Bcl-xL
Article Snippet: Then TUNEL staining was performed using an In Situ Cell Death Detection Kit (Roche). .. lncRNA-HEIH and miR-939 binding sites mutated lncRNA-HEIH (lncRNA-HEIH-mut) were in vitro transcribed from pSPT19-HEIH, and pSPT19-HEIH-mut, respectively, using the SP6 RNA polymerase (Roche) and biotinlabeled with the Biotin RNA Labeling Mix (Roche) in accordance with the manufacturer’s protocols. .. After being treated with RNase-free DNase I (Roche), the in vitro transcribed biotinylated RNA was purified using an RNeasy Mini Kit (Qiagen, Valencia, CA).

Article Title: Upregulation of LncRNA‐HIT promotes migration and invasion of non‐small cell lung cancer cells by association with ZEB1
Article Snippet: RNA pull‐down and deletion mapping were performed as described previously . .. Briefly, biotin‐labeled lncRNA‐HIT were in vitro transcribed with the Biotin RNA Labeling Mix (Roche, Cambridge, UK) and T7 RNA polymerase (Roche), treated with RNase‐free DNase I (Roche), and purified with the RNeasy Mini Kit (Qiagen, Dusseldolf, Germany). .. Cell nuclear proteins were extracted using the Cytoplasmic and Nuclear Protein Extraction Kit (Tiangen, Beijing China).

Article Title: Long noncoding RNA OCC-1 suppresses cell growth through destabilizing HuR protein in colorectal cancer
Article Snippet: RNA pull-down assay was carried out as previously described ( ). .. Briefly, the relative long 918-nucleotide OCC-1 3′UTR RNA was synthesized and labeled with Biotin RNA Labeling Mix (Roche) by in vitro transcription. .. The biotin-labeled RNA (1 μg) was first folded in RNA structure buffer (20 mM Tris–Cl [pH 7.0], 0.2 M KCl and 20 mM MgCl2 ) and then incubated with Caco-2 whole-cell lysate at 4°C for 1 h with rotation.

Article Title: Human colorectal cancer-specific CCAT1-L lncRNA regulates long-range chromatin interactions at the MYC locus
Article Snippet: Biotin-labeled RNAs pull-down assay was performed as described , , with minor modifications. .. Biotin-labeled CCAT1-L truncation probes were in vitro transcribed with the Biotin RNA Labeling Mix (Roche) and AmpliScripe T7/SP6-flash Transcription Kit (Epicentre). .. 4 μg biotinylated RNA was denatured for 5 min at 65 °C in PA buffer (10 mM Tris-HCl, pH 7.5, 10 mM MgCl2 , 100 mM NH4 Cl) and slowly cooled down to RT.

Article Title: The Staphylococcus aureus Protein-Coding Gene gdpS Modulates sarS Expression via mRNA-mRNA Interaction
Article Snippet: For the gel-shift assays, the sarS and spa promoters were identified as previously described ( ). .. In vitro transcription was carried out with RiboMAX large-scale RNA production systems (Promega). gdpS mRNA was amplified and biotin labeled with a Roche biotin RNA-labeling mix (Roche). .. Labeled probes were incubated with increasing amounts of sarS mRNA or spa mRNA at 85°C for 2 min and 37°C for 30 min. Then, the mixture was processed using a 4% native polyacrylamide gel and transferred to a charged nylon membrane.

Article Title: A Prader-Willi locus lncRNA cloud modulates diurnal genes and energy expenditure
Article Snippet: Paragraph title: In vitro biotinylated RNA pull-down of proteins ... Template DNA was linearized and 250 ng of DNA was IVT using Biotin RNA labeling Mix (Roche), DNAse I (NEB) treated and nucleotides removed using Micro-Biospin columns (Biorad).

Article Title: LncSHRG promotes hepatocellular carcinoma progression by activating HES6
Article Snippet: After transferred on a nylon membrane (Amersham Biosciences), the labeled DNA was cross-linked by UV, probed with streptavidin-HRP conjugate and then incubated with the detection substrate. .. For RNA pulldown assays, Biotin-labeled lncSHRG and anti-sense control of lncSHRG were obtained by T7 transcription in vitro using Biotin RNA labeling Mix (Roche) and added into cell lysates containing His-SATB1. .. After incubation overnight at 4°C, Streptavidin-magnetic C1 beads was added and incubated for another 4 h at 4°C.

Article Title: m6A-induced lncRNA RP11 triggers the dissemination of colorectal cancer cells via upregulation of Zeb1
Article Snippet: RNA expression was normalized to the total amount of RNA used for reverse transcription. .. LncRNA-RP11 and its antisense RNA were transcribed in vitro from the pGEM-T-RP11 vector, biotin-labelled with the Biotin RNA Labeling Mix (Roche Diagnostics, Indianapolis, IN, USA) and T7/SP6 RNA polymerase (Roche), treated with RNase-free DNase I (Roche), and purified with an RNeasy Mini Kit (Qiagen, Valencia, CA, USA). .. One milligram of protein from the extracts of HCT-15 cells stably transfected with pcDNA3.1-RP11 was then mixed with 50 pmoles of biotinylated RNA, incubated with streptavidin agarose beads (Invitrogen, Carlsbad, CA, USA), and washed.

Fluorescence In Situ Hybridization:

Article Title: Circular RNA hsa-circ-0012129 Promotes Cell Proliferation and Invasion in 30 Cases of Human Glioma and Human Glioma Cell Lines U373, A172, and SHG44, by Targeting MicroRNA-661 (miR-661)
Article Snippet: Paragraph title: Fluorescence in situ hybridization (FISH) assay ... PCR products were labeled with biotin-labeled RNA probes by using biotin RNA labeling mix (Roche Applied Science, Mannheim, Germany) (Cat. No: 11685597910) and T7 RNA polymerase (Cat. No: M0251L) according to the manufacturer’s instructions.

Spectrophotometry:

Article Title: A Prader-Willi locus lncRNA cloud modulates diurnal genes and energy expenditure
Article Snippet: Template DNA was linearized and 250 ng of DNA was IVT using Biotin RNA labeling Mix (Roche), DNAse I (NEB) treated and nucleotides removed using Micro-Biospin columns (Biorad). .. Template DNA was linearized and 250 ng of DNA was IVT using Biotin RNA labeling Mix (Roche), DNAse I (NEB) treated and nucleotides removed using Micro-Biospin columns (Biorad).

Immunoprecipitation:

Article Title: LncBRM initiates YAP1 signalling activation to drive self-renewal of liver cancer stem cells
Article Snippet: BRG1 and KLF4 EMSA assays were performed using standardized EMSA procedure, and KLF4 and BRG1 were obtained using immunoprecipitation from oncospheres . .. LncBRM-specific probes were labelled with Biotin using Biotin RNA Labeling Mix (Roche).

Article Title: linc-HOXA1 is a noncoding RNA that represses Hoxa1 transcription in cis
Article Snippet: Biotin-labeled RNA of the three isoforms of linc-HOXA1 , along with deletions of these isoforms and antisense versions, were prepared using the Biotin RNA labeling mix (Roche) and T7 RNA polymerase (Roche). .. Biotin-labeled RNA of the three isoforms of linc-HOXA1 , along with deletions of these isoforms and antisense versions, were prepared using the Biotin RNA labeling mix (Roche) and T7 RNA polymerase (Roche).

Staining:

Article Title: Involvement of KSRP in the post-transcriptional regulation of human iNOS expression-complex interplay of KSRP with TTP and HuR
Article Snippet: To generate biotinylated-RNA sense probes for the affinity purification 0.5–1 µg of the linearized plasmids pCR-iNOS 3′-UTR or pCR-iNOS 3′-UTR-non-AU ( ) were in vitro transcribed using the biotin RNA labeling mix (Roche Diagnostics, Mannheim, Germany) as described by the manufacturer. .. Isolated proteins were eluted from the beads by incubating with elution buffer [10 mM HEPES (pH 7.6), 3 mM MgCl2 , 5 mM EDTA, 2 mM DTT, 0.2% glycerol and 2 M KCl] for 20 min at 4°C.

Article Title: Circular RNA hsa-circ-0012129 Promotes Cell Proliferation and Invasion in 30 Cases of Human Glioma and Human Glioma Cell Lines U373, A172, and SHG44, by Targeting MicroRNA-661 (miR-661)
Article Snippet: PCR products were labeled with biotin-labeled RNA probes by using biotin RNA labeling mix (Roche Applied Science, Mannheim, Germany) (Cat. No: 11685597910) and T7 RNA polymerase (Cat. No: M0251L) according to the manufacturer’s instructions. .. PCR products were labeled with biotin-labeled RNA probes by using biotin RNA labeling mix (Roche Applied Science, Mannheim, Germany) (Cat. No: 11685597910) and T7 RNA polymerase (Cat. No: M0251L) according to the manufacturer’s instructions.

Article Title: Monitoring the Transcriptional Activity of Human Endogenous Retroviral HERV-W Family Using PNA Strand Invasion into Double-Stranded DNA
Article Snippet: After staining and digitalizing, DNA was electrotransferred onto a positively charged, nylon blotting membrane (Hybond-N+, GE Healthcare Life Sciences, Warsaw, Poland). .. An ERVWE1-specific, biotinylated RNA probe was synthetized using T3 RNA polymerase and Biotin RNA Labeling Mix (Roche Diagnostics, Warsaw, Poland); XbaI-linearized pSC-B-ERVWE1270 plasmid served as a template in transcription reaction.

Clear Native PAGE:

Article Title: LncBRM initiates YAP1 signalling activation to drive self-renewal of liver cancer stem cells
Article Snippet: LncBRM-specific probes were labelled with Biotin using Biotin RNA Labeling Mix (Roche). .. BRM nuclear extracts and biotin-labelled probe were incubated in 1 × REMSA binding buffer supplementing with Glycerol, transfer RNA and dithiothreitol for 30 min according to LightShift Chemiluminescent RNA EMSA Kit (Thermo Scientific).

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    Roche biotin rna labeling mix
    Depletion of KSRP enhances iNOS <t>3′-UTR</t> <t>RNA</t> stability in in vitro degradation assays. DLD-1 cells were preincubated for 18 h in medium without FCS and phenol red. Then the cells were incubated with the CM for 6 h and cytoplasmic proteins were isolated. Parts of these extracts were immunodepleted of KSRP by using a polyclonal anti-KSRP antibody (KSRP-depl). The extracts were incubated with radiolabeled 5′-capped and 3′-polyadenylated iNOS 3′-UTR RNA with (iNOS 3′-UTR) or without (non-AU) the AREs for different time periods from 0 to 30 min. Then the degradation of the RNA in the different samples was stopped by adding SDS. To normalize the subsequent purification steps in vitro transcribed radiolabeled β-actin RNA fragments were added. After phenol extraction and ethanol precipitation the material was separated on denaturing urea polyacrylamide gels. ( A ) Scheme of the human 5′-capped and 3′-polyadenylated iNOS 3′-UTR RNAs used for in vitro decay experiments. The positions of AU-repeats are indicated by arrowheads. ( B ) The degree of KSRP depletion of two different immunodepletion reactions (IP α-KSRP) were analyzed by western blotting using specific anti-KSRP- and anti-β-tubulin antibodies. For comparison the corresponding untreated samples were analyzed. The position of KSRP and β-tubulin is indicated. ( C ) Summary of densitometric analyses of eight different in vitro decay assays using 5′-capped and 3′-polyadenylated iNOS 3′-UTR RNAs containing the AREs (iNOS 3′-UTR). Data points (means ± SEM) represent relative iNOS 3′-UTR RNA levels (100% = 0 min incubation; circles: undepleted extracts from CM-treated cells; squares: KSRP-depleted extracts from CM-treated cells; ** P
    Biotin Rna Labeling Mix, supplied by Roche, used in various techniques. Bioz Stars score: 99/100, based on 226 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Depletion of KSRP enhances iNOS 3′-UTR RNA stability in in vitro degradation assays. DLD-1 cells were preincubated for 18 h in medium without FCS and phenol red. Then the cells were incubated with the CM for 6 h and cytoplasmic proteins were isolated. Parts of these extracts were immunodepleted of KSRP by using a polyclonal anti-KSRP antibody (KSRP-depl). The extracts were incubated with radiolabeled 5′-capped and 3′-polyadenylated iNOS 3′-UTR RNA with (iNOS 3′-UTR) or without (non-AU) the AREs for different time periods from 0 to 30 min. Then the degradation of the RNA in the different samples was stopped by adding SDS. To normalize the subsequent purification steps in vitro transcribed radiolabeled β-actin RNA fragments were added. After phenol extraction and ethanol precipitation the material was separated on denaturing urea polyacrylamide gels. ( A ) Scheme of the human 5′-capped and 3′-polyadenylated iNOS 3′-UTR RNAs used for in vitro decay experiments. The positions of AU-repeats are indicated by arrowheads. ( B ) The degree of KSRP depletion of two different immunodepletion reactions (IP α-KSRP) were analyzed by western blotting using specific anti-KSRP- and anti-β-tubulin antibodies. For comparison the corresponding untreated samples were analyzed. The position of KSRP and β-tubulin is indicated. ( C ) Summary of densitometric analyses of eight different in vitro decay assays using 5′-capped and 3′-polyadenylated iNOS 3′-UTR RNAs containing the AREs (iNOS 3′-UTR). Data points (means ± SEM) represent relative iNOS 3′-UTR RNA levels (100% = 0 min incubation; circles: undepleted extracts from CM-treated cells; squares: KSRP-depleted extracts from CM-treated cells; ** P

    Journal: Nucleic Acids Research

    Article Title: Involvement of KSRP in the post-transcriptional regulation of human iNOS expression-complex interplay of KSRP with TTP and HuR

    doi: 10.1093/nar/gki797

    Figure Lengend Snippet: Depletion of KSRP enhances iNOS 3′-UTR RNA stability in in vitro degradation assays. DLD-1 cells were preincubated for 18 h in medium without FCS and phenol red. Then the cells were incubated with the CM for 6 h and cytoplasmic proteins were isolated. Parts of these extracts were immunodepleted of KSRP by using a polyclonal anti-KSRP antibody (KSRP-depl). The extracts were incubated with radiolabeled 5′-capped and 3′-polyadenylated iNOS 3′-UTR RNA with (iNOS 3′-UTR) or without (non-AU) the AREs for different time periods from 0 to 30 min. Then the degradation of the RNA in the different samples was stopped by adding SDS. To normalize the subsequent purification steps in vitro transcribed radiolabeled β-actin RNA fragments were added. After phenol extraction and ethanol precipitation the material was separated on denaturing urea polyacrylamide gels. ( A ) Scheme of the human 5′-capped and 3′-polyadenylated iNOS 3′-UTR RNAs used for in vitro decay experiments. The positions of AU-repeats are indicated by arrowheads. ( B ) The degree of KSRP depletion of two different immunodepletion reactions (IP α-KSRP) were analyzed by western blotting using specific anti-KSRP- and anti-β-tubulin antibodies. For comparison the corresponding untreated samples were analyzed. The position of KSRP and β-tubulin is indicated. ( C ) Summary of densitometric analyses of eight different in vitro decay assays using 5′-capped and 3′-polyadenylated iNOS 3′-UTR RNAs containing the AREs (iNOS 3′-UTR). Data points (means ± SEM) represent relative iNOS 3′-UTR RNA levels (100% = 0 min incubation; circles: undepleted extracts from CM-treated cells; squares: KSRP-depleted extracts from CM-treated cells; ** P

    Article Snippet: To generate biotinylated-RNA sense probes for the affinity purification 0.5–1 µg of the linearized plasmids pCR-iNOS 3′-UTR or pCR-iNOS 3′-UTR-non-AU ( ) were in vitro transcribed using the biotin RNA labeling mix (Roche Diagnostics, Mannheim, Germany) as described by the manufacturer.

    Techniques: In Vitro, Incubation, Isolation, Purification, Ethanol Precipitation, Western Blot

    Cytokine incubation reduces intracellular binding of KSRP and enhances intracellular binding of HuR to the iNOS mRNA. DLD-1 cells were incubated for 4 h with or without CM. Cells were lyzed and RNA bound by KSRP or HuR protein was immunoprecipitated by specific antibodies. Immunoprecipitation with IgG was used as negative control. To normalize for the subsequent RNA purification steps 1 ng/sample of luciferase RNA transcribed in vitro was added before the RNA was isolated from immunoprecipitated proteins. The amount of iNOS mRNA bound by KSRP or HuR was determined by qRT–PCR using the luciferase RNA as normalization control. The values of the IgG controls were subtracted and the values for the iNOS mRNA bound by HuR were divided by the values for the iNOS mRNA bound by KSRP. A summary of 12 immunoprecipitation-qRT–PCR analyses is shown. Columns (means ± SEM) represent relative iNOS mRNA levels bound by HuR divided by the relative iNOS mRNA levels bound by KSRP ( ** P

    Journal: Nucleic Acids Research

    Article Title: Involvement of KSRP in the post-transcriptional regulation of human iNOS expression-complex interplay of KSRP with TTP and HuR

    doi: 10.1093/nar/gki797

    Figure Lengend Snippet: Cytokine incubation reduces intracellular binding of KSRP and enhances intracellular binding of HuR to the iNOS mRNA. DLD-1 cells were incubated for 4 h with or without CM. Cells were lyzed and RNA bound by KSRP or HuR protein was immunoprecipitated by specific antibodies. Immunoprecipitation with IgG was used as negative control. To normalize for the subsequent RNA purification steps 1 ng/sample of luciferase RNA transcribed in vitro was added before the RNA was isolated from immunoprecipitated proteins. The amount of iNOS mRNA bound by KSRP or HuR was determined by qRT–PCR using the luciferase RNA as normalization control. The values of the IgG controls were subtracted and the values for the iNOS mRNA bound by HuR were divided by the values for the iNOS mRNA bound by KSRP. A summary of 12 immunoprecipitation-qRT–PCR analyses is shown. Columns (means ± SEM) represent relative iNOS mRNA levels bound by HuR divided by the relative iNOS mRNA levels bound by KSRP ( ** P

    Article Snippet: To generate biotinylated-RNA sense probes for the affinity purification 0.5–1 µg of the linearized plasmids pCR-iNOS 3′-UTR or pCR-iNOS 3′-UTR-non-AU ( ) were in vitro transcribed using the biotin RNA labeling mix (Roche Diagnostics, Mannheim, Germany) as described by the manufacturer.

    Techniques: Incubation, Binding Assay, Immunoprecipitation, Negative Control, Purification, Luciferase, In Vitro, Isolation, Quantitative RT-PCR

    Binding of KSRP and HuR to the 3′-UTR of the human iNOS mRNA is mutually exclusive. 32 P-radiolabeled in vitro transcribed RNAs containing the sequence of subfragment C (387–477) were incubated in parallel with fixed amounts (0.6 µg) of GST-HuR or GST-KSRP and increasing amounts of GST-KSRP or GST-HuR (0 to 1.2 µg), respectively. After binding, proteins were UV crosslinked to the RNA and the complexes were digested with RNase. RNA–protein complexes were separated on SDS–polyacrylamide gels. The positions of RNA–protein complexes are indicated. (A) Radiolabeled fragment C-RNAs were incubated with 0.6 µg GST-HuR (0.6 µg) and 0 to 1.2 µg GST-KSRP. (B) Radiolabeled fragment C-RNAs were incubated with 0.6 µg GST-KSRP (0.6 µg) and 0–1.2 µg GST-HuR.

    Journal: Nucleic Acids Research

    Article Title: Involvement of KSRP in the post-transcriptional regulation of human iNOS expression-complex interplay of KSRP with TTP and HuR

    doi: 10.1093/nar/gki797

    Figure Lengend Snippet: Binding of KSRP and HuR to the 3′-UTR of the human iNOS mRNA is mutually exclusive. 32 P-radiolabeled in vitro transcribed RNAs containing the sequence of subfragment C (387–477) were incubated in parallel with fixed amounts (0.6 µg) of GST-HuR or GST-KSRP and increasing amounts of GST-KSRP or GST-HuR (0 to 1.2 µg), respectively. After binding, proteins were UV crosslinked to the RNA and the complexes were digested with RNase. RNA–protein complexes were separated on SDS–polyacrylamide gels. The positions of RNA–protein complexes are indicated. (A) Radiolabeled fragment C-RNAs were incubated with 0.6 µg GST-HuR (0.6 µg) and 0 to 1.2 µg GST-KSRP. (B) Radiolabeled fragment C-RNAs were incubated with 0.6 µg GST-KSRP (0.6 µg) and 0–1.2 µg GST-HuR.

    Article Snippet: To generate biotinylated-RNA sense probes for the affinity purification 0.5–1 µg of the linearized plasmids pCR-iNOS 3′-UTR or pCR-iNOS 3′-UTR-non-AU ( ) were in vitro transcribed using the biotin RNA labeling mix (Roche Diagnostics, Mannheim, Germany) as described by the manufacturer.

    Techniques: Binding Assay, In Vitro, Sequencing, Incubation

    Overexpression of a KSRP mutant which is not able to bind to RNA enhances iNOS expression. Plasmids allowing high level expression of a mutant KSRP protein not able to bind to RNA (KSRPdel) were stably transfected into DLD-1 cells. Cells transfected with the pcDNA3 vector backbone (pcDNA3) were used as controls. For analysis of iNOS expression pools of stable transfected cell were preincubated for 18 h in medium without FCS and phenol red. Then cells were incubated with (CM) or without (Co) the cytokine mixture for 6 h, RNA was isolated and iNOS and GAPDH mRNA expression was analyzed by real-time RT–PCR. To determine iNOS-mediated NO-production cells were incubated for 24 h with or without CM and the supernatant of the cells was analyzed for nitrite content. ( A ) Pooled populations of pcDNA3- or pcDNA-KSRPdel cells were analyzed for KSRP expression by western blots using specific anti-KSRP antibodies. One representative blot out of three is shown. ( B ) A summary of 5 qRT–PCR analyses is shown using RNAs from DLD-1-pcDNA3 (pcDNA3) or DLD-1-pcDNA3-KSRPdel (KSRPdel) cells. Data (means ± SEM) represent relative iNOS mRNA levels ( * P

    Journal: Nucleic Acids Research

    Article Title: Involvement of KSRP in the post-transcriptional regulation of human iNOS expression-complex interplay of KSRP with TTP and HuR

    doi: 10.1093/nar/gki797

    Figure Lengend Snippet: Overexpression of a KSRP mutant which is not able to bind to RNA enhances iNOS expression. Plasmids allowing high level expression of a mutant KSRP protein not able to bind to RNA (KSRPdel) were stably transfected into DLD-1 cells. Cells transfected with the pcDNA3 vector backbone (pcDNA3) were used as controls. For analysis of iNOS expression pools of stable transfected cell were preincubated for 18 h in medium without FCS and phenol red. Then cells were incubated with (CM) or without (Co) the cytokine mixture for 6 h, RNA was isolated and iNOS and GAPDH mRNA expression was analyzed by real-time RT–PCR. To determine iNOS-mediated NO-production cells were incubated for 24 h with or without CM and the supernatant of the cells was analyzed for nitrite content. ( A ) Pooled populations of pcDNA3- or pcDNA-KSRPdel cells were analyzed for KSRP expression by western blots using specific anti-KSRP antibodies. One representative blot out of three is shown. ( B ) A summary of 5 qRT–PCR analyses is shown using RNAs from DLD-1-pcDNA3 (pcDNA3) or DLD-1-pcDNA3-KSRPdel (KSRPdel) cells. Data (means ± SEM) represent relative iNOS mRNA levels ( * P

    Article Snippet: To generate biotinylated-RNA sense probes for the affinity purification 0.5–1 µg of the linearized plasmids pCR-iNOS 3′-UTR or pCR-iNOS 3′-UTR-non-AU ( ) were in vitro transcribed using the biotin RNA labeling mix (Roche Diagnostics, Mannheim, Germany) as described by the manufacturer.

    Techniques: Over Expression, Mutagenesis, Expressing, Stable Transfection, Transfection, Plasmid Preparation, Incubation, Isolation, Quantitative RT-PCR, Western Blot

    Analysis of the KSRP binding site in the human iNOS 3′-UTR RNA. Purified BSA, glutathione-S-transferase (GST), and GST-KSRP or GST-HuR fusion proteins were incubated with different radiolabeled RNAs generated by in vitro transcription using the different iNOS 3′-UTR fragments shown in Figure A. After binding, proteins were UV crosslinked to the RNA and the complexes were digested with RNase. RNA–protein complexes were separated on SDS–polyacrylamide gels. ( A ) Structure of the human iNOS 3′-UTR mRNA and fragments used in RNA binding studies. Scheme of the human iNOS 3′-UTR mRNA (477 nt) and transcripts used in RNA binding studies. The initial UGA nucleotide sequence (−3 to −1) corresponds to the translation termination codon. AUUUA and AUUUUA repeats are indicated by arrowheads. The sequences of different mutations in fragment C are shown. ( B ) KSRP binds to the AU-fragment of the human iNOS 3′-UTR. 32 P-radiolabeled RNA transcripts [3′-UTR; non-AU; AU; see (A)] were incubated with BSA, GST, GST-KSRP fusion protein (KSRP) or GST-HuR fusion protein (HuR). The positions of RNA–protein complexes are indicated. ( C ) KSRP binds to the most 3′-located ARE in the human iNOS 3′-UTR. 32 P-radiolabeled 3′-UTR, subfragment A (232–319, frag A), subfragment B (317–420; frag B) or subfragment C (387–477; frag C) were incubated with GST-KSRP (GST-KSRP) protein. The positions of RNA–protein complexes are indicated (left side). 32 P-radiolabeled RNA transcripts (5′-ARE mutated: mut 1; 3′-ARE mutated: mut 2; both AREs mutated: mut 1 + 2) were incubated with either GST or GST-KSRP fusion protein (KSRP). The positions of RNA–protein complexes are indicated (right side).

    Journal: Nucleic Acids Research

    Article Title: Involvement of KSRP in the post-transcriptional regulation of human iNOS expression-complex interplay of KSRP with TTP and HuR

    doi: 10.1093/nar/gki797

    Figure Lengend Snippet: Analysis of the KSRP binding site in the human iNOS 3′-UTR RNA. Purified BSA, glutathione-S-transferase (GST), and GST-KSRP or GST-HuR fusion proteins were incubated with different radiolabeled RNAs generated by in vitro transcription using the different iNOS 3′-UTR fragments shown in Figure A. After binding, proteins were UV crosslinked to the RNA and the complexes were digested with RNase. RNA–protein complexes were separated on SDS–polyacrylamide gels. ( A ) Structure of the human iNOS 3′-UTR mRNA and fragments used in RNA binding studies. Scheme of the human iNOS 3′-UTR mRNA (477 nt) and transcripts used in RNA binding studies. The initial UGA nucleotide sequence (−3 to −1) corresponds to the translation termination codon. AUUUA and AUUUUA repeats are indicated by arrowheads. The sequences of different mutations in fragment C are shown. ( B ) KSRP binds to the AU-fragment of the human iNOS 3′-UTR. 32 P-radiolabeled RNA transcripts [3′-UTR; non-AU; AU; see (A)] were incubated with BSA, GST, GST-KSRP fusion protein (KSRP) or GST-HuR fusion protein (HuR). The positions of RNA–protein complexes are indicated. ( C ) KSRP binds to the most 3′-located ARE in the human iNOS 3′-UTR. 32 P-radiolabeled 3′-UTR, subfragment A (232–319, frag A), subfragment B (317–420; frag B) or subfragment C (387–477; frag C) were incubated with GST-KSRP (GST-KSRP) protein. The positions of RNA–protein complexes are indicated (left side). 32 P-radiolabeled RNA transcripts (5′-ARE mutated: mut 1; 3′-ARE mutated: mut 2; both AREs mutated: mut 1 + 2) were incubated with either GST or GST-KSRP fusion protein (KSRP). The positions of RNA–protein complexes are indicated (right side).

    Article Snippet: To generate biotinylated-RNA sense probes for the affinity purification 0.5–1 µg of the linearized plasmids pCR-iNOS 3′-UTR or pCR-iNOS 3′-UTR-non-AU ( ) were in vitro transcribed using the biotin RNA labeling mix (Roche Diagnostics, Mannheim, Germany) as described by the manufacturer.

    Techniques: Binding Assay, Purification, Incubation, Generated, In Vitro, RNA Binding Assay, Sequencing

    Modulation of KSRP expression alters cytokine-induced iNOS mRNA expression and iNOS-dependent NO-production. Plasmid constructs allowing high level expression of sense (pcDNA-His-KSRP) or antisense (pcDNA-KSRPas) KSRP cDNA were stably transfected into DLD-1 cells. Cells transfected with the pcDNA3 vector backbone (pcDNA3) were used as controls. For analysis of iNOS expression pools of stable transfected cell were preincubated for 18 h in medium without FCS and phenol red. Then cells were incubated with (CM) or without (Co) the cytokine mixture for 6 h, RNA was isolated and iNOS and GAPDH mRNA expression was analyzed. To determine iNOS-mediated NO-production cells were incubated for 24 h with or without CM and the supernatant of the cells was analyzed for nitrite content. As another approach to downregulate KSRP expression the RNA interference technique was used. DLD-1 cells were transfected with siRNA directed against luciferase (control, siLUC) or KSRP (siKSRP). After 24 h the transfected cells were preincubated for 18 h in medium without FCS and phenol red. Then the cells were incubated with or without CM for 6 h, RNA was isolated and iNOS, KSRP and GAPDH mRNA expression was analyzed by real-time RT–PCR. ( A ) Western blots using specific anti-KSRP- and anti-β-tubulin antibodies and extracts from the stable transfected DLD-1 cell pools. The blots are representative of four other blots showing similar results. The positions of KSRP and β-tubulin are indicated. ( B ) A summary of 10 qRT–PCR analyses is shown using RNAs form DLD-1-pcDNA3 (pcDNA3), DLD-1-pcDNA-His-KSRP (pcDNA-His-KSRP) or DLD-1-pcDNA3-KSRPas (pcDNA-KSRPas) cells. Data (means ± SEM) represent relative iNOS mRNA levels ( *** P

    Journal: Nucleic Acids Research

    Article Title: Involvement of KSRP in the post-transcriptional regulation of human iNOS expression-complex interplay of KSRP with TTP and HuR

    doi: 10.1093/nar/gki797

    Figure Lengend Snippet: Modulation of KSRP expression alters cytokine-induced iNOS mRNA expression and iNOS-dependent NO-production. Plasmid constructs allowing high level expression of sense (pcDNA-His-KSRP) or antisense (pcDNA-KSRPas) KSRP cDNA were stably transfected into DLD-1 cells. Cells transfected with the pcDNA3 vector backbone (pcDNA3) were used as controls. For analysis of iNOS expression pools of stable transfected cell were preincubated for 18 h in medium without FCS and phenol red. Then cells were incubated with (CM) or without (Co) the cytokine mixture for 6 h, RNA was isolated and iNOS and GAPDH mRNA expression was analyzed. To determine iNOS-mediated NO-production cells were incubated for 24 h with or without CM and the supernatant of the cells was analyzed for nitrite content. As another approach to downregulate KSRP expression the RNA interference technique was used. DLD-1 cells were transfected with siRNA directed against luciferase (control, siLUC) or KSRP (siKSRP). After 24 h the transfected cells were preincubated for 18 h in medium without FCS and phenol red. Then the cells were incubated with or without CM for 6 h, RNA was isolated and iNOS, KSRP and GAPDH mRNA expression was analyzed by real-time RT–PCR. ( A ) Western blots using specific anti-KSRP- and anti-β-tubulin antibodies and extracts from the stable transfected DLD-1 cell pools. The blots are representative of four other blots showing similar results. The positions of KSRP and β-tubulin are indicated. ( B ) A summary of 10 qRT–PCR analyses is shown using RNAs form DLD-1-pcDNA3 (pcDNA3), DLD-1-pcDNA-His-KSRP (pcDNA-His-KSRP) or DLD-1-pcDNA3-KSRPas (pcDNA-KSRPas) cells. Data (means ± SEM) represent relative iNOS mRNA levels ( *** P

    Article Snippet: To generate biotinylated-RNA sense probes for the affinity purification 0.5–1 µg of the linearized plasmids pCR-iNOS 3′-UTR or pCR-iNOS 3′-UTR-non-AU ( ) were in vitro transcribed using the biotin RNA labeling mix (Roche Diagnostics, Mannheim, Germany) as described by the manufacturer.

    Techniques: Expressing, Plasmid Preparation, Construct, Stable Transfection, Transfection, Incubation, Isolation, Luciferase, Quantitative RT-PCR, Western Blot

    Purification of RNA-bps interacting with the 3′-UTR of the human iNOS mRNA. To purify proteins binding to the 3′-UTR of the human iNOS mRNA affinity chromatographies using biotinylated iNOS 3′-UTR RNA were performed ( 45 , 46 ) as described in Materials and Methods. DLD-1 cells were preincubated for 18 h in medium without FCS and phenol red. Then cells were incubated with the cytokine mixture for 6 h and protein extracts were isolated. These extracts were incubated with biotinylated iNOS 3′-UTR RNA (3′-UTR) or biotinylated iNOS 3′-UTR RNA without the ARE-sequences (3′-UTR Δ–ARE) and streptavidine-agarose beads. After several washing and centrifugation steps the RNA-bps were eluted by 2 M KCl. ( A ) Proofing the applicability of this method western blots using a specific anti-HuR antibody were performed, since HuR is known to interact with the human iNOS 3′-UTR ( 28 ). As a positive control bacterial expressed GST-HuR fusion protein was also loaded on the SDS gel. One representative blot of three different experiments were shown. ( B ) The eluates were tested for the presence of KSRP by western blots using a specific anti-KSRP antibody. As a positive control bacterial expressed His-KSRP fusion protein was also loaded on the SDS gel. One representative blot of three different experiments were shown.

    Journal: Nucleic Acids Research

    Article Title: Involvement of KSRP in the post-transcriptional regulation of human iNOS expression-complex interplay of KSRP with TTP and HuR

    doi: 10.1093/nar/gki797

    Figure Lengend Snippet: Purification of RNA-bps interacting with the 3′-UTR of the human iNOS mRNA. To purify proteins binding to the 3′-UTR of the human iNOS mRNA affinity chromatographies using biotinylated iNOS 3′-UTR RNA were performed ( 45 , 46 ) as described in Materials and Methods. DLD-1 cells were preincubated for 18 h in medium without FCS and phenol red. Then cells were incubated with the cytokine mixture for 6 h and protein extracts were isolated. These extracts were incubated with biotinylated iNOS 3′-UTR RNA (3′-UTR) or biotinylated iNOS 3′-UTR RNA without the ARE-sequences (3′-UTR Δ–ARE) and streptavidine-agarose beads. After several washing and centrifugation steps the RNA-bps were eluted by 2 M KCl. ( A ) Proofing the applicability of this method western blots using a specific anti-HuR antibody were performed, since HuR is known to interact with the human iNOS 3′-UTR ( 28 ). As a positive control bacterial expressed GST-HuR fusion protein was also loaded on the SDS gel. One representative blot of three different experiments were shown. ( B ) The eluates were tested for the presence of KSRP by western blots using a specific anti-KSRP antibody. As a positive control bacterial expressed His-KSRP fusion protein was also loaded on the SDS gel. One representative blot of three different experiments were shown.

    Article Snippet: To generate biotinylated-RNA sense probes for the affinity purification 0.5–1 µg of the linearized plasmids pCR-iNOS 3′-UTR or pCR-iNOS 3′-UTR-non-AU ( ) were in vitro transcribed using the biotin RNA labeling mix (Roche Diagnostics, Mannheim, Germany) as described by the manufacturer.

    Techniques: Purification, Binding Assay, Incubation, Isolation, Centrifugation, Western Blot, Positive Control, SDS-Gel

    Bcl-xL mRNA level is positively correlated with long noncoding RNA HEIH (lncRNA-HEIH) expression level in colorectal cancer (CRC) tissues. (A) Bcl-xL mRNA levels in 84 paired CRC and adjacent normal mucosa were detected by quantitative real-time polymerase chain reaction and normalized to glyceraldehyde 3-phosphate dehydrogenase. ***p

    Journal: Cancer Research and Treatment : Official Journal of Korean Cancer Association

    Article Title: Long Noncoding RNA HEIH Promotes Colorectal Cancer Tumorigenesis via Counteracting miR-939‒Mediated Transcriptional Repression of Bcl-xL

    doi: 10.4143/crt.2017.226

    Figure Lengend Snippet: Bcl-xL mRNA level is positively correlated with long noncoding RNA HEIH (lncRNA-HEIH) expression level in colorectal cancer (CRC) tissues. (A) Bcl-xL mRNA levels in 84 paired CRC and adjacent normal mucosa were detected by quantitative real-time polymerase chain reaction and normalized to glyceraldehyde 3-phosphate dehydrogenase. ***p

    Article Snippet: lncRNA-HEIH and miR-939 binding sites mutated lncRNA-HEIH (lncRNA-HEIH-mut) were in vitro transcribed from pSPT19-HEIH, and pSPT19-HEIH-mut, respectively, using the SP6 RNA polymerase (Roche) and biotinlabeled with the Biotin RNA Labeling Mix (Roche) in accordance with the manufacturer’s protocols.

    Techniques: Expressing, Real-time Polymerase Chain Reaction

    Long noncoding RNA HEIH (lncRNA-HEIH) counteracts miR-939‒mediated transcriptional repression of Bcl-xL. (A) After transient transfection of lncRNA-HEIH or lncRNA-HEIH-mut into HT-29 cells, chromatin immunoprecipitation (ChIP) assays with nuclear factor κB (NF-κB) specific antibody were performed, and the retrieved DNA was detected by quantitative real-time polymerase chain reaction (qRT-PCR) and normalized to input. (B) After co-transfection of lncRNA-HEIH knockdown plasmid and miR-939 inhibitors into LoVo cells, ChIP assays with NF-κB specific antibody were performed, and the retrieved DNA was detected by qRT-PCR and normalized to input. (C) After co-transfection of lncRNA-HEIH or lncRNA-HEIH-mut and luciferase reporter containing Bcl-xL promoterinto HT-29 cells, the luciferase activities were measured. Results are shown as the relative ratio of firefly luciferase activity to Renilla luciferase activity. (D) After co-transfection of lncRNA-HEIH knockdown plasmid, miR-939 inhibitors, and luciferase reporter containing Bcl-xL promoter into LoVo cells, the luciferase activities were measured. Results are shown as in panel C. (E) After transient transfection of lncRNA-HEIH or lncRNA-HEIH-mut into HT-29 cells, Bcl-xL mRNA levels were detected by qRT-PCR and normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH). (F) After co-transfection of lncRNA-HEIH knockdown plasmid and miR-939 inhibitors into LoVo cells, Bcl-xL mRNA levels were detected by qRT-PCR and normalized to GAPDH. (G) After transient transfection of lncRNA-HEIH or lncRNA-HEIH-mut into HT-29 cells, Bcl-xL protein levels were detected by western blot and normalized to GAPDH. (H) After co-transfection of lncRNA-HEIH knockdown plasmid and miR-939 inhibitors into LoVo cells, Bcl-xL protein levels were detected by western blot and normalized to GAPDH. Results are shown as mean±standard deviation from three independent experiments. **p

    Journal: Cancer Research and Treatment : Official Journal of Korean Cancer Association

    Article Title: Long Noncoding RNA HEIH Promotes Colorectal Cancer Tumorigenesis via Counteracting miR-939‒Mediated Transcriptional Repression of Bcl-xL

    doi: 10.4143/crt.2017.226

    Figure Lengend Snippet: Long noncoding RNA HEIH (lncRNA-HEIH) counteracts miR-939‒mediated transcriptional repression of Bcl-xL. (A) After transient transfection of lncRNA-HEIH or lncRNA-HEIH-mut into HT-29 cells, chromatin immunoprecipitation (ChIP) assays with nuclear factor κB (NF-κB) specific antibody were performed, and the retrieved DNA was detected by quantitative real-time polymerase chain reaction (qRT-PCR) and normalized to input. (B) After co-transfection of lncRNA-HEIH knockdown plasmid and miR-939 inhibitors into LoVo cells, ChIP assays with NF-κB specific antibody were performed, and the retrieved DNA was detected by qRT-PCR and normalized to input. (C) After co-transfection of lncRNA-HEIH or lncRNA-HEIH-mut and luciferase reporter containing Bcl-xL promoterinto HT-29 cells, the luciferase activities were measured. Results are shown as the relative ratio of firefly luciferase activity to Renilla luciferase activity. (D) After co-transfection of lncRNA-HEIH knockdown plasmid, miR-939 inhibitors, and luciferase reporter containing Bcl-xL promoter into LoVo cells, the luciferase activities were measured. Results are shown as in panel C. (E) After transient transfection of lncRNA-HEIH or lncRNA-HEIH-mut into HT-29 cells, Bcl-xL mRNA levels were detected by qRT-PCR and normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH). (F) After co-transfection of lncRNA-HEIH knockdown plasmid and miR-939 inhibitors into LoVo cells, Bcl-xL mRNA levels were detected by qRT-PCR and normalized to GAPDH. (G) After transient transfection of lncRNA-HEIH or lncRNA-HEIH-mut into HT-29 cells, Bcl-xL protein levels were detected by western blot and normalized to GAPDH. (H) After co-transfection of lncRNA-HEIH knockdown plasmid and miR-939 inhibitors into LoVo cells, Bcl-xL protein levels were detected by western blot and normalized to GAPDH. Results are shown as mean±standard deviation from three independent experiments. **p

    Article Snippet: lncRNA-HEIH and miR-939 binding sites mutated lncRNA-HEIH (lncRNA-HEIH-mut) were in vitro transcribed from pSPT19-HEIH, and pSPT19-HEIH-mut, respectively, using the SP6 RNA polymerase (Roche) and biotinlabeled with the Biotin RNA Labeling Mix (Roche) in accordance with the manufacturer’s protocols.

    Techniques: Transfection, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Cotransfection, Plasmid Preparation, Luciferase, Activity Assay, Western Blot, Standard Deviation

    Long noncoding RNA HEIH (lncRNA-HEIH) physically binds to miR-939 and counteracts the binding between miR-939 and nuclear factor κB (NF-κB). (A) Schematic outlining the predicted miR-939 binding sites on lncRNA-HEIH. The red nucleotides indict the seed sequences of miR-939. (B) HT-29 cell lysates were incubated with biotinylated lncRNA-HEIH or the miR-939 binding sites mutated lncRNA-HEIH (lncRNA-HEIH-mut); after pull-down, RNAs were retrieved and detected by quantitative real-time polymerase chain reaction (qRT-PCR), and normalized to input. (C) After transient transfection of lncRNA-HEIH or lncRNA-HEIH-mut into HT-29 cells, RNA immunoprecipitation assay (RIP) assays with NF-κB specific antibody were performed, and the retrieved RNA was detected by qRT-PCR and normalized to input. (D) After co-transfection of lncRNA-HEIH knockdown plasmid and miR-939 inhibitors into LoVo cells, RIP assays with NF-κB specific antibody were performed, and the retrieved RNA was detected by qRT-PCR and normalized to input. Results are shown as mean±standard deviation from three independent experiments. **p

    Journal: Cancer Research and Treatment : Official Journal of Korean Cancer Association

    Article Title: Long Noncoding RNA HEIH Promotes Colorectal Cancer Tumorigenesis via Counteracting miR-939‒Mediated Transcriptional Repression of Bcl-xL

    doi: 10.4143/crt.2017.226

    Figure Lengend Snippet: Long noncoding RNA HEIH (lncRNA-HEIH) physically binds to miR-939 and counteracts the binding between miR-939 and nuclear factor κB (NF-κB). (A) Schematic outlining the predicted miR-939 binding sites on lncRNA-HEIH. The red nucleotides indict the seed sequences of miR-939. (B) HT-29 cell lysates were incubated with biotinylated lncRNA-HEIH or the miR-939 binding sites mutated lncRNA-HEIH (lncRNA-HEIH-mut); after pull-down, RNAs were retrieved and detected by quantitative real-time polymerase chain reaction (qRT-PCR), and normalized to input. (C) After transient transfection of lncRNA-HEIH or lncRNA-HEIH-mut into HT-29 cells, RNA immunoprecipitation assay (RIP) assays with NF-κB specific antibody were performed, and the retrieved RNA was detected by qRT-PCR and normalized to input. (D) After co-transfection of lncRNA-HEIH knockdown plasmid and miR-939 inhibitors into LoVo cells, RIP assays with NF-κB specific antibody were performed, and the retrieved RNA was detected by qRT-PCR and normalized to input. Results are shown as mean±standard deviation from three independent experiments. **p

    Article Snippet: lncRNA-HEIH and miR-939 binding sites mutated lncRNA-HEIH (lncRNA-HEIH-mut) were in vitro transcribed from pSPT19-HEIH, and pSPT19-HEIH-mut, respectively, using the SP6 RNA polymerase (Roche) and biotinlabeled with the Biotin RNA Labeling Mix (Roche) in accordance with the manufacturer’s protocols.

    Techniques: Binding Assay, Incubation, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Transfection, Immunoprecipitation, Cotransfection, Plasmid Preparation, Standard Deviation

    Long noncoding RNA HEIH (lncRNA-HEIH) is up-regulated in colorectal cancer (CRC) and indicts poor prognosis of CRC patients. (A) The expression of lncRNA-HEIH in 84 paired CRC and adjacent normal mucosa was detected by quantitative real-time polymerase chain reaction (qRT-PCR) and normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH). ***p

    Journal: Cancer Research and Treatment : Official Journal of Korean Cancer Association

    Article Title: Long Noncoding RNA HEIH Promotes Colorectal Cancer Tumorigenesis via Counteracting miR-939‒Mediated Transcriptional Repression of Bcl-xL

    doi: 10.4143/crt.2017.226

    Figure Lengend Snippet: Long noncoding RNA HEIH (lncRNA-HEIH) is up-regulated in colorectal cancer (CRC) and indicts poor prognosis of CRC patients. (A) The expression of lncRNA-HEIH in 84 paired CRC and adjacent normal mucosa was detected by quantitative real-time polymerase chain reaction (qRT-PCR) and normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH). ***p

    Article Snippet: lncRNA-HEIH and miR-939 binding sites mutated lncRNA-HEIH (lncRNA-HEIH-mut) were in vitro transcribed from pSPT19-HEIH, and pSPT19-HEIH-mut, respectively, using the SP6 RNA polymerase (Roche) and biotinlabeled with the Biotin RNA Labeling Mix (Roche) in accordance with the manufacturer’s protocols.

    Techniques: Expressing, Real-time Polymerase Chain Reaction, Quantitative RT-PCR

    The mutation of miR-939 binding sites on long noncoding RNA HEIH (lncRNA-HEIH) abolished the effects of lncRNA-HEIH on colorectal cancer tumorigenesis. (A) The expression of lncRNA-HEIH in lncRNA-HEIH or lncRNA-HEIH-mut stably overexpressed and control HT-29 cells was detected by quantitative real-time polymerase chain reaction and normalized to glyceraldehyde 3-phosphate dehydrogenase. (B) Cell proliferation rate of lncRNA-HEIH or lncRNA-HEIH-mut stably overexpressed and control HT-29 cells were detected by the Cell Counting Kit-8 assays. (C) Proliferative cells of lncRNA-HEIH or lncRNA-HEIH-mut stably overexpressed and control HT-29 were labeled with ethynyl deoxyuridine (EdU). Red color indicts EdU-positive cells. Scale bars=100 μm. (D) The level of apoptosis in lncRNA-HEIH or lncRNA-HEIH-mut stably overexpressed and control HT-29 cells was detected by TdT-mediated dUTP nick end labeling (TUNEL) staining. Blue color indicts TUNEL-positive cells. Scale bars=100 μm. For A-D, results are shown as mean±standard deviation (SD) from three independent experiments. **p

    Journal: Cancer Research and Treatment : Official Journal of Korean Cancer Association

    Article Title: Long Noncoding RNA HEIH Promotes Colorectal Cancer Tumorigenesis via Counteracting miR-939‒Mediated Transcriptional Repression of Bcl-xL

    doi: 10.4143/crt.2017.226

    Figure Lengend Snippet: The mutation of miR-939 binding sites on long noncoding RNA HEIH (lncRNA-HEIH) abolished the effects of lncRNA-HEIH on colorectal cancer tumorigenesis. (A) The expression of lncRNA-HEIH in lncRNA-HEIH or lncRNA-HEIH-mut stably overexpressed and control HT-29 cells was detected by quantitative real-time polymerase chain reaction and normalized to glyceraldehyde 3-phosphate dehydrogenase. (B) Cell proliferation rate of lncRNA-HEIH or lncRNA-HEIH-mut stably overexpressed and control HT-29 cells were detected by the Cell Counting Kit-8 assays. (C) Proliferative cells of lncRNA-HEIH or lncRNA-HEIH-mut stably overexpressed and control HT-29 were labeled with ethynyl deoxyuridine (EdU). Red color indicts EdU-positive cells. Scale bars=100 μm. (D) The level of apoptosis in lncRNA-HEIH or lncRNA-HEIH-mut stably overexpressed and control HT-29 cells was detected by TdT-mediated dUTP nick end labeling (TUNEL) staining. Blue color indicts TUNEL-positive cells. Scale bars=100 μm. For A-D, results are shown as mean±standard deviation (SD) from three independent experiments. **p

    Article Snippet: lncRNA-HEIH and miR-939 binding sites mutated lncRNA-HEIH (lncRNA-HEIH-mut) were in vitro transcribed from pSPT19-HEIH, and pSPT19-HEIH-mut, respectively, using the SP6 RNA polymerase (Roche) and biotinlabeled with the Biotin RNA Labeling Mix (Roche) in accordance with the manufacturer’s protocols.

    Techniques: Mutagenesis, Binding Assay, Expressing, Stable Transfection, Real-time Polymerase Chain Reaction, Cell Counting, Labeling, End Labeling, TUNEL Assay, Staining, Standard Deviation

    Knockdown of long noncoding RNA HEIH (lncRNA-HEIH) inhibits colorectal cancer tumorigenesis. (A) The expression of lncRNA-HEIH in lncRNA-HEIH stably knocked-down and control LoVo cells was detected by quantitative real-time polymerase chain reaction and normalized to glyceraldehyde 3-phosphate dehydrogenase. (B) Cell proliferation rate of lncRNA-HEIH stably knocked-down and control LoVo cells were detected by the Cell Counting Kit-8 assays. (C) Proliferative cells of lncRNA-HEIH stably knocked-down and control LoVo were labeled with ethynyl deoxyuridine (EdU). Red color indicts EdU-positive cells. Scale bars=100 μm. (D) The level of apoptosis in lncRNA-HEIH stably knocked-down and control LoVo cells was detected by TdT-mediated dUTP nick end labeling (TUNEL) staining. Blue color indicts TUNEL-positive cells. Scale bars=100 μm. For A-D, results are shown as mean±standard deviation (SD) from three independent experiments. *p

    Journal: Cancer Research and Treatment : Official Journal of Korean Cancer Association

    Article Title: Long Noncoding RNA HEIH Promotes Colorectal Cancer Tumorigenesis via Counteracting miR-939‒Mediated Transcriptional Repression of Bcl-xL

    doi: 10.4143/crt.2017.226

    Figure Lengend Snippet: Knockdown of long noncoding RNA HEIH (lncRNA-HEIH) inhibits colorectal cancer tumorigenesis. (A) The expression of lncRNA-HEIH in lncRNA-HEIH stably knocked-down and control LoVo cells was detected by quantitative real-time polymerase chain reaction and normalized to glyceraldehyde 3-phosphate dehydrogenase. (B) Cell proliferation rate of lncRNA-HEIH stably knocked-down and control LoVo cells were detected by the Cell Counting Kit-8 assays. (C) Proliferative cells of lncRNA-HEIH stably knocked-down and control LoVo were labeled with ethynyl deoxyuridine (EdU). Red color indicts EdU-positive cells. Scale bars=100 μm. (D) The level of apoptosis in lncRNA-HEIH stably knocked-down and control LoVo cells was detected by TdT-mediated dUTP nick end labeling (TUNEL) staining. Blue color indicts TUNEL-positive cells. Scale bars=100 μm. For A-D, results are shown as mean±standard deviation (SD) from three independent experiments. *p

    Article Snippet: lncRNA-HEIH and miR-939 binding sites mutated lncRNA-HEIH (lncRNA-HEIH-mut) were in vitro transcribed from pSPT19-HEIH, and pSPT19-HEIH-mut, respectively, using the SP6 RNA polymerase (Roche) and biotinlabeled with the Biotin RNA Labeling Mix (Roche) in accordance with the manufacturer’s protocols.

    Techniques: Expressing, Stable Transfection, Real-time Polymerase Chain Reaction, Cell Counting, Labeling, End Labeling, TUNEL Assay, Staining, Standard Deviation

    Enhanced expression of long noncoding RNA HEIH (lncRNA-HEIH) promotes colorectal cancer tumorigenesis. (A) The expression of lncRNA-HEIH in lncRNA-HEIH stably overexpressed and control HT-29 cells was detected by quantitative real-time polymerase chain reaction and normalized to glyceraldehyde 3-phosphat e dehydrogenase. (B) Cell proliferation rate of lncRNA-HEIH stably overexpressed and control HT-29 cells were detected by the Cell Counting Kit-8 assays. (C) Proliferative cells of lncRNA-HEIH stably overexpressed and control HT-29 were labeled with ethynyl deoxyuridine (EdU). Red color indicts EdU-positive cells. Scale bars=100 μm. (D) The level of apoptosis in lncRNA-HEIH stably overexpressed and control HT-29 cells was detected by TdT-mediated dUTP nick end labeling (TUNEL) staining. Blue color indicts TUNEL-positive cells. Scale bars=100 μm. For A-D, results are shown as mean±standard deviation (SD). from three independent experiments. **p

    Journal: Cancer Research and Treatment : Official Journal of Korean Cancer Association

    Article Title: Long Noncoding RNA HEIH Promotes Colorectal Cancer Tumorigenesis via Counteracting miR-939‒Mediated Transcriptional Repression of Bcl-xL

    doi: 10.4143/crt.2017.226

    Figure Lengend Snippet: Enhanced expression of long noncoding RNA HEIH (lncRNA-HEIH) promotes colorectal cancer tumorigenesis. (A) The expression of lncRNA-HEIH in lncRNA-HEIH stably overexpressed and control HT-29 cells was detected by quantitative real-time polymerase chain reaction and normalized to glyceraldehyde 3-phosphat e dehydrogenase. (B) Cell proliferation rate of lncRNA-HEIH stably overexpressed and control HT-29 cells were detected by the Cell Counting Kit-8 assays. (C) Proliferative cells of lncRNA-HEIH stably overexpressed and control HT-29 were labeled with ethynyl deoxyuridine (EdU). Red color indicts EdU-positive cells. Scale bars=100 μm. (D) The level of apoptosis in lncRNA-HEIH stably overexpressed and control HT-29 cells was detected by TdT-mediated dUTP nick end labeling (TUNEL) staining. Blue color indicts TUNEL-positive cells. Scale bars=100 μm. For A-D, results are shown as mean±standard deviation (SD). from three independent experiments. **p

    Article Snippet: lncRNA-HEIH and miR-939 binding sites mutated lncRNA-HEIH (lncRNA-HEIH-mut) were in vitro transcribed from pSPT19-HEIH, and pSPT19-HEIH-mut, respectively, using the SP6 RNA polymerase (Roche) and biotinlabeled with the Biotin RNA Labeling Mix (Roche) in accordance with the manufacturer’s protocols.

    Techniques: Expressing, Stable Transfection, Real-time Polymerase Chain Reaction, Cell Counting, Labeling, End Labeling, TUNEL Assay, Staining, Standard Deviation

    lnc RNA ‐ HIT promotes migration and invasion via regulation of ZEB 1 expression. (A) ZEB 1 protein expression in A549 control and lnc RNA ‐ HIT knockdown cells expressing control and ZEB 1. (B) Migration of A549 cells expressing control and lnc RNA ‐ HIT sh RNA s with and without ZEB 1. (C) Invasion of A549 cells expressing control and lnc RNA ‐ HIT sh RNA s with and without ZEB 1. Data are shown as mean ± SD. * P

    Journal: Cancer Medicine

    Article Title: Upregulation of LncRNA‐HIT promotes migration and invasion of non‐small cell lung cancer cells by association with ZEB1

    doi: 10.1002/cam4.948

    Figure Lengend Snippet: lnc RNA ‐ HIT promotes migration and invasion via regulation of ZEB 1 expression. (A) ZEB 1 protein expression in A549 control and lnc RNA ‐ HIT knockdown cells expressing control and ZEB 1. (B) Migration of A549 cells expressing control and lnc RNA ‐ HIT sh RNA s with and without ZEB 1. (C) Invasion of A549 cells expressing control and lnc RNA ‐ HIT sh RNA s with and without ZEB 1. Data are shown as mean ± SD. * P

    Article Snippet: Briefly, biotin‐labeled lncRNA‐HIT were in vitro transcribed with the Biotin RNA Labeling Mix (Roche, Cambridge, UK) and T7 RNA polymerase (Roche), treated with RNase‐free DNase I (Roche), and purified with the RNeasy Mini Kit (Qiagen, Dusseldolf, Germany).

    Techniques: Migration, Expressing

    lnc RNA ‐ HIT is upregulated in NSCLC tissues and cell lines and correlates with prognosis.(A) The relative expression of lnc RNA ‐ HIT in different NSCLC cell lines and normal lung epithelial cells ( NLEC ). (B) The relative expression of lnc RNA ‐ HIT in NSCLC tumor tissues (T) compared to matched adjacent nontumor tissues ( NT ) from 60 NSCLC patients. (C) Kaplan–Meier analyses of the correlations between lnc RNA ‐ HIT expression level and overall survival of 60 patients with NSCLC . The cut point of high and low Lnc RNA ‐ HIT expressers is the median. Data are shown as mean ± SD. * P

    Journal: Cancer Medicine

    Article Title: Upregulation of LncRNA‐HIT promotes migration and invasion of non‐small cell lung cancer cells by association with ZEB1

    doi: 10.1002/cam4.948

    Figure Lengend Snippet: lnc RNA ‐ HIT is upregulated in NSCLC tissues and cell lines and correlates with prognosis.(A) The relative expression of lnc RNA ‐ HIT in different NSCLC cell lines and normal lung epithelial cells ( NLEC ). (B) The relative expression of lnc RNA ‐ HIT in NSCLC tumor tissues (T) compared to matched adjacent nontumor tissues ( NT ) from 60 NSCLC patients. (C) Kaplan–Meier analyses of the correlations between lnc RNA ‐ HIT expression level and overall survival of 60 patients with NSCLC . The cut point of high and low Lnc RNA ‐ HIT expressers is the median. Data are shown as mean ± SD. * P

    Article Snippet: Briefly, biotin‐labeled lncRNA‐HIT were in vitro transcribed with the Biotin RNA Labeling Mix (Roche, Cambridge, UK) and T7 RNA polymerase (Roche), treated with RNase‐free DNase I (Roche), and purified with the RNeasy Mini Kit (Qiagen, Dusseldolf, Germany).

    Techniques: Expressing

    Silence of lnc RNA ‐ HIT inhibits the migration and invasion of NSCLC cells. (A) The relative expression of lnc RNA ‐ HIT in control and lnc RNA ‐ HIT knockdown cells. (B) The EMT markers detected by western blot in control and lnc RNA ‐ HIT knockdown cells. (C) Knockdown of lnc RNA ‐ HIT suppressed migration in NSCLC cells. (D) Knockdown of lnc RNA ‐ HIT suppressed invasion in NSCLC cells. Data are shown as mean ± SD . * P

    Journal: Cancer Medicine

    Article Title: Upregulation of LncRNA‐HIT promotes migration and invasion of non‐small cell lung cancer cells by association with ZEB1

    doi: 10.1002/cam4.948

    Figure Lengend Snippet: Silence of lnc RNA ‐ HIT inhibits the migration and invasion of NSCLC cells. (A) The relative expression of lnc RNA ‐ HIT in control and lnc RNA ‐ HIT knockdown cells. (B) The EMT markers detected by western blot in control and lnc RNA ‐ HIT knockdown cells. (C) Knockdown of lnc RNA ‐ HIT suppressed migration in NSCLC cells. (D) Knockdown of lnc RNA ‐ HIT suppressed invasion in NSCLC cells. Data are shown as mean ± SD . * P

    Article Snippet: Briefly, biotin‐labeled lncRNA‐HIT were in vitro transcribed with the Biotin RNA Labeling Mix (Roche, Cambridge, UK) and T7 RNA polymerase (Roche), treated with RNase‐free DNase I (Roche), and purified with the RNeasy Mini Kit (Qiagen, Dusseldolf, Germany).

    Techniques: Migration, Expressing, Western Blot

    lnc RNA ‐ HIT associates with ZEB 1. (A) RIP assays for lnc RNA ‐ HIT were performed using indicated antibodies. (B) Deletion mapping of ZEB 1‐binding domain in lnc RNA ‐ HIT (Up). Western blot for ZEB 1 in samples pulled down by different lnc RNA ‐ HIT fragments (Down). Antisense lnc RNA ‐ HIT was used as a negative control. (C) The relative ZEB 1 mRNA expression in control and lnc RNA ‐ HIT knockdown cells. (D) The ZEB 1 protein level in control and lnc RNA ‐ HIT knockdown cells. (E) The relative ZEB 1 mRNA expression in control and lnc RNA ‐ HIT overexpressed cells. (F) The ZEB 1 protein level in control and lnc RNA ‐ HIT overexpressed cells. (G) The stability of ZEB 1 protein over time was measured by western blot relative to time 0 after blocking new protein synthesis with 100 mg/ml CHX in control and lnc RNA ‐ HIT knockdown A549 cells. (H) ZEB 1 protein expression in control and lnc RNA ‐ HIT knockdown A549 cells treated with vehicle control ( DMSO ) or 10 μ mol/L MG 132 for 12 h. (I) The occupancy of ZEB 1 in the promoter of CDH 1 was measured by ZEB 1 Ch IP assay followed by qRT ‐ PCR in control and lnc RNA ‐ HIT knockdown A549 cells. (J) The occupancy of ZEB 1 in the promoter of CDH 1 was measured by ZEB 1 Ch IP assay followed by qRT ‐ PCR in control and lnc RNA ‐ HIT overexpressed A549 cells. Data are shown as mean ± SD. * P

    Journal: Cancer Medicine

    Article Title: Upregulation of LncRNA‐HIT promotes migration and invasion of non‐small cell lung cancer cells by association with ZEB1

    doi: 10.1002/cam4.948

    Figure Lengend Snippet: lnc RNA ‐ HIT associates with ZEB 1. (A) RIP assays for lnc RNA ‐ HIT were performed using indicated antibodies. (B) Deletion mapping of ZEB 1‐binding domain in lnc RNA ‐ HIT (Up). Western blot for ZEB 1 in samples pulled down by different lnc RNA ‐ HIT fragments (Down). Antisense lnc RNA ‐ HIT was used as a negative control. (C) The relative ZEB 1 mRNA expression in control and lnc RNA ‐ HIT knockdown cells. (D) The ZEB 1 protein level in control and lnc RNA ‐ HIT knockdown cells. (E) The relative ZEB 1 mRNA expression in control and lnc RNA ‐ HIT overexpressed cells. (F) The ZEB 1 protein level in control and lnc RNA ‐ HIT overexpressed cells. (G) The stability of ZEB 1 protein over time was measured by western blot relative to time 0 after blocking new protein synthesis with 100 mg/ml CHX in control and lnc RNA ‐ HIT knockdown A549 cells. (H) ZEB 1 protein expression in control and lnc RNA ‐ HIT knockdown A549 cells treated with vehicle control ( DMSO ) or 10 μ mol/L MG 132 for 12 h. (I) The occupancy of ZEB 1 in the promoter of CDH 1 was measured by ZEB 1 Ch IP assay followed by qRT ‐ PCR in control and lnc RNA ‐ HIT knockdown A549 cells. (J) The occupancy of ZEB 1 in the promoter of CDH 1 was measured by ZEB 1 Ch IP assay followed by qRT ‐ PCR in control and lnc RNA ‐ HIT overexpressed A549 cells. Data are shown as mean ± SD. * P

    Article Snippet: Briefly, biotin‐labeled lncRNA‐HIT were in vitro transcribed with the Biotin RNA Labeling Mix (Roche, Cambridge, UK) and T7 RNA polymerase (Roche), treated with RNase‐free DNase I (Roche), and purified with the RNeasy Mini Kit (Qiagen, Dusseldolf, Germany).

    Techniques: Binding Assay, Western Blot, Negative Control, Expressing, Blocking Assay, Quantitative RT-PCR

    Overexpression of lnc RNA ‐ HIT promotes the migration and invasion of NSCLC cells. (A) The relative expression of lnc RNA ‐ HIT in control and lnc RNA ‐ HIT overexpressed cells. (B) The EMT markers detected by western blot in control and lnc RNA ‐ HIT overexpressed cells. (C) Overexpression of lnc RNA ‐ HIT promoted migration in NSCLC cells. (D) Overexpression of lnc RNA ‐ HIT promoted invasion in NSCLC cells. Data are shown as mean ± SD. * P

    Journal: Cancer Medicine

    Article Title: Upregulation of LncRNA‐HIT promotes migration and invasion of non‐small cell lung cancer cells by association with ZEB1

    doi: 10.1002/cam4.948

    Figure Lengend Snippet: Overexpression of lnc RNA ‐ HIT promotes the migration and invasion of NSCLC cells. (A) The relative expression of lnc RNA ‐ HIT in control and lnc RNA ‐ HIT overexpressed cells. (B) The EMT markers detected by western blot in control and lnc RNA ‐ HIT overexpressed cells. (C) Overexpression of lnc RNA ‐ HIT promoted migration in NSCLC cells. (D) Overexpression of lnc RNA ‐ HIT promoted invasion in NSCLC cells. Data are shown as mean ± SD. * P

    Article Snippet: Briefly, biotin‐labeled lncRNA‐HIT were in vitro transcribed with the Biotin RNA Labeling Mix (Roche, Cambridge, UK) and T7 RNA polymerase (Roche), treated with RNase‐free DNase I (Roche), and purified with the RNeasy Mini Kit (Qiagen, Dusseldolf, Germany).

    Techniques: Over Expression, Migration, Expressing, Western Blot

    OCC-1 plays a tumor suppressive role in CRC. ( A ) Analysis of two independent gene expression data sets of clinical samples revealed that OCC-1 expression was significantly downregulated in tumors with advanced tumor (T) stage in CRC. Tis, carcinoma in situ . ( B ) RNA FISH revealed that OCC-1 is predominantly localized in the cytoplasm of Caco-2 and HCT116 cells (Scale bar, 20 μm). ( C ) RT-qPCR analysis showed that OCC-1 RNA level was efficiently reduced by two independent shRNAs in Caco-2 and HCT116 cells. shCTRL, a non-target shRNA control. ( D ) CCK-8 assay demonstrated that cell proliferation was significantly increased after knockdown of OCC-1 in Caco-2 and HCT116 cells. ( E ) Ki67 staining showed that the proportions of Ki67-positive proliferating cells were significantly higher in OCC-1 knockdown cells compared to their control cells. ( F ) FACS analysis revealed the cell cycle distribution of Caco-2 cells after OCC-1 knockdown. ( G ) Colony formation assay showed that OCC-1 knockdown cells formed more colonies than the control cells. Images are the result of one representative experiment. Data are presented as mean± standard deviation of three independent experiments. ** P

    Journal: Nucleic Acids Research

    Article Title: Long noncoding RNA OCC-1 suppresses cell growth through destabilizing HuR protein in colorectal cancer

    doi: 10.1093/nar/gky214

    Figure Lengend Snippet: OCC-1 plays a tumor suppressive role in CRC. ( A ) Analysis of two independent gene expression data sets of clinical samples revealed that OCC-1 expression was significantly downregulated in tumors with advanced tumor (T) stage in CRC. Tis, carcinoma in situ . ( B ) RNA FISH revealed that OCC-1 is predominantly localized in the cytoplasm of Caco-2 and HCT116 cells (Scale bar, 20 μm). ( C ) RT-qPCR analysis showed that OCC-1 RNA level was efficiently reduced by two independent shRNAs in Caco-2 and HCT116 cells. shCTRL, a non-target shRNA control. ( D ) CCK-8 assay demonstrated that cell proliferation was significantly increased after knockdown of OCC-1 in Caco-2 and HCT116 cells. ( E ) Ki67 staining showed that the proportions of Ki67-positive proliferating cells were significantly higher in OCC-1 knockdown cells compared to their control cells. ( F ) FACS analysis revealed the cell cycle distribution of Caco-2 cells after OCC-1 knockdown. ( G ) Colony formation assay showed that OCC-1 knockdown cells formed more colonies than the control cells. Images are the result of one representative experiment. Data are presented as mean± standard deviation of three independent experiments. ** P

    Article Snippet: Briefly, the relative long 918-nucleotide OCC-1 3′UTR RNA was synthesized and labeled with Biotin RNA Labeling Mix (Roche) by in vitro transcription.

    Techniques: Expressing, In Situ, Fluorescence In Situ Hybridization, Quantitative RT-PCR, shRNA, CCK-8 Assay, Staining, FACS, Colony Assay, Standard Deviation

    OCC-1 RNA associates with HuR protein. ( A ) RNA pull-down assay followed by western blot confirmed HuR as a protein partner binding specifically to OCC-1 3′UTR. EGFP RNA was used as a RNA control. ACTB and GAPDH are protein controls. ( B ) RIP confirmed the association between OCC-1 and HuR in Caco-2 cells. GAPDH mRNA was used as a non-HuR target control. ( C ) Schematic of the 3′UTRs of OCC-1 orthologues showing the HuR-binding motifs. The HuR-binding sites (HuR CLIP site) identified previously by a CLIP experiment in human OCC-1 3′UTR were also indicated. ( D ) Venn diagram demonstrating the significant enrichment of OCC-1-repressed genes in the set of HuR targets determined by the previous CLIP experiment. About 74% of OCC-1-repressed genes identified by microarray analysis were also HuR targets. ( E ) RIP assay revealed that the mRNAs of the six selected OCC-1-repressed genes also interact with HuR in Caco-2 cells. ( F ) The mRNA and protein level of HuR were markedly reduced by a HuR-targeting shRNA as determined by RT-qPCR ( left ) and western blot ( right ). The density of protein bands was measured by Image J software and the relative level of HuR protein was calculated after normalizing to ACTB protein. ( G ) RT-qPCR analysis revealed that OCC-1 and all six selected OCC-1-repressed genes were downregulated by HuR knockdown. Images are the result of one representative experiment. Data are presented as mean± standard deviation of three independent experiments. ** P

    Journal: Nucleic Acids Research

    Article Title: Long noncoding RNA OCC-1 suppresses cell growth through destabilizing HuR protein in colorectal cancer

    doi: 10.1093/nar/gky214

    Figure Lengend Snippet: OCC-1 RNA associates with HuR protein. ( A ) RNA pull-down assay followed by western blot confirmed HuR as a protein partner binding specifically to OCC-1 3′UTR. EGFP RNA was used as a RNA control. ACTB and GAPDH are protein controls. ( B ) RIP confirmed the association between OCC-1 and HuR in Caco-2 cells. GAPDH mRNA was used as a non-HuR target control. ( C ) Schematic of the 3′UTRs of OCC-1 orthologues showing the HuR-binding motifs. The HuR-binding sites (HuR CLIP site) identified previously by a CLIP experiment in human OCC-1 3′UTR were also indicated. ( D ) Venn diagram demonstrating the significant enrichment of OCC-1-repressed genes in the set of HuR targets determined by the previous CLIP experiment. About 74% of OCC-1-repressed genes identified by microarray analysis were also HuR targets. ( E ) RIP assay revealed that the mRNAs of the six selected OCC-1-repressed genes also interact with HuR in Caco-2 cells. ( F ) The mRNA and protein level of HuR were markedly reduced by a HuR-targeting shRNA as determined by RT-qPCR ( left ) and western blot ( right ). The density of protein bands was measured by Image J software and the relative level of HuR protein was calculated after normalizing to ACTB protein. ( G ) RT-qPCR analysis revealed that OCC-1 and all six selected OCC-1-repressed genes were downregulated by HuR knockdown. Images are the result of one representative experiment. Data are presented as mean± standard deviation of three independent experiments. ** P

    Article Snippet: Briefly, the relative long 918-nucleotide OCC-1 3′UTR RNA was synthesized and labeled with Biotin RNA Labeling Mix (Roche) by in vitro transcription.

    Techniques: Pull Down Assay, Western Blot, Binding Assay, Cross-linking Immunoprecipitation, Microarray, shRNA, Quantitative RT-PCR, Software, Standard Deviation

    Overxpression of OCC-1 RNA suppressed cell growth in OCC-1 knockdown cells. ( A ) RT-qPCR analysis showed that the shOCC-1-1-resistant forms of OCC-1 FL and FS RNA (FL + and FS + ) were successfully overexpressed in shOCC-1-1 Caco-2 cells. EV, empty vector. ( B ) CCK-8 assay demonstrated that cell proliferation was significantly suppressed by the expression of OCC-1 FL + and FS + RNA but not by the ORF in shOCC-1-1 Caco-2 cells. The fold changes of absorbances at day 4 relative to day 1 were shown. ( C ) FACS analysis revealed the cell cycle distribution of shOCC-1-1 Caco-2 cells after re-introduction of OCC-1 RNAs. ( D ) Colony formation assay showed that the shOCC-1-1 Caco-2 cells expressing OCC-1 FL + and FS + RNA formed less colonies than the shOCC-1-1 cells expressing the ORF and the EV control. Images are the result of one representative experiment. Data are presented as mean± standard deviation of three independent experiments. * P

    Journal: Nucleic Acids Research

    Article Title: Long noncoding RNA OCC-1 suppresses cell growth through destabilizing HuR protein in colorectal cancer

    doi: 10.1093/nar/gky214

    Figure Lengend Snippet: Overxpression of OCC-1 RNA suppressed cell growth in OCC-1 knockdown cells. ( A ) RT-qPCR analysis showed that the shOCC-1-1-resistant forms of OCC-1 FL and FS RNA (FL + and FS + ) were successfully overexpressed in shOCC-1-1 Caco-2 cells. EV, empty vector. ( B ) CCK-8 assay demonstrated that cell proliferation was significantly suppressed by the expression of OCC-1 FL + and FS + RNA but not by the ORF in shOCC-1-1 Caco-2 cells. The fold changes of absorbances at day 4 relative to day 1 were shown. ( C ) FACS analysis revealed the cell cycle distribution of shOCC-1-1 Caco-2 cells after re-introduction of OCC-1 RNAs. ( D ) Colony formation assay showed that the shOCC-1-1 Caco-2 cells expressing OCC-1 FL + and FS + RNA formed less colonies than the shOCC-1-1 cells expressing the ORF and the EV control. Images are the result of one representative experiment. Data are presented as mean± standard deviation of three independent experiments. * P

    Article Snippet: Briefly, the relative long 918-nucleotide OCC-1 3′UTR RNA was synthesized and labeled with Biotin RNA Labeling Mix (Roche) by in vitro transcription.

    Techniques: Quantitative RT-PCR, Plasmid Preparation, CCK-8 Assay, Expressing, FACS, Colony Assay, Standard Deviation

    OCC-1 promotes the ubiquitination and degradation of HuR by enhancing its binding to the ubiquitin E3 ligase β-TrCP1. ( A ) Western blot analysis showed that knockdown of OCC-1 led to the increase of HuR in Caco-2 cells. ( B ) Overxpression of OCC-1 FL + and FS + RNA but not the ORF reduced HuR level in shOCC-1-1 Caco-2 cells. EV, empty vector. ( C ) Cells were treated with CHX to inhibit protein synthesis and the remaining of HuR was measured by western blot in Caco-2 cells after OCC-1 knockdown. The percent of remaining HuR in the CHX treated cells (+) relative to the control cells treated with DMSO (–) was indicated. ( D ) Western blot analysis of HuR in the OCC-1 knockdown cells treated with MG132 (+) or DMSO (–). The density of protein bands was measured by Image J software and the relative level of HuR protein was calculated after normalizing to ACTB protein. ( E ) The OCC-1 knockdown cells were co-transfected with a plasmid expressing a HA-tagged ubiquitin (HA-Ub) and a plasmid expressing a FLAG-tagged HuR (FLAG-HuR). After MG132 treatment, cell lysates were prepared and subjected to IP using an anti-HA antibody. The ubiquitinated FLAG-HuR was further detected by western blot using an anti-FLAG antibody. ( F ) Co-IP experiments showed that the interaction between HuR and the ubiquitin E3 ligase β-TrCP1 was attenuated after OCC-1 knockdown in Caco-2 cells.

    Journal: Nucleic Acids Research

    Article Title: Long noncoding RNA OCC-1 suppresses cell growth through destabilizing HuR protein in colorectal cancer

    doi: 10.1093/nar/gky214

    Figure Lengend Snippet: OCC-1 promotes the ubiquitination and degradation of HuR by enhancing its binding to the ubiquitin E3 ligase β-TrCP1. ( A ) Western blot analysis showed that knockdown of OCC-1 led to the increase of HuR in Caco-2 cells. ( B ) Overxpression of OCC-1 FL + and FS + RNA but not the ORF reduced HuR level in shOCC-1-1 Caco-2 cells. EV, empty vector. ( C ) Cells were treated with CHX to inhibit protein synthesis and the remaining of HuR was measured by western blot in Caco-2 cells after OCC-1 knockdown. The percent of remaining HuR in the CHX treated cells (+) relative to the control cells treated with DMSO (–) was indicated. ( D ) Western blot analysis of HuR in the OCC-1 knockdown cells treated with MG132 (+) or DMSO (–). The density of protein bands was measured by Image J software and the relative level of HuR protein was calculated after normalizing to ACTB protein. ( E ) The OCC-1 knockdown cells were co-transfected with a plasmid expressing a HA-tagged ubiquitin (HA-Ub) and a plasmid expressing a FLAG-tagged HuR (FLAG-HuR). After MG132 treatment, cell lysates were prepared and subjected to IP using an anti-HA antibody. The ubiquitinated FLAG-HuR was further detected by western blot using an anti-FLAG antibody. ( F ) Co-IP experiments showed that the interaction between HuR and the ubiquitin E3 ligase β-TrCP1 was attenuated after OCC-1 knockdown in Caco-2 cells.

    Article Snippet: Briefly, the relative long 918-nucleotide OCC-1 3′UTR RNA was synthesized and labeled with Biotin RNA Labeling Mix (Roche) by in vitro transcription.

    Techniques: Binding Assay, Western Blot, Plasmid Preparation, Software, Transfection, Expressing, Hemagglutination Assay, Co-Immunoprecipitation Assay