Structured Review

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Western blot analysis of the protein expressions of Notch, CTNNB1, Bax, <t>Bcl-2,</t> BIM, Hes1, Runx2, and osteocalcin in each transfected group ( A ) Relative protein expression of Notch, CTNNB1, Bax, Bcl-2, BIM, Hes1, Runx2, and osteocalcin in each group. ( B ) Protein bands of Notch, CTNNB1, Bax, Bcl-2, BIM, Hes1, Runx2, and osteocalcin by Western blot analysis; * P
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Images

1) Product Images from "Up-regulation of microRNA-340 promotes osteosarcoma cell apoptosis while suppressing proliferation, migration, and invasion by inactivating the CTNNB1-mediated Notch signaling pathway"

Article Title: Up-regulation of microRNA-340 promotes osteosarcoma cell apoptosis while suppressing proliferation, migration, and invasion by inactivating the CTNNB1-mediated Notch signaling pathway

Journal: Bioscience Reports

doi: 10.1042/BSR20171615

Western blot analysis of the protein expressions of Notch, CTNNB1, Bax, Bcl-2, BIM, Hes1, Runx2, and osteocalcin in each transfected group ( A ) Relative protein expression of Notch, CTNNB1, Bax, Bcl-2, BIM, Hes1, Runx2, and osteocalcin in each group. ( B ) Protein bands of Notch, CTNNB1, Bax, Bcl-2, BIM, Hes1, Runx2, and osteocalcin by Western blot analysis; * P
Figure Legend Snippet: Western blot analysis of the protein expressions of Notch, CTNNB1, Bax, Bcl-2, BIM, Hes1, Runx2, and osteocalcin in each transfected group ( A ) Relative protein expression of Notch, CTNNB1, Bax, Bcl-2, BIM, Hes1, Runx2, and osteocalcin in each group. ( B ) Protein bands of Notch, CTNNB1, Bax, Bcl-2, BIM, Hes1, Runx2, and osteocalcin by Western blot analysis; * P

Techniques Used: Western Blot, Transfection, Expressing

RT-qPCR detection of miR-340 expression and relative mRNA expression of Notch, CTNNB1, Bax, Bcl-2, BIM, Hes1, Runx2, and osteocalcin in each transfected group * P
Figure Legend Snippet: RT-qPCR detection of miR-340 expression and relative mRNA expression of Notch, CTNNB1, Bax, Bcl-2, BIM, Hes1, Runx2, and osteocalcin in each transfected group * P

Techniques Used: Quantitative RT-PCR, Expressing, Transfection

Immunohistochemistry and positive expression rate of CTNNB1 and Bcl-2 protein in OS and normal bone tissue ( A ) Immunohistochemical images (×200) of CTNNB1 in normal bone and OS tissues. ( B ) Positive expression rate of CTNNB1 in normal bone and OS tissues. ( C ) Immunohistochemical images (×200) of Bcl-2 in normal bone and OS tissues. ( D ) Positive expression rate of Bcl-2 in normal bone and OS tissues; * P
Figure Legend Snippet: Immunohistochemistry and positive expression rate of CTNNB1 and Bcl-2 protein in OS and normal bone tissue ( A ) Immunohistochemical images (×200) of CTNNB1 in normal bone and OS tissues. ( B ) Positive expression rate of CTNNB1 in normal bone and OS tissues. ( C ) Immunohistochemical images (×200) of Bcl-2 in normal bone and OS tissues. ( D ) Positive expression rate of Bcl-2 in normal bone and OS tissues; * P

Techniques Used: Immunohistochemistry, Expressing

RT-qPCR detection of miR-340 expression and relative mRNA expression of Notch, CTNNB1, Bax, Bcl-2, BIM, Hes1, Runx2, and osteocalcin in OS and normal bone tissues * P
Figure Legend Snippet: RT-qPCR detection of miR-340 expression and relative mRNA expression of Notch, CTNNB1, Bax, Bcl-2, BIM, Hes1, Runx2, and osteocalcin in OS and normal bone tissues * P

Techniques Used: Quantitative RT-PCR, Expressing

Western blot analysis of the relative protein expression of Notch, CTNNB1, Bax, Bcl-2, BIM, Hes1, Runx2, and osteocalcin in OS and normal bone tissues ( A ) Relative protein expression of Notch, CTNNB1, Bax, Bcl-2, BIM, Hes1, Runx2, and osteocalcin. ( B ) Protein bands of Notch, CTNNB1, Bax, Bcl-2, BIM, Hes1, Runx2, and osteocalcin by Western blot analysis; * P
Figure Legend Snippet: Western blot analysis of the relative protein expression of Notch, CTNNB1, Bax, Bcl-2, BIM, Hes1, Runx2, and osteocalcin in OS and normal bone tissues ( A ) Relative protein expression of Notch, CTNNB1, Bax, Bcl-2, BIM, Hes1, Runx2, and osteocalcin. ( B ) Protein bands of Notch, CTNNB1, Bax, Bcl-2, BIM, Hes1, Runx2, and osteocalcin by Western blot analysis; * P

Techniques Used: Western Blot, Expressing

2) Product Images from "Arabidopsis EMB1990 Encoding a Plastid-Targeted YlmG Protein Is Required for Chloroplast Biogenesis and Embryo Development"

Article Title: Arabidopsis EMB1990 Encoding a Plastid-Targeted YlmG Protein Is Required for Chloroplast Biogenesis and Embryo Development

Journal: Frontiers in Plant Science

doi: 10.3389/fpls.2018.00181

Expression pattern of EMB1990/YLMG1-1 gene in Arabidopsis tissues and organs. (A) Expression levels of EMB1990/YLMG1-1 genes in different tissues by qPCR assay. R, root; S, stem; L, leave; Sl, seedling; In, inflorescence; F, flower; 1Si, 1 DAP silique; 2Si, 2 DAP silique; 3Si, 3 DAP silique; 4Si, 4 DAP silique; 5Si, 5 DAP silique; 6Si, 6 DAP silique; 7Si, 7 DAP silique. (B–F) GUS activity in pYLMG1-1::GUS transgenic plants. (B) Flower; (C) inflorescence; (D) 7 DAG seedling; (E) 14 DAG seedling; (F) rosette leave. Scale bars = 2mm. (G–J) Fluorescence analysis of embryos at different stage from pYLMG1-1::YLMG1-1-Venus transgenic plants. (G) Globular stage; (H) Heart stage; (G) torpedo stage; (G) bent cotyledon stage. Scale bars = 20 μm.
Figure Legend Snippet: Expression pattern of EMB1990/YLMG1-1 gene in Arabidopsis tissues and organs. (A) Expression levels of EMB1990/YLMG1-1 genes in different tissues by qPCR assay. R, root; S, stem; L, leave; Sl, seedling; In, inflorescence; F, flower; 1Si, 1 DAP silique; 2Si, 2 DAP silique; 3Si, 3 DAP silique; 4Si, 4 DAP silique; 5Si, 5 DAP silique; 6Si, 6 DAP silique; 7Si, 7 DAP silique. (B–F) GUS activity in pYLMG1-1::GUS transgenic plants. (B) Flower; (C) inflorescence; (D) 7 DAG seedling; (E) 14 DAG seedling; (F) rosette leave. Scale bars = 2mm. (G–J) Fluorescence analysis of embryos at different stage from pYLMG1-1::YLMG1-1-Venus transgenic plants. (G) Globular stage; (H) Heart stage; (G) torpedo stage; (G) bent cotyledon stage. Scale bars = 20 μm.

Techniques Used: Expressing, Real-time Polymerase Chain Reaction, Activity Assay, Transgenic Assay, Fluorescence

Characterization and complementation of Arabidopsis emb1990 mutants. (A) Schematic diagrams of EMB1990/YLMG1-1 gene. The positions of T-DNA insertions in emb1990-1 and emb1990-2 mutants are shown in the schematic diagrams. Arrowheads indicate the positions of primers used for genotyping. (B–F) Seed development in siliques of wild-type, mutants, and complemented plants. White arrows highlight the aborted white ovules, and the siliques were placed as morphological apical to basal from left to right. Bars = 1 mm.
Figure Legend Snippet: Characterization and complementation of Arabidopsis emb1990 mutants. (A) Schematic diagrams of EMB1990/YLMG1-1 gene. The positions of T-DNA insertions in emb1990-1 and emb1990-2 mutants are shown in the schematic diagrams. Arrowheads indicate the positions of primers used for genotyping. (B–F) Seed development in siliques of wild-type, mutants, and complemented plants. White arrows highlight the aborted white ovules, and the siliques were placed as morphological apical to basal from left to right. Bars = 1 mm.

Techniques Used:

3) Product Images from "Circular RNA AKT3 upregulates PIK3R1 to enhance cisplatin resistance in gastric cancer via miR-198 suppression"

Article Title: Circular RNA AKT3 upregulates PIK3R1 to enhance cisplatin resistance in gastric cancer via miR-198 suppression

Journal: Molecular Cancer

doi: 10.1186/s12943-019-0969-3

circAKT3 exerts its function by sponging miR-198. a b Schematic illustration showing the overlap of the target miRNAs of circAKT3 predicted by miRanda, PITA and RNAhybrid. c d Lysates prepared from SGC7901CDDP and BGC823CDDP cells stably transfected with circAKT3 or vector were subjected to RNA pull-down and tested by RT-PCR (C) and RT-qPCR (D). The relative level of circAKT3 was normalized to the input. GAPDH served as a negative control. e f The relative levels of 11 miRNA candidates in SGC7901CDDP and BGC823CDDP lysates were detected by RT-qPCR. Multiple miRNAs were pulled down by circAKT3, and miR-198 was pulled down by circAKT3 in both cell lines. g Schematic illustration showing the 3′UTR of luciferase reporters containing the complete circAKT3 sequence (luc-wt) or the circAKT3 sequence with deletions of miR-198 (luc-m1-m8) binding sites. h Reporter assays showing the luciferase activity of luc-wt and luc-m1-m8 in 293 T cells cotransfected with miR-198 mimics or a scrambled oligonucleotide (control). i FISH showing the colocalization of circAKT3 and miR-198 in SGC7901CDDP cells. Nuclei were stained with DAPI. Scale bar, 10 μm. The results are presented as the mean ± SEM. * P
Figure Legend Snippet: circAKT3 exerts its function by sponging miR-198. a b Schematic illustration showing the overlap of the target miRNAs of circAKT3 predicted by miRanda, PITA and RNAhybrid. c d Lysates prepared from SGC7901CDDP and BGC823CDDP cells stably transfected with circAKT3 or vector were subjected to RNA pull-down and tested by RT-PCR (C) and RT-qPCR (D). The relative level of circAKT3 was normalized to the input. GAPDH served as a negative control. e f The relative levels of 11 miRNA candidates in SGC7901CDDP and BGC823CDDP lysates were detected by RT-qPCR. Multiple miRNAs were pulled down by circAKT3, and miR-198 was pulled down by circAKT3 in both cell lines. g Schematic illustration showing the 3′UTR of luciferase reporters containing the complete circAKT3 sequence (luc-wt) or the circAKT3 sequence with deletions of miR-198 (luc-m1-m8) binding sites. h Reporter assays showing the luciferase activity of luc-wt and luc-m1-m8 in 293 T cells cotransfected with miR-198 mimics or a scrambled oligonucleotide (control). i FISH showing the colocalization of circAKT3 and miR-198 in SGC7901CDDP cells. Nuclei were stained with DAPI. Scale bar, 10 μm. The results are presented as the mean ± SEM. * P

Techniques Used: Stable Transfection, Transfection, Plasmid Preparation, Reverse Transcription Polymerase Chain Reaction, Quantitative RT-PCR, Negative Control, Luciferase, Sequencing, Binding Assay, Activity Assay, Fluorescence In Situ Hybridization, Staining

circAKT3 expression is increased in CDDP-resistant GC cells and tissues. a Validated expression of 10 circRNAs in the tissues from 44 GC patients using RT-qPCR. b Expression levels of circAKT3 in CDDP-resistant and their matched sensitive parental cell lines (SGC7901CDDP, BGC823CDDP, SGC7901 and BGC823) normalized to GAPDH expression. c The existence of circAKT3 was validated by Sanger sequencing. The red arrow shows the “head-to-tail” splicing sites of circAKT3. d The existence of circAKT3 was validated in SGC7901CDDP and BGC823CDDP cell lines by RT-PCR. Divergent primers amplified circAKT3 in cDNA but not in genomic DNA (gDNA). GAPDH served as a negative control. e RNA from SGC7901CDDP and BGC823CDDP cells was treated with or without RNase R for RT-qPCR. The relative levels of circAKT3 and AKT3 mRNA were normalized to the values measured in the mock-treated cells. f Levels of small nucleolar RNA (U6, as a positive control for the nuclear fraction), GAPDH (positive control for cytoplasmic fraction), AKT3 mRNA and circRNAs from the nuclear and cytoplasmic fractions of SGC7901CDDP cells. g RNA stability of circular and linear transcripts of AKT3 and of 18S rRNA in SGC7901CDDP cells. h Representative images of RNA FISH of circAKT3 expression in SGC7901CDDP cells, which show that circAKT3 is predominantly localized to the cytoplasm. Nuclei were stained with DAPI. Scale bar, 10 μm. The results are presented as the mean ± SEM. * P
Figure Legend Snippet: circAKT3 expression is increased in CDDP-resistant GC cells and tissues. a Validated expression of 10 circRNAs in the tissues from 44 GC patients using RT-qPCR. b Expression levels of circAKT3 in CDDP-resistant and their matched sensitive parental cell lines (SGC7901CDDP, BGC823CDDP, SGC7901 and BGC823) normalized to GAPDH expression. c The existence of circAKT3 was validated by Sanger sequencing. The red arrow shows the “head-to-tail” splicing sites of circAKT3. d The existence of circAKT3 was validated in SGC7901CDDP and BGC823CDDP cell lines by RT-PCR. Divergent primers amplified circAKT3 in cDNA but not in genomic DNA (gDNA). GAPDH served as a negative control. e RNA from SGC7901CDDP and BGC823CDDP cells was treated with or without RNase R for RT-qPCR. The relative levels of circAKT3 and AKT3 mRNA were normalized to the values measured in the mock-treated cells. f Levels of small nucleolar RNA (U6, as a positive control for the nuclear fraction), GAPDH (positive control for cytoplasmic fraction), AKT3 mRNA and circRNAs from the nuclear and cytoplasmic fractions of SGC7901CDDP cells. g RNA stability of circular and linear transcripts of AKT3 and of 18S rRNA in SGC7901CDDP cells. h Representative images of RNA FISH of circAKT3 expression in SGC7901CDDP cells, which show that circAKT3 is predominantly localized to the cytoplasm. Nuclei were stained with DAPI. Scale bar, 10 μm. The results are presented as the mean ± SEM. * P

Techniques Used: Expressing, Quantitative RT-PCR, Sequencing, Reverse Transcription Polymerase Chain Reaction, Amplification, Negative Control, Positive Control, Fluorescence In Situ Hybridization, Staining

4) Product Images from "Long non-coding RNA CASC2 suppresses pulmonary artery smooth muscle cell proliferation and phenotypic switch in hypoxia-induced pulmonary hypertension"

Article Title: Long non-coding RNA CASC2 suppresses pulmonary artery smooth muscle cell proliferation and phenotypic switch in hypoxia-induced pulmonary hypertension

Journal: Respiratory Research

doi: 10.1186/s12931-019-1018-x

LncRNA CASC2 inhibited hypoxia-induced cell proliferation in rat pulmonary arterial tissues. ( a ) The mRNA expression of PCNA was detected by qRT-PCR in rats′ PAs tissues. ( b , c ) The protein expression of PCNA was detected by western blot analysis in rat pulmonary arterial tissues. ## p
Figure Legend Snippet: LncRNA CASC2 inhibited hypoxia-induced cell proliferation in rat pulmonary arterial tissues. ( a ) The mRNA expression of PCNA was detected by qRT-PCR in rats′ PAs tissues. ( b , c ) The protein expression of PCNA was detected by western blot analysis in rat pulmonary arterial tissues. ## p

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

Up-regulation of lncRNA CASC2 inhibited proliferation in hypoxia-induced PASMCs. ( a ) The expression of lncRNA CASC2 was detected by qRT-PCR in PASMCs. ( b ) Cell number of PASMCs was counted by CyQuant cell proliferation assay kit. ( c ) The mRNA expression of PCNA was detected by qRT-PCR in PASMCs. ( d ) The protein expression of PCNA was detected by western blot analysis in PASMCs. ( e , f ) Cell proliferation status of PASMCs was assessed by EdU staining assay. ### p
Figure Legend Snippet: Up-regulation of lncRNA CASC2 inhibited proliferation in hypoxia-induced PASMCs. ( a ) The expression of lncRNA CASC2 was detected by qRT-PCR in PASMCs. ( b ) Cell number of PASMCs was counted by CyQuant cell proliferation assay kit. ( c ) The mRNA expression of PCNA was detected by qRT-PCR in PASMCs. ( d ) The protein expression of PCNA was detected by western blot analysis in PASMCs. ( e , f ) Cell proliferation status of PASMCs was assessed by EdU staining assay. ### p

Techniques Used: Expressing, Quantitative RT-PCR, CyQUANT Assay, Proliferation Assay, Western Blot, Staining

LncRNA CASC2 was down-regulated in hypoxia-induced rats and reversed the increase of mPAP and progression of right ventricular hypertrophy caused by hypoxia. ( a ) The expression of lncRNA CASC2 was detected by qRT-PCR in rat pulmonary arterial tissues. ( b ) Systemic blood pressure (SBP) was assessed in normoxia and hypoxia-induced rats. ( c ) Mean pulmonary arterial pressure (mPAP) was detected in normoxia and hypoxia-induced rats. ( d ) The right ventricular hypertrophy index (RVI) was calculated in normoxia and hypoxia-induced rats. # p
Figure Legend Snippet: LncRNA CASC2 was down-regulated in hypoxia-induced rats and reversed the increase of mPAP and progression of right ventricular hypertrophy caused by hypoxia. ( a ) The expression of lncRNA CASC2 was detected by qRT-PCR in rat pulmonary arterial tissues. ( b ) Systemic blood pressure (SBP) was assessed in normoxia and hypoxia-induced rats. ( c ) Mean pulmonary arterial pressure (mPAP) was detected in normoxia and hypoxia-induced rats. ( d ) The right ventricular hypertrophy index (RVI) was calculated in normoxia and hypoxia-induced rats. # p

Techniques Used: Expressing, Quantitative RT-PCR

5) Product Images from "Lentivirus-mediated LIGHT overexpression inhibits human colorectal carcinoma cell growth in vitro and in vivo"

Article Title: Lentivirus-mediated LIGHT overexpression inhibits human colorectal carcinoma cell growth in vitro and in vivo

Journal: Oncology Letters

doi: 10.3892/ol.2013.1505

Effects of overexpressing LIGHT on the mRNA level of LIGHT. (A) Electrophoresis and semiquantitative analysis of PCR products. The mRNA level of LIGHT was higher in the HCT116/LIGHT cells compared with the HCT116/GFP and HCT116 cells. Lanes 1 and 4, HCT116; lanes 2 and 5, HCT116/GFP; lanes 3 and 6, HCT116/LIGHT; M, DL1000 DNA marker. (B) Statistical analysis of the mRNA level of LIGHT relative to that of GAPDH. LIGHT, lymphotoxin-related inducible ligand that competes for glycoprotein D binding to herpesvirus entry mediator on T cells.
Figure Legend Snippet: Effects of overexpressing LIGHT on the mRNA level of LIGHT. (A) Electrophoresis and semiquantitative analysis of PCR products. The mRNA level of LIGHT was higher in the HCT116/LIGHT cells compared with the HCT116/GFP and HCT116 cells. Lanes 1 and 4, HCT116; lanes 2 and 5, HCT116/GFP; lanes 3 and 6, HCT116/LIGHT; M, DL1000 DNA marker. (B) Statistical analysis of the mRNA level of LIGHT relative to that of GAPDH. LIGHT, lymphotoxin-related inducible ligand that competes for glycoprotein D binding to herpesvirus entry mediator on T cells.

Techniques Used: Electrophoresis, Polymerase Chain Reaction, Marker, Binding Assay

Green fluorescent protein (GFP) visualization of HCT116 cells transduced with Lenti-GFP. HCT116 cells were transduced with Lenti-GFP at different multiplicities of infection (MOI). At 72 h post-transduction, GFP-expressing cells were imaged by fluorescence microscopy (magnification, ×200; left column, bright field; right column, fluorescence vision). (A) MOI, 2. (B) MOI, 5. (C) MOI, 8.
Figure Legend Snippet: Green fluorescent protein (GFP) visualization of HCT116 cells transduced with Lenti-GFP. HCT116 cells were transduced with Lenti-GFP at different multiplicities of infection (MOI). At 72 h post-transduction, GFP-expressing cells were imaged by fluorescence microscopy (magnification, ×200; left column, bright field; right column, fluorescence vision). (A) MOI, 2. (B) MOI, 5. (C) MOI, 8.

Techniques Used: Transduction, Infection, Expressing, Fluorescence, Microscopy

Comparison of the size of the harvested implanted tumors in nude BALBC/c mice. (A) Tumors transplanted with the HCT116/LIGHT cells. (B) Tumors transplanted with the HCT116/GFP cells. (C) Tumors transplanted with the HCT116 cells. LIGHT, lymphotoxin-related inducible ligand that competes for glycoprotein D binding to herpesvirus entry mediator on T cells.
Figure Legend Snippet: Comparison of the size of the harvested implanted tumors in nude BALBC/c mice. (A) Tumors transplanted with the HCT116/LIGHT cells. (B) Tumors transplanted with the HCT116/GFP cells. (C) Tumors transplanted with the HCT116 cells. LIGHT, lymphotoxin-related inducible ligand that competes for glycoprotein D binding to herpesvirus entry mediator on T cells.

Techniques Used: Mouse Assay, Binding Assay

LIGHT transfection reduced caspase-3 and Bcl-2 protein levels in HCT116 cells. (A) Western blot showing caspase-3, Bcl-2 and GAPDH (loading control) staining for untreated HCT116 (lane 1), Lenti-GFP (lane 2) or Lenti-LIGHT-treated HCT116 cells (lane 3). Band signals from caspase-3 and Bcl-2 were normalized to those from GAPDH. (B) Relative expression of caspase-3 and Bcl-2 in the three groups. Lenti-LIGHT-treated HCT116 cells showed higher caspase-3 and lower Bcl-2 protein expression compared with untreated and Lenti-GFP-treated HCT116 cells (P
Figure Legend Snippet: LIGHT transfection reduced caspase-3 and Bcl-2 protein levels in HCT116 cells. (A) Western blot showing caspase-3, Bcl-2 and GAPDH (loading control) staining for untreated HCT116 (lane 1), Lenti-GFP (lane 2) or Lenti-LIGHT-treated HCT116 cells (lane 3). Band signals from caspase-3 and Bcl-2 were normalized to those from GAPDH. (B) Relative expression of caspase-3 and Bcl-2 in the three groups. Lenti-LIGHT-treated HCT116 cells showed higher caspase-3 and lower Bcl-2 protein expression compared with untreated and Lenti-GFP-treated HCT116 cells (P

Techniques Used: Transfection, Western Blot, Staining, Expressing

6) Product Images from "Genotype-4 hepatitis E in a human after ingesting roe deer meat in South Korea"

Article Title: Genotype-4 hepatitis E in a human after ingesting roe deer meat in South Korea

Journal: Clinical and molecular hepatology

doi: 10.3350/cmh.2013.19.3.309

A phylogenetic tree constructed by the neighbor-joining method based on the ORF2 sequence of the Korean HEV in question, 2010-JKSH-CYY (HM769726), and 42 HEV reference strains with genotypes 1 - 4 (G1-G4). All strains were separated into four groups according to their genotype. H: isolated from humans; P: isolated from pigs. Sequence analysis was conducted using the software VectorNTI and MEGA 5.04. Bootstrap values are indicated for the major nodes as a percentage of the data obtained from 1000 resamplings.
Figure Legend Snippet: A phylogenetic tree constructed by the neighbor-joining method based on the ORF2 sequence of the Korean HEV in question, 2010-JKSH-CYY (HM769726), and 42 HEV reference strains with genotypes 1 - 4 (G1-G4). All strains were separated into four groups according to their genotype. H: isolated from humans; P: isolated from pigs. Sequence analysis was conducted using the software VectorNTI and MEGA 5.04. Bootstrap values are indicated for the major nodes as a percentage of the data obtained from 1000 resamplings.

Techniques Used: Construct, Sequencing, Isolation, Software

7) Product Images from "The Promoter Structure Differentiation of a MYB Transcription Factor RLC1 Causes Red Leaf Coloration in Empire Red Leaf Cotton under Light"

Article Title: The Promoter Structure Differentiation of a MYB Transcription Factor RLC1 Causes Red Leaf Coloration in Empire Red Leaf Cotton under Light

Journal: PLoS ONE

doi: 10.1371/journal.pone.0077891

Analysis of RLC1 and gene expression levels in cotton by RT-PCR. UBI 7 was used as a positive control. A , RLC1 expression analysis was performed in roots, seedlings, leaves, and mature petals of ERLC and CCRI 24 cultivars grown in light by semi-quantitative RT-PCR. B , Comparison of the expression levels of RLC1 in mature leaves of ERLC grown in shade, light, and combined conditions. C , Comparison of color accumulation in transformed hairy roots of CCRI 24. a , Hairy root transformed with pBI121; b , Hairy root transformed with pBI35S:: RLC1. Scale bar indicates 1 cm. D , Expression analysis of structural genes in hairy roots of transformed CCRI 24 with the negative controls pBI121 and pBI35S:: RLC1 , respectively.
Figure Legend Snippet: Analysis of RLC1 and gene expression levels in cotton by RT-PCR. UBI 7 was used as a positive control. A , RLC1 expression analysis was performed in roots, seedlings, leaves, and mature petals of ERLC and CCRI 24 cultivars grown in light by semi-quantitative RT-PCR. B , Comparison of the expression levels of RLC1 in mature leaves of ERLC grown in shade, light, and combined conditions. C , Comparison of color accumulation in transformed hairy roots of CCRI 24. a , Hairy root transformed with pBI121; b , Hairy root transformed with pBI35S:: RLC1. Scale bar indicates 1 cm. D , Expression analysis of structural genes in hairy roots of transformed CCRI 24 with the negative controls pBI121 and pBI35S:: RLC1 , respectively.

Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction, Positive Control, Quantitative RT-PCR, Transformation Assay

Comparison of leaf colors and analysis of total anthocyanin concentrations of leaves from ERLC and CCRI 24 cultivars grown in light and shade conditions. A, Comparison of leaf colors. The cultivars are indicated on the left and conditions are indicated above. The bar indicates 1; b,A mature leaf of ERLC grown in shade; c, A mature leaf of CCRI 24 grown in light; d, A mature leaf of CCRI 24 grown in shade. B, Total anthocyanin extracted from three fully opened young leaves of each cultivar, respectively, measured using a UV spectrometer. Means of three replicates with error bars indicating standard error (± SD). C, Transient analysis was performed on the leaves of CCRI 24, the Agrobacterium strain GV3101/pBI35S:: ROSEA1 (left), and the negative control GV3101/pBI121 (right). The treated cotton leaves were cultured at 25°C in, 16 h light for three days, and observed for color accumulation by microscopy. Scale bar is 0.4 cm.
Figure Legend Snippet: Comparison of leaf colors and analysis of total anthocyanin concentrations of leaves from ERLC and CCRI 24 cultivars grown in light and shade conditions. A, Comparison of leaf colors. The cultivars are indicated on the left and conditions are indicated above. The bar indicates 1; b,A mature leaf of ERLC grown in shade; c, A mature leaf of CCRI 24 grown in light; d, A mature leaf of CCRI 24 grown in shade. B, Total anthocyanin extracted from three fully opened young leaves of each cultivar, respectively, measured using a UV spectrometer. Means of three replicates with error bars indicating standard error (± SD). C, Transient analysis was performed on the leaves of CCRI 24, the Agrobacterium strain GV3101/pBI35S:: ROSEA1 (left), and the negative control GV3101/pBI121 (right). The treated cotton leaves were cultured at 25°C in, 16 h light for three days, and observed for color accumulation by microscopy. Scale bar is 0.4 cm.

Techniques Used: Negative Control, Cell Culture, Microscopy

Phenotypic comparison between Empire red leaf cotton (ERLC) (upper panel) and green leaf cotton CCRI 24 (bottom panel) cultivars grown in light. A, A 7-day-old young seedling of ERLC;B, A young leaf from a 4-week-old ERLC plant; C, stems of ERLC; D, A mature flower of ERLC; E, A boll of ERLC; F, A 7-day-old seedling of CCRI 24; G, A young leaf from a 4-week-old plant of CCRI 24; H, stems of CCRI 24; I, A mature flower of CCRI 24; J, A boll of CCRI 24. Scale bar indicates 1 cm.
Figure Legend Snippet: Phenotypic comparison between Empire red leaf cotton (ERLC) (upper panel) and green leaf cotton CCRI 24 (bottom panel) cultivars grown in light. A, A 7-day-old young seedling of ERLC;B, A young leaf from a 4-week-old ERLC plant; C, stems of ERLC; D, A mature flower of ERLC; E, A boll of ERLC; F, A 7-day-old seedling of CCRI 24; G, A young leaf from a 4-week-old plant of CCRI 24; H, stems of CCRI 24; I, A mature flower of CCRI 24; J, A boll of CCRI 24. Scale bar indicates 1 cm.

Techniques Used: Empire Assay

Comparison of the RLC1 alleles between ERLC and CCRI 24. A, The coding sequence is shown in gray boxes and the non-coding sequence is shown as a black line. Location of the putative TATA box is shown by an empty square. Repeat fragments (R) are indicated by black squares. The exons, introns, and promoter region are labeled. The location of a nucleotide change site is also indicated in exon 2 of CCRI 24. Numbers refer to the position relative to the first nucleotide of the start codon. B, DNA sequence of the 228-bp fragment, with the location of putative I-box and G-box (underlined). C, Functional analysis of the RLC1a region of CCRI 24 (Fig. 6A, bottom) in the hairy roots of A. majus by A. rhizhogenes- mediated transformation. The pBI35S::RLC1a expression vector contained the RLC1a region on pBI121 driven by the cauliflower mosaic virus 35S promoter. a, a red pigmented mass developed on the end of hypocotyl segment of A. majus two weeks after infection; b, red pigmented hairy root developed from the ends of hypocotyl segments four weeks after infection. The pigmented mass (a) and hairy roots (b) are indicated by small arrows.
Figure Legend Snippet: Comparison of the RLC1 alleles between ERLC and CCRI 24. A, The coding sequence is shown in gray boxes and the non-coding sequence is shown as a black line. Location of the putative TATA box is shown by an empty square. Repeat fragments (R) are indicated by black squares. The exons, introns, and promoter region are labeled. The location of a nucleotide change site is also indicated in exon 2 of CCRI 24. Numbers refer to the position relative to the first nucleotide of the start codon. B, DNA sequence of the 228-bp fragment, with the location of putative I-box and G-box (underlined). C, Functional analysis of the RLC1a region of CCRI 24 (Fig. 6A, bottom) in the hairy roots of A. majus by A. rhizhogenes- mediated transformation. The pBI35S::RLC1a expression vector contained the RLC1a region on pBI121 driven by the cauliflower mosaic virus 35S promoter. a, a red pigmented mass developed on the end of hypocotyl segment of A. majus two weeks after infection; b, red pigmented hairy root developed from the ends of hypocotyl segments four weeks after infection. The pigmented mass (a) and hairy roots (b) are indicated by small arrows.

Techniques Used: Sequencing, Labeling, Functional Assay, Transformation Assay, Expressing, Plasmid Preparation, Infection

Analysis of RLC1 promoter activity by infiltration. A , Diagrams of constructs for the analysis of RLC1 promoter activity. R −pro : 2300-bp promoter region of RLC1 of ERLC; G −pro : 2080-bp promoter region of RLC1 of CCRI 24 ( Fig. 6 ). B , promoter activity tests were performed using mature young leaves of CCRI 24 using the combination of expression vectors described above. Treated cotton leaves were cultured at 25°C with 16-h light periods for three days, and the leaves were used for color observation or GUS staining. a , A leaf infiltrated with 35:: RLC1 and cultured for three days in light; b , A leaf infiltrated with R−pro:: RLC1 and cultured for three days in light; c , A leaf infiltrated with G − pro:: RLC1 and cultured for three days in light; d , A GUS-stained leaf infiltrated with pBI121 and cultured for three days in light; e , A GUS stained leaf infiltrated with R−pro:: GUS and cultured for three days in light; f , A leaf infiltrated with G − pro:: GUS and cultured for three days in light. Scale bar is 0.1 cm.
Figure Legend Snippet: Analysis of RLC1 promoter activity by infiltration. A , Diagrams of constructs for the analysis of RLC1 promoter activity. R −pro : 2300-bp promoter region of RLC1 of ERLC; G −pro : 2080-bp promoter region of RLC1 of CCRI 24 ( Fig. 6 ). B , promoter activity tests were performed using mature young leaves of CCRI 24 using the combination of expression vectors described above. Treated cotton leaves were cultured at 25°C with 16-h light periods for three days, and the leaves were used for color observation or GUS staining. a , A leaf infiltrated with 35:: RLC1 and cultured for three days in light; b , A leaf infiltrated with R−pro:: RLC1 and cultured for three days in light; c , A leaf infiltrated with G − pro:: RLC1 and cultured for three days in light; d , A GUS-stained leaf infiltrated with pBI121 and cultured for three days in light; e , A GUS stained leaf infiltrated with R−pro:: GUS and cultured for three days in light; f , A leaf infiltrated with G − pro:: GUS and cultured for three days in light. Scale bar is 0.1 cm.

Techniques Used: Activity Assay, Construct, Expressing, Cell Culture, Staining

8) Product Images from "The Molecular Basis of Inactivation of Metronidazole-Resistant Helicobacter pylori Using Polyethyleneimine Functionalized Zinc Oxide Nanoparticles"

Article Title: The Molecular Basis of Inactivation of Metronidazole-Resistant Helicobacter pylori Using Polyethyleneimine Functionalized Zinc Oxide Nanoparticles

Journal: PLoS ONE

doi: 10.1371/journal.pone.0070776

Morphological transition and rRNA degradation. H. pylori incubated without or with ZnO-PEI NP for 3 h was visualized by (a) SEM and (b) TEM. (c) RNA was extracted from ZnO-PEI NP treated and untreated H. pylori and electrophoresed on 7M urea-6% polyacrylamide gel. In (a), (b) and (c) the left panel shows the control and the right panel shows treated cells. (d) 16S and 23S rRNA was estimated in the RNA samples by qRT-PCR (list of primers are provided in Table S2 ). * P
Figure Legend Snippet: Morphological transition and rRNA degradation. H. pylori incubated without or with ZnO-PEI NP for 3 h was visualized by (a) SEM and (b) TEM. (c) RNA was extracted from ZnO-PEI NP treated and untreated H. pylori and electrophoresed on 7M urea-6% polyacrylamide gel. In (a), (b) and (c) the left panel shows the control and the right panel shows treated cells. (d) 16S and 23S rRNA was estimated in the RNA samples by qRT-PCR (list of primers are provided in Table S2 ). * P

Techniques Used: Incubation, Transmission Electron Microscopy, Quantitative RT-PCR

9) Product Images from "Class I/II hybrid inhibitory oligodeoxynucleotide exerts Th1 and Th2 double immunosuppression"

Article Title: Class I/II hybrid inhibitory oligodeoxynucleotide exerts Th1 and Th2 double immunosuppression

Journal: FEBS Open Bio

doi: 10.1016/j.fob.2012.11.002

Real-time quantitative PCR analysis of IL-4, IL-13, IL-6, IL-12p35, IL-12p40, and IFNγ mRNA levels in splenocytes isolated from OVA-immunized mice that were subsequently treated with 10 μg/mL OVA + 3.0 μM ODN 1612 , ODN 1555 , A151, H154, or iSG3. Mouse splenocytes were pre-incubated in medium for 3 h prior to exposure to OVA and ODNs for 72 h. Expression of IL-4 (A), IL-13 (B), IL-6 (C), IL-12p35 (D), IL-12p40 (E), and IFNγ (F) mRNAs were determined with real-time quantitative PCR. Results are shown as the ratio of cytokine mRNA levels (normalized to β-actin; see Section 2 ) in stimulated versus non-treated cells, as well as the level in the medium control. Values represent means, and error bars indicate the SD of three independent experiments. Values with different letters ( i.e., a, b, c, d, and e) were significantly different ( P
Figure Legend Snippet: Real-time quantitative PCR analysis of IL-4, IL-13, IL-6, IL-12p35, IL-12p40, and IFNγ mRNA levels in splenocytes isolated from OVA-immunized mice that were subsequently treated with 10 μg/mL OVA + 3.0 μM ODN 1612 , ODN 1555 , A151, H154, or iSG3. Mouse splenocytes were pre-incubated in medium for 3 h prior to exposure to OVA and ODNs for 72 h. Expression of IL-4 (A), IL-13 (B), IL-6 (C), IL-12p35 (D), IL-12p40 (E), and IFNγ (F) mRNAs were determined with real-time quantitative PCR. Results are shown as the ratio of cytokine mRNA levels (normalized to β-actin; see Section 2 ) in stimulated versus non-treated cells, as well as the level in the medium control. Values represent means, and error bars indicate the SD of three independent experiments. Values with different letters ( i.e., a, b, c, d, and e) were significantly different ( P

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

10) Product Images from "Novel Identification of Dermacentor variabilis Arp2/3 Complex and Its Role in Rickettsial Infection of the Arthropod Vector"

Article Title: Novel Identification of Dermacentor variabilis Arp2/3 Complex and Its Role in Rickettsial Infection of the Arthropod Vector

Journal: PLoS ONE

doi: 10.1371/journal.pone.0093768

Effect of Arp2/3 complex inhibitor on R. montanensis invasion of D. variabilis tissues. Tick tissues including midgut, ovary, and salivary glands were dissected out prior to infection with R. montanensis (8×10 7 per tissue). After 1 h, rickettsiae were removed and the tissues were washed once with PBS and rickettsiae and tick cells were quantified by qPCR. The experiments were performed in quadruplicate for each treatment group and the results were the combination of the three independent experiments. The asterisk indicates a significant difference between treatment and inhibitor vehicle control.
Figure Legend Snippet: Effect of Arp2/3 complex inhibitor on R. montanensis invasion of D. variabilis tissues. Tick tissues including midgut, ovary, and salivary glands were dissected out prior to infection with R. montanensis (8×10 7 per tissue). After 1 h, rickettsiae were removed and the tissues were washed once with PBS and rickettsiae and tick cells were quantified by qPCR. The experiments were performed in quadruplicate for each treatment group and the results were the combination of the three independent experiments. The asterisk indicates a significant difference between treatment and inhibitor vehicle control.

Techniques Used: Infection, Real-time Polymerase Chain Reaction

Transcriptional profile of Arp2/3 complex (all subunits) in D. variabilis tissues. R. montanensis was used to infect tick midgut, ovary, and salivary glands (8×10 7 rickettsiae per tissue) for 1 h. After removal of rickettsiae, tick tissues were washed and collected by low-speed centrifugation. Total RNA was then extracted from the tissues and the levels of Dv Arp2/3 complex mRNA were measured by qRT-PCR. Dv GAPDH mRNA was used to normalize the differences among samples. Data shown are mean (±SEM) relative expression from two independent experiments. The asterisk denotes a significant difference between treatment groups (unexposed- or Rickettsia- exposed group) in the same tissue. For each subunit, different letters above bars represents significance differences between tissues.
Figure Legend Snippet: Transcriptional profile of Arp2/3 complex (all subunits) in D. variabilis tissues. R. montanensis was used to infect tick midgut, ovary, and salivary glands (8×10 7 rickettsiae per tissue) for 1 h. After removal of rickettsiae, tick tissues were washed and collected by low-speed centrifugation. Total RNA was then extracted from the tissues and the levels of Dv Arp2/3 complex mRNA were measured by qRT-PCR. Dv GAPDH mRNA was used to normalize the differences among samples. Data shown are mean (±SEM) relative expression from two independent experiments. The asterisk denotes a significant difference between treatment groups (unexposed- or Rickettsia- exposed group) in the same tissue. For each subunit, different letters above bars represents significance differences between tissues.

Techniques Used: Centrifugation, Quantitative RT-PCR, Expressing

11) Product Images from "A Broadly Reactive One-Step SYBR Green I Real-Time RT-PCR Assay for Rapid Detection of Murine Norovirus"

Article Title: A Broadly Reactive One-Step SYBR Green I Real-Time RT-PCR Assay for Rapid Detection of Murine Norovirus

Journal: PLoS ONE

doi: 10.1371/journal.pone.0098108

SYBR Green I assay followed by melting curve analysis of 7 (A) Amplification kinetics results with 1000 copies of the 7 MNV plasmids, and (B) melting curve analysis results of 7 MNV PCR products. 1.CW1, blue; Apo960, red; Berlin/04/06, green; KHU-1, magenta; S7-PP3, black; TW2006, yellow; TW2007, cyan. As negative controls, pUC19 DNA (purple) and molecular grade water (gray) are shown in A. All samples were tested in triplicates.
Figure Legend Snippet: SYBR Green I assay followed by melting curve analysis of 7 (A) Amplification kinetics results with 1000 copies of the 7 MNV plasmids, and (B) melting curve analysis results of 7 MNV PCR products. 1.CW1, blue; Apo960, red; Berlin/04/06, green; KHU-1, magenta; S7-PP3, black; TW2006, yellow; TW2007, cyan. As negative controls, pUC19 DNA (purple) and molecular grade water (gray) are shown in A. All samples were tested in triplicates.

Techniques Used: SYBR Green Assay, Amplification, Polymerase Chain Reaction

12) Product Images from "Puerarin protects brain tissue against cerebral ischemia/reperfusion injury by inhibiting the inflammatory response"

Article Title: Puerarin protects brain tissue against cerebral ischemia/reperfusion injury by inhibiting the inflammatory response

Journal: Neural Regeneration Research

doi: 10.4103/1673-5374.147934

Detection of mRNA expression of Toll-like receptor 4, myeloid differentiation factor 88, nuclear factor kappa B and tumor necrosis factor-α in ischemic brain tissue by real-time reverse transcription-PCR (RT-PCR)
Figure Legend Snippet: Detection of mRNA expression of Toll-like receptor 4, myeloid differentiation factor 88, nuclear factor kappa B and tumor necrosis factor-α in ischemic brain tissue by real-time reverse transcription-PCR (RT-PCR)

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

13) Product Images from "Inhibitory effect of insulin-like growth factor-binding protein-7 (IGFBP7) on in vitro angiogenesis of vascular endothelial cells in the rat corpus luteum"

Article Title: Inhibitory effect of insulin-like growth factor-binding protein-7 (IGFBP7) on in vitro angiogenesis of vascular endothelial cells in the rat corpus luteum

Journal: The Journal of Reproduction and Development

doi: 10.1262/jrd.2014-069

Effects of IGFBP7 on VEGFA-stimulated cyclooxygenase-2 (COX-2) mRNA expression and prostaglandin E 2 (PGE 2 ) production in LECs. (A) LECs were incubated with IGFBP7 (2.5–160 ng/ml) for 2 h in the presence or absence of VEGFA (10 ng/ml). RNA was analyzed with semiquantitative RT-PCR for VEGFA and COX-2. (B) LECs were incubated for 24 h with IGFBP7 in the presence or absence of VEGFA (10 ng/ml). The culture medium was collected for the PGE 2 assay. The results are expressed as means ± SEM. *P
Figure Legend Snippet: Effects of IGFBP7 on VEGFA-stimulated cyclooxygenase-2 (COX-2) mRNA expression and prostaglandin E 2 (PGE 2 ) production in LECs. (A) LECs were incubated with IGFBP7 (2.5–160 ng/ml) for 2 h in the presence or absence of VEGFA (10 ng/ml). RNA was analyzed with semiquantitative RT-PCR for VEGFA and COX-2. (B) LECs were incubated for 24 h with IGFBP7 in the presence or absence of VEGFA (10 ng/ml). The culture medium was collected for the PGE 2 assay. The results are expressed as means ± SEM. *P

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

14) Product Images from "MiR-138 Suppresses Cell Proliferation by Targeting Bag-1 in Gallbladder Carcinoma"

Article Title: MiR-138 Suppresses Cell Proliferation by Targeting Bag-1 in Gallbladder Carcinoma

Journal: PLoS ONE

doi: 10.1371/journal.pone.0126499

Expression of miR-138 and Bag-1 in gallbladder carcinoma specimens. (A) The expression of miR-138 was determined in gallbladder carcinoma tissues compared with matched normal adjacent gallbladder tissues using qRT-PCR. (B) Relative expression of Bag-1 at mRNA level was examined in gallbladder carcinoma tissues compared with matched normal adjacent gallbladder tissues using qRT-PCR. (C) Inverse correlation between miR-138 and Bag-1 expression in gallbladder carcinoma tissues using Pearson’s correlation coefficient. The expression of miR-138 was normalized to that of U6, and the expression of Bag-1 mRNA was normalized to that of β-actin in each sample.
Figure Legend Snippet: Expression of miR-138 and Bag-1 in gallbladder carcinoma specimens. (A) The expression of miR-138 was determined in gallbladder carcinoma tissues compared with matched normal adjacent gallbladder tissues using qRT-PCR. (B) Relative expression of Bag-1 at mRNA level was examined in gallbladder carcinoma tissues compared with matched normal adjacent gallbladder tissues using qRT-PCR. (C) Inverse correlation between miR-138 and Bag-1 expression in gallbladder carcinoma tissues using Pearson’s correlation coefficient. The expression of miR-138 was normalized to that of U6, and the expression of Bag-1 mRNA was normalized to that of β-actin in each sample.

Techniques Used: Expressing, Quantitative RT-PCR

Effect of miR-138 on gallbladder carcinoma cell proliferation and apoptosis. (A) The qRT-PCR analysis confirmed that the expression of miR-138 was clearly increased in cells transduced with miR-138 compared with the control vector. (B and C) Effect of miR-138 on cell proliferation was measured using MTT assay in OCUG-1 and NOZ cells transduced with miR-138 or the control vector. (D) Flow cytometric analysis of the effect of miR-138 on apoptosis of OCUG-1 and NOZ cells. Error bars represented the SD from three independent trials. ** P
Figure Legend Snippet: Effect of miR-138 on gallbladder carcinoma cell proliferation and apoptosis. (A) The qRT-PCR analysis confirmed that the expression of miR-138 was clearly increased in cells transduced with miR-138 compared with the control vector. (B and C) Effect of miR-138 on cell proliferation was measured using MTT assay in OCUG-1 and NOZ cells transduced with miR-138 or the control vector. (D) Flow cytometric analysis of the effect of miR-138 on apoptosis of OCUG-1 and NOZ cells. Error bars represented the SD from three independent trials. ** P

Techniques Used: Quantitative RT-PCR, Expressing, Transduction, Plasmid Preparation, MTT Assay, Flow Cytometry

15) Product Images from "Genetic diversity of human RNase 8"

Article Title: Genetic diversity of human RNase 8

Journal: BMC Genomics

doi: 10.1186/1471-2164-13-40

Identification and characterization of RNase 8 transcript . (A) ~ 800 and ~1000 bp transcripts encoding RNase 8 were isolated from human spleen cDNA using a nested rapid amplification of cDNA ends (RACE); +, including adapter primer;-without adapter primer. (B) Findings from RACE confirmed by RT-PCR using two unique sources of poly A + RNA; +, including reverse transcriptase;-without reverse transcriptase. (C) RNase 8 on human chromosome 14; black bar, coding sequence originally described [ 25 ] green bar, novel amino terminal extension. RACE amplification products are shown above extending 0.8 and 1 kb from primer within the RNase 8 coding sequence; nested RT-PCR amplification product (0.5 kb) is also as shown below. The GenBank accession number for the spleen cDNA RNase 8 transcript sequences is JQ353679 .
Figure Legend Snippet: Identification and characterization of RNase 8 transcript . (A) ~ 800 and ~1000 bp transcripts encoding RNase 8 were isolated from human spleen cDNA using a nested rapid amplification of cDNA ends (RACE); +, including adapter primer;-without adapter primer. (B) Findings from RACE confirmed by RT-PCR using two unique sources of poly A + RNA; +, including reverse transcriptase;-without reverse transcriptase. (C) RNase 8 on human chromosome 14; black bar, coding sequence originally described [ 25 ] green bar, novel amino terminal extension. RACE amplification products are shown above extending 0.8 and 1 kb from primer within the RNase 8 coding sequence; nested RT-PCR amplification product (0.5 kb) is also as shown below. The GenBank accession number for the spleen cDNA RNase 8 transcript sequences is JQ353679 .

Techniques Used: Isolation, Rapid Amplification of cDNA Ends, Reverse Transcription Polymerase Chain Reaction, Sequencing, Amplification

16) Product Images from "Change of dopamine receptor mRNA expression in lymphocyte of schizophrenic patients"

Article Title: Change of dopamine receptor mRNA expression in lymphocyte of schizophrenic patients

Journal: BMC Medical Genetics

doi: 10.1186/1471-2350-2-3

Examples of quantitative RT-PCR of D3R mRNA in one patient. Upper band represents the D3R mutant that competes with D3R cDNA (lower band). This figure shows lane 3 has approximately equal concentrations of D3R and D3R mutant.
Figure Legend Snippet: Examples of quantitative RT-PCR of D3R mRNA in one patient. Upper band represents the D3R mutant that competes with D3R cDNA (lower band). This figure shows lane 3 has approximately equal concentrations of D3R and D3R mutant.

Techniques Used: Quantitative RT-PCR, Mutagenesis

17) Product Images from "Retigeric Acid B Exhibits Antitumor Activity through Suppression of Nuclear Factor-?B Signaling in Prostate Cancer Cells in Vitro and in Vivo"

Article Title: Retigeric Acid B Exhibits Antitumor Activity through Suppression of Nuclear Factor-?B Signaling in Prostate Cancer Cells in Vitro and in Vivo

Journal: PLoS ONE

doi: 10.1371/journal.pone.0038000

RELA is involved in RB-induced apoptotic cell death. (A) and (B) RELA overexpression by transfection of a RELA expression vector partially abolished cytotoxicity in PC3 and DU145 cells. Cells were transfected with siRNA of RELA (RELAi) or with a negative control siRNA (NCi), using a 50 nmol/L final siRNA concentration. After 48 h transfection for RELA overexpression, the effect of RB on p65, p–p65, and PARP expression in RELA transfected cells was determined by western blot; cell viability was also determined after additional exposure for 24 h to RB. (C) and (D) Silencing of RELA expression by siRNA increased cytotoxicity induced by RB. Similar procedure was performed for the studies on RELA. The details are described in Material and Methods . In (A) and (C), the protein levels of p65 and p–p65 were normalized with GAPDH. Results shown are representatives of three independent experiments. In (B) and (D), p
Figure Legend Snippet: RELA is involved in RB-induced apoptotic cell death. (A) and (B) RELA overexpression by transfection of a RELA expression vector partially abolished cytotoxicity in PC3 and DU145 cells. Cells were transfected with siRNA of RELA (RELAi) or with a negative control siRNA (NCi), using a 50 nmol/L final siRNA concentration. After 48 h transfection for RELA overexpression, the effect of RB on p65, p–p65, and PARP expression in RELA transfected cells was determined by western blot; cell viability was also determined after additional exposure for 24 h to RB. (C) and (D) Silencing of RELA expression by siRNA increased cytotoxicity induced by RB. Similar procedure was performed for the studies on RELA. The details are described in Material and Methods . In (A) and (C), the protein levels of p65 and p–p65 were normalized with GAPDH. Results shown are representatives of three independent experiments. In (B) and (D), p

Techniques Used: Over Expression, Transfection, Expressing, Plasmid Preparation, Negative Control, Concentration Assay, Western Blot

The effect of RB on p65 expression and phosphorylation in PCa cells. (A) and (B) RB slightly inhibited expression of p65 in PC3 and DU145 cells, while significantly for the Ser-536 phosphorylation of p65 ( p–p65 ), in both dosage-dependent and time-dependent manner. Lysates from whole cells treated with RB of different concentrations for 24 h (A) or with 10 µM RB for indicated times were used for western blot (B). GAPDH served as the loading control. Protein amount was normalized to the amount of GAPDH, and was quantified by densitometry of X-ray films. Results of one of at least three independent experiments are shown. (C) and (D) RB moderately down-regulated p65 mRNA level in PC3 and DU145 cells as detected by QRT-PCR assay. The procedure was performed as described in Materials and Methods . Results shown are representatives of three independent experiments, p
Figure Legend Snippet: The effect of RB on p65 expression and phosphorylation in PCa cells. (A) and (B) RB slightly inhibited expression of p65 in PC3 and DU145 cells, while significantly for the Ser-536 phosphorylation of p65 ( p–p65 ), in both dosage-dependent and time-dependent manner. Lysates from whole cells treated with RB of different concentrations for 24 h (A) or with 10 µM RB for indicated times were used for western blot (B). GAPDH served as the loading control. Protein amount was normalized to the amount of GAPDH, and was quantified by densitometry of X-ray films. Results of one of at least three independent experiments are shown. (C) and (D) RB moderately down-regulated p65 mRNA level in PC3 and DU145 cells as detected by QRT-PCR assay. The procedure was performed as described in Materials and Methods . Results shown are representatives of three independent experiments, p

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

The effects of RB on function of NF-κB in vitro . (A) RB dosage-dependently inhibited the nuclear localization of p65 Lysates from cytoplasm and nucleus respectively after treating of PC3 cells with RB for 24 h were used for western blot. GAPDH and H1 respectively served as the loading control. (B) Immunofluorescence analysis of the inhibitory nuclear localization of p65 by RB-treatment for 12 h in PC3 cells. For confocal microscopy, α-tubulin and p-p65 were immunostained, with nuclei stained with DAPI. (Scale bar, 10 μm). (C) Pretreatment of PC3 cells with RB inhibited binding of nuclear extracts to the NF-κB binding site, as detected by electrophoretic mobility shift assay. Lysates from nuclei after treating of PC3 cells with RB for 24 h were used for EMSA. (D), (E) and (F) RB decreased NF-κB activation in PC3 and DU145 cells, and inhibited LPS-induced NF-κB activation in LNCaP cells. The inhibition was more significant when pNF-κB-Luciferase and RELA expression vector co-transfected. Transiently transfected cells were preincubated with RB for 24 h. The luciferase assay was done as described in Materials and Methods . In (D)–(F), results are the mean ± S.E. of three independent experiments, each performed in triplicate. p
Figure Legend Snippet: The effects of RB on function of NF-κB in vitro . (A) RB dosage-dependently inhibited the nuclear localization of p65 Lysates from cytoplasm and nucleus respectively after treating of PC3 cells with RB for 24 h were used for western blot. GAPDH and H1 respectively served as the loading control. (B) Immunofluorescence analysis of the inhibitory nuclear localization of p65 by RB-treatment for 12 h in PC3 cells. For confocal microscopy, α-tubulin and p-p65 were immunostained, with nuclei stained with DAPI. (Scale bar, 10 μm). (C) Pretreatment of PC3 cells with RB inhibited binding of nuclear extracts to the NF-κB binding site, as detected by electrophoretic mobility shift assay. Lysates from nuclei after treating of PC3 cells with RB for 24 h were used for EMSA. (D), (E) and (F) RB decreased NF-κB activation in PC3 and DU145 cells, and inhibited LPS-induced NF-κB activation in LNCaP cells. The inhibition was more significant when pNF-κB-Luciferase and RELA expression vector co-transfected. Transiently transfected cells were preincubated with RB for 24 h. The luciferase assay was done as described in Materials and Methods . In (D)–(F), results are the mean ± S.E. of three independent experiments, each performed in triplicate. p

Techniques Used: In Vitro, Western Blot, Immunofluorescence, Confocal Microscopy, Staining, Binding Assay, Electrophoretic Mobility Shift Assay, Activation Assay, Inhibition, Luciferase, Expressing, Plasmid Preparation, Transfection

RB inhibits phosphorylation and degradation of IκBα. (A) Western blot analysis of expression of total IκBα and phosphor-IκBα ( p–IκBα, Ser32/36)p-IκBα. PC3 and DU145 cells were treated with RB of different doses as indicated. (B) Western blot analysis of expression of total IκBα and p-IκBα. PC3 and DU145 cells were treated with RB of different times as indicated. In (A) and (B), equal protein loading was evaluated by GAPDH. Protein amount was normalized to the amount of GAPDH, and was quantified by densitometry of X-ray films. Results of one of at least three independent experiments are shown. (C) The effect of RB on the purified 20S proteasome in vitro . MG132 served as positive control. Results are the mean ± SD of three independent experiments, p
Figure Legend Snippet: RB inhibits phosphorylation and degradation of IκBα. (A) Western blot analysis of expression of total IκBα and phosphor-IκBα ( p–IκBα, Ser32/36)p-IκBα. PC3 and DU145 cells were treated with RB of different doses as indicated. (B) Western blot analysis of expression of total IκBα and p-IκBα. PC3 and DU145 cells were treated with RB of different times as indicated. In (A) and (B), equal protein loading was evaluated by GAPDH. Protein amount was normalized to the amount of GAPDH, and was quantified by densitometry of X-ray films. Results of one of at least three independent experiments are shown. (C) The effect of RB on the purified 20S proteasome in vitro . MG132 served as positive control. Results are the mean ± SD of three independent experiments, p

Techniques Used: Western Blot, Expressing, Purification, In Vitro, Positive Control

18) Product Images from "Low Levels of Antibody-Dependent Enhancement in Vitro Using Viruses and Plasma from Dengue Patients"

Article Title: Low Levels of Antibody-Dependent Enhancement in Vitro Using Viruses and Plasma from Dengue Patients

Journal: PLoS ONE

doi: 10.1371/journal.pone.0092173

Construction of recombinant DENVs based on virus samples from patient plasma. RNA was extracted from the plasma of patient D30 and the prM-E encoding region of DENV was amplified by RT-PCR and cloned into the pCR4Blunt-TOPO vector for DNA sequence analysis. Representative prM-E region variants were selected and used to construct full-length DENV cDNA clones based on plasmid pmMW/R05-624. The resultant full-length cDNAs containing D30-derived variants were used as templates for RNA synthesis, and in vitro -transcribed viral RNAs were transfected into C6/36 cells. Supernatants from transfected cells were passaged once in C6/36 to obtain adequate quantities of viruses. EDI, envelope domain I. EDII, envelope domain II. EDIII, envelope domain III. TM, transmembrane. The recombinant DENV clone in brackets was excluded from further study due to an inadequate titer.
Figure Legend Snippet: Construction of recombinant DENVs based on virus samples from patient plasma. RNA was extracted from the plasma of patient D30 and the prM-E encoding region of DENV was amplified by RT-PCR and cloned into the pCR4Blunt-TOPO vector for DNA sequence analysis. Representative prM-E region variants were selected and used to construct full-length DENV cDNA clones based on plasmid pmMW/R05-624. The resultant full-length cDNAs containing D30-derived variants were used as templates for RNA synthesis, and in vitro -transcribed viral RNAs were transfected into C6/36 cells. Supernatants from transfected cells were passaged once in C6/36 to obtain adequate quantities of viruses. EDI, envelope domain I. EDII, envelope domain II. EDIII, envelope domain III. TM, transmembrane. The recombinant DENV clone in brackets was excluded from further study due to an inadequate titer.

Techniques Used: Recombinant, Amplification, Reverse Transcription Polymerase Chain Reaction, Clone Assay, Plasmid Preparation, Sequencing, Construct, Derivative Assay, In Vitro, Transfection

19) Product Images from "Contribution of Eukaryotic-Type Serine/Threonine Kinase to Stress Response and Virulence of Streptococcus suis"

Article Title: Contribution of Eukaryotic-Type Serine/Threonine Kinase to Stress Response and Virulence of Streptococcus suis

Journal: PLoS ONE

doi: 10.1371/journal.pone.0091971

Virulence gene expression of different strains in vitro . Total RNA was extracted from SS2-1, Δ stk and C Δ stk grown in THY medium at an OD600 of 0.6–0.8 and used for qRT-PCR analysis. The mRNA level of each gene was normalized to that of 16S rRNA . Results are shown as relative expression ratios compared to expression in the parental strain SS2-1. Data from three independent assays are presented as the means±SE. Differences between SS2-1 and Δ stk was determined by two-way ANOVA. (**p
Figure Legend Snippet: Virulence gene expression of different strains in vitro . Total RNA was extracted from SS2-1, Δ stk and C Δ stk grown in THY medium at an OD600 of 0.6–0.8 and used for qRT-PCR analysis. The mRNA level of each gene was normalized to that of 16S rRNA . Results are shown as relative expression ratios compared to expression in the parental strain SS2-1. Data from three independent assays are presented as the means±SE. Differences between SS2-1 and Δ stk was determined by two-way ANOVA. (**p

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

20) Product Images from "Natural HCV variants with increased replicative fitness due to NS3 helicase mutations in the C-terminal helix α18"

Article Title: Natural HCV variants with increased replicative fitness due to NS3 helicase mutations in the C-terminal helix α18

Journal: Scientific Reports

doi: 10.1038/srep19526

Impact of helicase mutations on HCV RNA replication. HCV RNA determined by two-step reverse transcription quantitative real-time PCR (RT-PCR), normalized to that of the wild type Con1 RNA from each helicase mutant. Bars are color coded as follows: cyan – increased replicative fitness; grey – not different from wild type; magenta – decreased replicative fitness. The data shown represent the mean ± SD from at least ten independent experiments. “wt” refers to wild type. Y axis shows relative replication to wt as fold change (FC). An asterisk designates mutants for which the fold change compared to wt is significant by Student t-test (P
Figure Legend Snippet: Impact of helicase mutations on HCV RNA replication. HCV RNA determined by two-step reverse transcription quantitative real-time PCR (RT-PCR), normalized to that of the wild type Con1 RNA from each helicase mutant. Bars are color coded as follows: cyan – increased replicative fitness; grey – not different from wild type; magenta – decreased replicative fitness. The data shown represent the mean ± SD from at least ten independent experiments. “wt” refers to wild type. Y axis shows relative replication to wt as fold change (FC). An asterisk designates mutants for which the fold change compared to wt is significant by Student t-test (P

Techniques Used: Real-time Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, Mutagenesis

21) Product Images from "Role of Nox2 and p22phox in Persistent Postoperative Hypertension in Aldosterone-Producing Adenoma Patients after Adrenalectomy"

Article Title: Role of Nox2 and p22phox in Persistent Postoperative Hypertension in Aldosterone-Producing Adenoma Patients after Adrenalectomy

Journal: International Journal of Endocrinology

doi: 10.1155/2016/2395634

Pathological and immunohistochemical analyses of p22phox in normal adrenocortical tissue, APA, and NFA. (a–i) p22phox presents a gradient of expression in the entire adrenal cortex (100x), and its localization was restricted to the cell membrane (400x). (j) Graphical illustration of the statistical distributions of p22phox in normal adrenocortical tissue, APA, and NFA. The p22phox IRS was higher in APA compared to that in normal adrenal tissue.
Figure Legend Snippet: Pathological and immunohistochemical analyses of p22phox in normal adrenocortical tissue, APA, and NFA. (a–i) p22phox presents a gradient of expression in the entire adrenal cortex (100x), and its localization was restricted to the cell membrane (400x). (j) Graphical illustration of the statistical distributions of p22phox in normal adrenocortical tissue, APA, and NFA. The p22phox IRS was higher in APA compared to that in normal adrenal tissue.

Techniques Used: Immunohistochemistry, Expressing

Activity and expression of Nox in different adrenal tissues. (a) Nox activity was detected in normal adrenocortical tissue, APA, and NFA by lucigenin chemiluminescence. (b) Expression of Nox1 – Nox5 , Duox1 , Duox2 , and p22phox mRNA in normal adrenocortical tissue and APA by RT-PCR. Signals for Nox1 , Nox2 , Nox4 , Duox1 , and p22phox were detected in normal adrenal tissue and APA. (c–h) Relative expression of Nox1 , Nox2 , Nox4 , Duox1 , p22phox , and CYP11B2 in normal adrenocortical tissue, APA, and NFA measured by Q-PCR. Nox2 , p22phox , and CYP11B2 were primarily expressed in APA. (i-j) Correlation between CYP11B2 mRNA with Nox2 , CYP11B2 , and p22phox mRNA in APA. A positive correlation between Nox2 and CYP11B2 mRNA was found. (k–n) Western blot analysis of Nox2 and p22phox in normal adrenocortical tissue, APA, and NFA. Nox2 and p22phox were enhanced in APA compared with normal adrenocortical tissue and NFA. Results of densitometric analysis of Nox2 and p22phox proteins normalized to GAPDH were shown.
Figure Legend Snippet: Activity and expression of Nox in different adrenal tissues. (a) Nox activity was detected in normal adrenocortical tissue, APA, and NFA by lucigenin chemiluminescence. (b) Expression of Nox1 – Nox5 , Duox1 , Duox2 , and p22phox mRNA in normal adrenocortical tissue and APA by RT-PCR. Signals for Nox1 , Nox2 , Nox4 , Duox1 , and p22phox were detected in normal adrenal tissue and APA. (c–h) Relative expression of Nox1 , Nox2 , Nox4 , Duox1 , p22phox , and CYP11B2 in normal adrenocortical tissue, APA, and NFA measured by Q-PCR. Nox2 , p22phox , and CYP11B2 were primarily expressed in APA. (i-j) Correlation between CYP11B2 mRNA with Nox2 , CYP11B2 , and p22phox mRNA in APA. A positive correlation between Nox2 and CYP11B2 mRNA was found. (k–n) Western blot analysis of Nox2 and p22phox in normal adrenocortical tissue, APA, and NFA. Nox2 and p22phox were enhanced in APA compared with normal adrenocortical tissue and NFA. Results of densitometric analysis of Nox2 and p22phox proteins normalized to GAPDH were shown.

Techniques Used: Activity Assay, Expressing, Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction, Western Blot

Pathological and immunohistochemical findings of Nox2 in normal adrenocortical tissue, APA, and NFA. (a, d, g, j, and m) HES staining of normal adrenocortical tissue (a), APA (d and g), and NFA (j and m). (b and c) Nox2 and Dab2 were expressed in normal adrenocortical ZG cells. However, Nox2 was also restricted to the ZR. (d–o) Nox2 and Dab2 expression were detectable and visible beneath the capsule (h, i, k, and l) and along the vasculature (e, f, n, and o) in APA and NFA as indicated by HES. Moreover, higher magnification revealed predominant cytoplasmic localization of Nox2 and Dab2. (p) Graphical illustration of statistical distributions of Nox2 in normal adrenocortical tissue, APA, and NFA. The Nox2 IRS (immunoreactive score) was higher in APA compared to those in normal adrenal tissue and NFA. Bar, 200 μ m on low magnification (100x) and 50 μ m on high magnification (400x).
Figure Legend Snippet: Pathological and immunohistochemical findings of Nox2 in normal adrenocortical tissue, APA, and NFA. (a, d, g, j, and m) HES staining of normal adrenocortical tissue (a), APA (d and g), and NFA (j and m). (b and c) Nox2 and Dab2 were expressed in normal adrenocortical ZG cells. However, Nox2 was also restricted to the ZR. (d–o) Nox2 and Dab2 expression were detectable and visible beneath the capsule (h, i, k, and l) and along the vasculature (e, f, n, and o) in APA and NFA as indicated by HES. Moreover, higher magnification revealed predominant cytoplasmic localization of Nox2 and Dab2. (p) Graphical illustration of statistical distributions of Nox2 in normal adrenocortical tissue, APA, and NFA. The Nox2 IRS (immunoreactive score) was higher in APA compared to those in normal adrenal tissue and NFA. Bar, 200 μ m on low magnification (100x) and 50 μ m on high magnification (400x).

Techniques Used: Immunohistochemistry, Staining, Expressing

22) Product Images from "Lysine-specific demethylase 1 promotes tumorigenesis and predicts prognosis in gallbladder cancer"

Article Title: Lysine-specific demethylase 1 promotes tumorigenesis and predicts prognosis in gallbladder cancer

Journal: Oncotarget

doi:

Knock-down of LSD1 inhibit the invasion and metastasis in GBC cell lines Comparison among the MOCK (untransfected), NC (transfected with the PWPXL-GFP plasmid) and LSD1 knockdown/RNAi (transfected with the PWPXL-sh-LSD1 plasmid) groups of GBC-SD and NOZ cells on A. Transwell assay, B. wound-healing assay, C. proliferation assay using CCK-8 and D. plate colony formation assay. P value was significant at less than 0.05.
Figure Legend Snippet: Knock-down of LSD1 inhibit the invasion and metastasis in GBC cell lines Comparison among the MOCK (untransfected), NC (transfected with the PWPXL-GFP plasmid) and LSD1 knockdown/RNAi (transfected with the PWPXL-sh-LSD1 plasmid) groups of GBC-SD and NOZ cells on A. Transwell assay, B. wound-healing assay, C. proliferation assay using CCK-8 and D. plate colony formation assay. P value was significant at less than 0.05.

Techniques Used: Transfection, Plasmid Preparation, Transwell Assay, Wound Healing Assay, Proliferation Assay, CCK-8 Assay, Colony Assay

Overexpression of LSD1 promotes the invasion and metastasis in GBC cell lines Comparison among the MOCK (untransfected), NC (transfected with the PWPXL-GFP plasmid) and LSD1 overexpression (transfected with the PWPXL-LSD1 plasmid) groups of GBC-SD and NOZ cells on A. Transwell assay, B. wound-healing assay, C. proliferation assay using CCK-8 and D. plate colony formation assay. P value was significant at less than 0.05.
Figure Legend Snippet: Overexpression of LSD1 promotes the invasion and metastasis in GBC cell lines Comparison among the MOCK (untransfected), NC (transfected with the PWPXL-GFP plasmid) and LSD1 overexpression (transfected with the PWPXL-LSD1 plasmid) groups of GBC-SD and NOZ cells on A. Transwell assay, B. wound-healing assay, C. proliferation assay using CCK-8 and D. plate colony formation assay. P value was significant at less than 0.05.

Techniques Used: Over Expression, Transfection, Plasmid Preparation, Transwell Assay, Wound Healing Assay, Proliferation Assay, CCK-8 Assay, Colony Assay

23) Product Images from "Roles of Long Non-Coding RNA CCAT2 in Cervical Cancer Cell Growth and Apoptosis"

Article Title: Roles of Long Non-Coding RNA CCAT2 in Cervical Cancer Cell Growth and Apoptosis

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

doi: 10.12659/MSM.897754

Relative expression of CCAT2 in 3 cervical cancer cell lines was carried out by qRT-PCR ( A ). Knockdown effects of siRNA CCAT2 in HeLa cell line ( B ). CCK8 assay to test the function of lncRNA CCAT2. Downregulation of CCAT2 inhibited HeLa cells growth ( C ). Statistical analyses were performed with the independent samples t test. All data are shown as the mean ±SD.
Figure Legend Snippet: Relative expression of CCAT2 in 3 cervical cancer cell lines was carried out by qRT-PCR ( A ). Knockdown effects of siRNA CCAT2 in HeLa cell line ( B ). CCK8 assay to test the function of lncRNA CCAT2. Downregulation of CCAT2 inhibited HeLa cells growth ( C ). Statistical analyses were performed with the independent samples t test. All data are shown as the mean ±SD.

Techniques Used: Expressing, Quantitative RT-PCR, CCK-8 Assay

24) Product Images from "Comprehensive Identification and Bread-Making Quality Evaluation of Common Wheat Somatic Variation Line AS208 on Glutenin Composition"

Article Title: Comprehensive Identification and Bread-Making Quality Evaluation of Common Wheat Somatic Variation Line AS208 on Glutenin Composition

Journal: PLoS ONE

doi: 10.1371/journal.pone.0146933

Expression profile of the 1Bx20 gene at different development stages in LX987 and AS208 assessed by qRT-PCR. The 1Bx20 gene was not expressed at any stages during grain development in AS208 (A8-A29). Its expression in LX987 started from the 11 th day since anthesis (L11), reached its highest level at the 19 th day post anthesis, and then gradually declined as filling progressed (L21-L29).
Figure Legend Snippet: Expression profile of the 1Bx20 gene at different development stages in LX987 and AS208 assessed by qRT-PCR. The 1Bx20 gene was not expressed at any stages during grain development in AS208 (A8-A29). Its expression in LX987 started from the 11 th day since anthesis (L11), reached its highest level at the 19 th day post anthesis, and then gradually declined as filling progressed (L21-L29).

Techniques Used: Expressing, Quantitative RT-PCR

Identification of seed storage proteins in AS208, LX987, and CS by RP-HPLC. The peaks for albumin (a), globulin (b), and gliadin (c) in AS208 and LX987 were very similar, but were different from those of CS. However, the peak patterns for glutenin (d) were different among AS208, LX987, and CS, in which the three wheat accessions all had peaks for 1Dx2 and 1Dy12, while AS208 was missing peaks for 1Bx20 and 1By20 compared to its progenitor genotype LX987. The grains were harvested in Beijing in 2013.
Figure Legend Snippet: Identification of seed storage proteins in AS208, LX987, and CS by RP-HPLC. The peaks for albumin (a), globulin (b), and gliadin (c) in AS208 and LX987 were very similar, but were different from those of CS. However, the peak patterns for glutenin (d) were different among AS208, LX987, and CS, in which the three wheat accessions all had peaks for 1Dx2 and 1Dy12, while AS208 was missing peaks for 1Bx20 and 1By20 compared to its progenitor genotype LX987. The grains were harvested in Beijing in 2013.

Techniques Used: High Performance Liquid Chromatography

25) Product Images from "miR-98 suppresses melanoma metastasis through a negative feedback loop with its target gene IL-6"

Article Title: miR-98 suppresses melanoma metastasis through a negative feedback loop with its target gene IL-6

Journal: Experimental & Molecular Medicine

doi: 10.1038/emm.2014.63

In vivo validation of the relationship between miR-98 and interleukin-6 (IL-6) production. ( a ) Metastatic tumor size of both the miRNA-negative control (miR-NC) and miR-98 groups over time. Data are presented as mean±s.d. ( n =3 in each group). *A statistically significant difference between the miR-98 and miR-NC groups, P ⩽0.018. ( b ) Representative gross image of lung metastases at day 35. ( c ) Kaplan–Meier survival curve, the dots in the figure indicate the censored cases. Log-rank tests indicate a significant difference between the groups, P =0.076. ( d ) Relative serum IL-6 levels after mice were treated with miRNA-NC or miR-98; n =3 for each group. ( e ) Western blot analysis of total Stat3 and p64 and phosphorylated Stat3 and p65 after 35 days treatment with miR-98 or miRNA-NC in tumor samples at day 35. ( f ) Relative lin28B mRNA levels in tumor samples at day 35 after mice were treated with miRNA-NC or miR-98. Data are presented as mean±s.d. ( n =3 in each group).
Figure Legend Snippet: In vivo validation of the relationship between miR-98 and interleukin-6 (IL-6) production. ( a ) Metastatic tumor size of both the miRNA-negative control (miR-NC) and miR-98 groups over time. Data are presented as mean±s.d. ( n =3 in each group). *A statistically significant difference between the miR-98 and miR-NC groups, P ⩽0.018. ( b ) Representative gross image of lung metastases at day 35. ( c ) Kaplan–Meier survival curve, the dots in the figure indicate the censored cases. Log-rank tests indicate a significant difference between the groups, P =0.076. ( d ) Relative serum IL-6 levels after mice were treated with miRNA-NC or miR-98; n =3 for each group. ( e ) Western blot analysis of total Stat3 and p64 and phosphorylated Stat3 and p65 after 35 days treatment with miR-98 or miRNA-NC in tumor samples at day 35. ( f ) Relative lin28B mRNA levels in tumor samples at day 35 after mice were treated with miRNA-NC or miR-98. Data are presented as mean±s.d. ( n =3 in each group).

Techniques Used: In Vivo, Negative Control, Mouse Assay, Western Blot

26) Product Images from "Retinoic Acid Induces Embryonic Stem Cell Differentiation by Altering Both Encoding RNA and microRNA Expression"

Article Title: Retinoic Acid Induces Embryonic Stem Cell Differentiation by Altering Both Encoding RNA and microRNA Expression

Journal: PLoS ONE

doi: 10.1371/journal.pone.0132566

Changes in the expression of genes involved in ESC self-renewal and differentiation, following retinoic acid (RA) treatment. (A) Alkaline phosphatase staining of J1 mESCs cultured on gelatin-coated plates without LIF and treated with 1 μM RA or DMSO for 24 h. Alkaline phosphatase staining is an indicator of pluripotency. Scale bars = 100 μm. (B, C, D) qPCR, immunofluorescence, and western blot analysis of Oct4 and Nanog expression in J1 mESCs. DAPI was used for nuclear staining. Scale bars represent 100 μm. (E) Expression pattern of the representative upregulated differentiation-associated genes and downregulated pluripotency-related genes. Low intensities are shown in green and high intensities in red. mESCs were treated with DMSO or RA. (F) Representative differentiation-associated genes. The Hoxb gene was upregulated significantly by RA treatment. (G) Representative pluripotency-related genes downregulated in the presence of RA were validated by qPCR. (H) Heatmap diagram of three germ cell marker genes treated with or without RA. Epiblast marker expression was significantly altered by RA. (I) qPCR analysis validated these trends for the altered expression of genes related to growth and development of mESCs. J1 mESCs cultured with DMSO (vehicle) or 1 μM RA for 24 h. Data are presented as the mean ± SEM of three independent experiments (*p
Figure Legend Snippet: Changes in the expression of genes involved in ESC self-renewal and differentiation, following retinoic acid (RA) treatment. (A) Alkaline phosphatase staining of J1 mESCs cultured on gelatin-coated plates without LIF and treated with 1 μM RA or DMSO for 24 h. Alkaline phosphatase staining is an indicator of pluripotency. Scale bars = 100 μm. (B, C, D) qPCR, immunofluorescence, and western blot analysis of Oct4 and Nanog expression in J1 mESCs. DAPI was used for nuclear staining. Scale bars represent 100 μm. (E) Expression pattern of the representative upregulated differentiation-associated genes and downregulated pluripotency-related genes. Low intensities are shown in green and high intensities in red. mESCs were treated with DMSO or RA. (F) Representative differentiation-associated genes. The Hoxb gene was upregulated significantly by RA treatment. (G) Representative pluripotency-related genes downregulated in the presence of RA were validated by qPCR. (H) Heatmap diagram of three germ cell marker genes treated with or without RA. Epiblast marker expression was significantly altered by RA. (I) qPCR analysis validated these trends for the altered expression of genes related to growth and development of mESCs. J1 mESCs cultured with DMSO (vehicle) or 1 μM RA for 24 h. Data are presented as the mean ± SEM of three independent experiments (*p

Techniques Used: Expressing, Staining, Cell Culture, Real-time Polymerase Chain Reaction, Immunofluorescence, Western Blot, Marker

Regulation of epigenetic regulatory gene expression by RA treatment. (A) Expression pattern of representative epigenetic-associated genes identified by microarray data in J1 mESCs. High expression levels are shown in green and low in red. The control group was treated with DMSO. (B) Representative epigenetic-related genes differently expressed in the presence of 1 μM RA were validated by qPCR. (C) Immunostaining assay of global DNA methylation by measurement of levels of 5-mC and 5-hmC in J1 mESCs. DAPI was used for nuclear staining. Scale bars represent 100 μm. (D) Immunostaining assay of H3K9Ac in J1 mESCs. DAPI was used for nuclear staining. Scale bars represent 100 μm. J1 mESCs were cultured in the presence or absence of 1 μM RA for 24 h. Data are presented as the mean ± SEM of three independent experiments (*p
Figure Legend Snippet: Regulation of epigenetic regulatory gene expression by RA treatment. (A) Expression pattern of representative epigenetic-associated genes identified by microarray data in J1 mESCs. High expression levels are shown in green and low in red. The control group was treated with DMSO. (B) Representative epigenetic-related genes differently expressed in the presence of 1 μM RA were validated by qPCR. (C) Immunostaining assay of global DNA methylation by measurement of levels of 5-mC and 5-hmC in J1 mESCs. DAPI was used for nuclear staining. Scale bars represent 100 μm. (D) Immunostaining assay of H3K9Ac in J1 mESCs. DAPI was used for nuclear staining. Scale bars represent 100 μm. J1 mESCs were cultured in the presence or absence of 1 μM RA for 24 h. Data are presented as the mean ± SEM of three independent experiments (*p

Techniques Used: Expressing, Microarray, Real-time Polymerase Chain Reaction, Immunostaining, DNA Methylation Assay, Staining, Cell Culture

27) Product Images from "Quantitative Proteomic Analysis of Wheat Seeds during Artificial Ageing and Priming Using the Isobaric Tandem Mass Tag Labeling"

Article Title: Quantitative Proteomic Analysis of Wheat Seeds during Artificial Ageing and Priming Using the Isobaric Tandem Mass Tag Labeling

Journal: PLoS ONE

doi: 10.1371/journal.pone.0162851

Comparisons of the protein and mRNA expression patterns of three representative DEPs at four artificial ageing stages (WH98, WH50, WH20, and WH01) by iTRAQ and qRT-PCR. Solid lines represent mRNA expression patterns, and dotted lines represent protein expression patterns.
Figure Legend Snippet: Comparisons of the protein and mRNA expression patterns of three representative DEPs at four artificial ageing stages (WH98, WH50, WH20, and WH01) by iTRAQ and qRT-PCR. Solid lines represent mRNA expression patterns, and dotted lines represent protein expression patterns.

Techniques Used: Expressing, Quantitative RT-PCR

28) Product Images from "A virus-like particle-based connective tissue growth factor vaccine suppresses carbon tetrachloride-induced hepatic fibrosis in mice"

Article Title: A virus-like particle-based connective tissue growth factor vaccine suppresses carbon tetrachloride-induced hepatic fibrosis in mice

Journal: Scientific Reports

doi: 10.1038/srep32155

Vaccination against CTGF reduced the expression of profibrogenic factors in the fibrotic livers. Western blotting detection of TGF-β1 ( a ), PDGF-B ( b ), TIMP-1 ( c ) and RQ-PCR evaluation of CTGF mRNA ( d ) revealed that vaccination against CTGF decreased the expressions of TGF-β1, PDGF-B, TIMP-1 and CTGF in CCl 4 -induced fibrotic mouse livers. Western blotting evaluation of the hepatic tissue p-Smad2/Smad2 ratio revealed that the vaccination inhibited Smad2 phosphorylation ( e ). Following Western blotting, bands were quantified with Image-J software. Relative protein abundance in each sample was normalized to that of β-actin. The RQ-PCR results were represented as the means ± SEM of three independent experiments, each of which was performed in triplicate reactions. Error bars indicate SEM. TGF-β1, transforming growth factor β1; PDGF-B, platelet-derived growth factor B; TIMP-1, tissue inhibitor of metalloproteinase-1; RQ-PCR, Real-time quantitative reverse transcriptase polymerase chain reaction.
Figure Legend Snippet: Vaccination against CTGF reduced the expression of profibrogenic factors in the fibrotic livers. Western blotting detection of TGF-β1 ( a ), PDGF-B ( b ), TIMP-1 ( c ) and RQ-PCR evaluation of CTGF mRNA ( d ) revealed that vaccination against CTGF decreased the expressions of TGF-β1, PDGF-B, TIMP-1 and CTGF in CCl 4 -induced fibrotic mouse livers. Western blotting evaluation of the hepatic tissue p-Smad2/Smad2 ratio revealed that the vaccination inhibited Smad2 phosphorylation ( e ). Following Western blotting, bands were quantified with Image-J software. Relative protein abundance in each sample was normalized to that of β-actin. The RQ-PCR results were represented as the means ± SEM of three independent experiments, each of which was performed in triplicate reactions. Error bars indicate SEM. TGF-β1, transforming growth factor β1; PDGF-B, platelet-derived growth factor B; TIMP-1, tissue inhibitor of metalloproteinase-1; RQ-PCR, Real-time quantitative reverse transcriptase polymerase chain reaction.

Techniques Used: Expressing, Western Blot, Polymerase Chain Reaction, Software, Derivative Assay

29) Product Images from "MicroRNA-449a enhances radiosensitivity by downregulation of c-Myc in prostate cancer cells"

Article Title: MicroRNA-449a enhances radiosensitivity by downregulation of c-Myc in prostate cancer cells

Journal: Scientific Reports

doi: 10.1038/srep27346

MiR-449a suppresses c-Myc expression by targeting the 3′-UTR of c-Myc mRNA. ( a ) Comparison of human miR-449a and miR-34a, b and c sequences. Seed sequences are shown in bold. ( b ) Putative miR-449a binding site within the human c-Myc 3′-UTR is shown at the top. The sequences of mature miR-449a aligned to the target site and the 3′-UTR mutated in the miR-449a seed-pairing sequence was shown below. ( c ) Luciferase reporter assay was performed 48 h after co-transfection in LNCaP cells with Wt c-Myc or Mut c-Myc vectors together with miR-449a or negative control. ( d ) qRT-PCR analysis of miR-449a expression at the indicated time points after transfection with miR-449a into LNCaP cells. ( e ) c-Myc expression is repressed by miR-449a at the mRNA level. qRT-PCR was conducted to quantify c-Myc expression at the indicated time point after transfection with miR-449a into LNCaP cells. ( f ) c-Myc expression is repressed by miR-449a at the protein level. Western blotting was performed at the indicated time points after transfection with miR-449a into LNCaP cells. Data are representative of at least three independent experiments. *p
Figure Legend Snippet: MiR-449a suppresses c-Myc expression by targeting the 3′-UTR of c-Myc mRNA. ( a ) Comparison of human miR-449a and miR-34a, b and c sequences. Seed sequences are shown in bold. ( b ) Putative miR-449a binding site within the human c-Myc 3′-UTR is shown at the top. The sequences of mature miR-449a aligned to the target site and the 3′-UTR mutated in the miR-449a seed-pairing sequence was shown below. ( c ) Luciferase reporter assay was performed 48 h after co-transfection in LNCaP cells with Wt c-Myc or Mut c-Myc vectors together with miR-449a or negative control. ( d ) qRT-PCR analysis of miR-449a expression at the indicated time points after transfection with miR-449a into LNCaP cells. ( e ) c-Myc expression is repressed by miR-449a at the mRNA level. qRT-PCR was conducted to quantify c-Myc expression at the indicated time point after transfection with miR-449a into LNCaP cells. ( f ) c-Myc expression is repressed by miR-449a at the protein level. Western blotting was performed at the indicated time points after transfection with miR-449a into LNCaP cells. Data are representative of at least three independent experiments. *p

Techniques Used: Expressing, Binding Assay, Sequencing, Luciferase, Reporter Assay, Cotransfection, Negative Control, Quantitative RT-PCR, Transfection, Western Blot

30) Product Images from "Down-regulation of microRNA-216b inhibits IL-1β-induced chondrocyte injury by up-regulation of Smad3"

Article Title: Down-regulation of microRNA-216b inhibits IL-1β-induced chondrocyte injury by up-regulation of Smad3

Journal: Bioscience Reports

doi: 10.1042/BSR20160588

Effects of miR-216 on IL-1β-induced inhibition of aggrecan and type II collagen synthesis in SW1353 cells SW1353 cells were treated with IL-1β (5 ng/ml) for 24 h after transfection with miR-216b mimic or inhibitor. The mRNA levels of aggrecan and type II collagen were determined by qRT-PCR. The data shown are mean ± S.E.M., n =4; ** P
Figure Legend Snippet: Effects of miR-216 on IL-1β-induced inhibition of aggrecan and type II collagen synthesis in SW1353 cells SW1353 cells were treated with IL-1β (5 ng/ml) for 24 h after transfection with miR-216b mimic or inhibitor. The mRNA levels of aggrecan and type II collagen were determined by qRT-PCR. The data shown are mean ± S.E.M., n =4; ** P

Techniques Used: Inhibition, Transfection, Quantitative RT-PCR

Smad3 was involved in the effects of miR-216b on IL-1β-induced cartilage degradation in SW1353 cells SW1353 cells were transfected with either miR-216b inhibitor or si-Smad3, and then treated with IL-1β (5 ng/ml) for 24 h. ( A ) The mRNA and protein levels of Smad3 were determined by qRT-PCR or Western blot respectively. Smad3 expression was normalized to β-actin. ( B ) Cell proliferation was assessed by BrdU-ELISA assay. ( C ) The mRNA levels of aggrecan, type II collagen, and MMP-13 were determined by qRT-PCR. β-Actin was detected as a loading control. All data are presented as mean ± S.E.M., n =4; ** P
Figure Legend Snippet: Smad3 was involved in the effects of miR-216b on IL-1β-induced cartilage degradation in SW1353 cells SW1353 cells were transfected with either miR-216b inhibitor or si-Smad3, and then treated with IL-1β (5 ng/ml) for 24 h. ( A ) The mRNA and protein levels of Smad3 were determined by qRT-PCR or Western blot respectively. Smad3 expression was normalized to β-actin. ( B ) Cell proliferation was assessed by BrdU-ELISA assay. ( C ) The mRNA levels of aggrecan, type II collagen, and MMP-13 were determined by qRT-PCR. β-Actin was detected as a loading control. All data are presented as mean ± S.E.M., n =4; ** P

Techniques Used: Transfection, Quantitative RT-PCR, Western Blot, Expressing, Enzyme-linked Immunosorbent Assay

31) Product Images from "MicroRNA-27a-mediated repression of cysteine-rich secretory protein 2 translation in asthenoteratozoospermic patients"

Article Title: MicroRNA-27a-mediated repression of cysteine-rich secretory protein 2 translation in asthenoteratozoospermic patients

Journal: Asian Journal of Andrology

doi: 10.4103/1008-682X.185001

The expression of CRISP2 in the ejaculated spermatozoa between asthenoteratozoospermic patients and normozoospermic volunteers. ( a ) The expression level of CRISP2 mRNA is detected by qRT-PCR in the ejaculated spermatozoa between ATZ patients and normozoospermic volunteers (Student's t -test, data are shown as mean ± s.e.m.). ( b ) The CRISP2 protein expression is detected by Western blot analysis in 24 (12 Norm vs 12 ATZ) randomly selected ejaculated spermatozoa. CRISP2: cysteine-rich secretory protein 2; Norm: normozoospermic control group; ATZ: asthenoteratozoospermic patients group; s.e.m.: standard error of the mean.
Figure Legend Snippet: The expression of CRISP2 in the ejaculated spermatozoa between asthenoteratozoospermic patients and normozoospermic volunteers. ( a ) The expression level of CRISP2 mRNA is detected by qRT-PCR in the ejaculated spermatozoa between ATZ patients and normozoospermic volunteers (Student's t -test, data are shown as mean ± s.e.m.). ( b ) The CRISP2 protein expression is detected by Western blot analysis in 24 (12 Norm vs 12 ATZ) randomly selected ejaculated spermatozoa. CRISP2: cysteine-rich secretory protein 2; Norm: normozoospermic control group; ATZ: asthenoteratozoospermic patients group; s.e.m.: standard error of the mean.

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

32) Product Images from "Effect of Wnt Signaling on the Differentiation of Islet β-Cells from Adipose-Derived Stem Cells"

Article Title: Effect of Wnt Signaling on the Differentiation of Islet β-Cells from Adipose-Derived Stem Cells

Journal: BioMed Research International

doi: 10.1155/2017/2501578

(a) Dvl-2 , (b) LRP5 , (c) GSK3β , (d) β-catenin , (e) TCF7L2 , (f) insulin , (g) Gcg , (h) Gck , (i) GLUT2 , (j) Irs1 , and (k) Irs2 mRNA expression in rat ADSCs induced into insulin-producing cells over time by real-time PCR. Data are presented as the mean ± standard deviation. ∗ P
Figure Legend Snippet: (a) Dvl-2 , (b) LRP5 , (c) GSK3β , (d) β-catenin , (e) TCF7L2 , (f) insulin , (g) Gcg , (h) Gck , (i) GLUT2 , (j) Irs1 , and (k) Irs2 mRNA expression in rat ADSCs induced into insulin-producing cells over time by real-time PCR. Data are presented as the mean ± standard deviation. ∗ P

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

33) Product Images from "Enhanced Responses to Angiogenic Cues Underlie the Pathogenesis of Hereditary Hemorrhagic Telangiectasia 2"

Article Title: Enhanced Responses to Angiogenic Cues Underlie the Pathogenesis of Hereditary Hemorrhagic Telangiectasia 2

Journal: PLoS ONE

doi: 10.1371/journal.pone.0063138

Alk1 -null pulmonary ECs displayed elevated migratory index upon angiogenic factor challenge. A. Time-lapse images of scratch-wound closure of 2f/1f- and 1f/1f-ECs in response to bFGF (50 ng/ml). At 12 hours post-wounding, mutant cells almost completely closed the wound, while the wound was still present in the control culture. B. Statistical analysis shows that the rate of wound closure in 1f/1f-ECs was higher than that in 2f/1f-ECs. C. Migration distance of 1f/1f-ECs at the 12-hour time point was higher than that of 2f/1f-ECs. D. In the 3D modified Boyden chamber assay, significantly more migrating 1f/1f-ECs were counted in six randomly chosen fields as compared with 2f/1f-ECs. Note that all data represent means from three independent experiments. Error bars show standard errors. *p
Figure Legend Snippet: Alk1 -null pulmonary ECs displayed elevated migratory index upon angiogenic factor challenge. A. Time-lapse images of scratch-wound closure of 2f/1f- and 1f/1f-ECs in response to bFGF (50 ng/ml). At 12 hours post-wounding, mutant cells almost completely closed the wound, while the wound was still present in the control culture. B. Statistical analysis shows that the rate of wound closure in 1f/1f-ECs was higher than that in 2f/1f-ECs. C. Migration distance of 1f/1f-ECs at the 12-hour time point was higher than that of 2f/1f-ECs. D. In the 3D modified Boyden chamber assay, significantly more migrating 1f/1f-ECs were counted in six randomly chosen fields as compared with 2f/1f-ECs. Note that all data represent means from three independent experiments. Error bars show standard errors. *p

Techniques Used: Mutagenesis, Migration, Modification, Boyden Chamber Assay

BMP-9-induced SMAD1/5/8 phosphorylation is impaired in both 2f/1f- and 1f/1f-ECs. Western blot showing SMAD1/5/8 phosphorylation, total SMAD1 protein, and β-actin in response to various concentrations of BMP-9 (A, B) and BMP-4 (C, D). A. Comparison of SMAD1/5/8 phosphorylation induced by BMP-9 shows no significant difference between 2f/1f and 1f/1f derived from 2f/1f-ECs. B. 2f/2f-ECs are 10-fold more sensitive to BMP-9 than 1f/1f-ECs derived from 2f/2f, suggesting that BMP-9-SMAD1/5/8 signaling is impaired in the 1f/1f-ECs. C and D. There is no difference in SMAD1/5/8 phosphorylation in response to BMP-4 in both 2f/1f (C) and 1f/1f (D) ECs. β-Actin was used as a loading control. Histogram shows phospho-SMAD1/5/8 normalized by total SMAD1 and β-Actin relative to the value indicated by asterisks. Data in all graphs represent means of values measured by densitometry from three separate blots. Error bars are SDs (* p
Figure Legend Snippet: BMP-9-induced SMAD1/5/8 phosphorylation is impaired in both 2f/1f- and 1f/1f-ECs. Western blot showing SMAD1/5/8 phosphorylation, total SMAD1 protein, and β-actin in response to various concentrations of BMP-9 (A, B) and BMP-4 (C, D). A. Comparison of SMAD1/5/8 phosphorylation induced by BMP-9 shows no significant difference between 2f/1f and 1f/1f derived from 2f/1f-ECs. B. 2f/2f-ECs are 10-fold more sensitive to BMP-9 than 1f/1f-ECs derived from 2f/2f, suggesting that BMP-9-SMAD1/5/8 signaling is impaired in the 1f/1f-ECs. C and D. There is no difference in SMAD1/5/8 phosphorylation in response to BMP-4 in both 2f/1f (C) and 1f/1f (D) ECs. β-Actin was used as a loading control. Histogram shows phospho-SMAD1/5/8 normalized by total SMAD1 and β-Actin relative to the value indicated by asterisks. Data in all graphs represent means of values measured by densitometry from three separate blots. Error bars are SDs (* p

Techniques Used: Western Blot, Derivative Assay

Establishment of R26 CreER/+ ; Alk1 2f/1f pulmonary endothelial cell line, and effective deletion of the Alk1 gene by 4TM treatment. A. Schematic diagram of the Alk1 wild-type, Alk1 2loxP (2f), and Alk1 1loxP (1f) alleles. Exons and loxP sequences are denoted by boxes and arrowheads, respectively. Locations of primer pairs detecting specific regions containing loxP sequences for the genomic PCR analysis and the probe recognizing the 5′ region of the Alk1 gene which was used for the genomic Southern blot analysis are indicated. Note that exon 5 encodes the transmembrane domain (indicated by asterisk) of the Alk1 gene. B. After two rounds of FACS with DioAcLDL, about 98% of the sorted immortalized cells were positive for DioAcLDL. C and D. The sorted cells exhibited EC-characteristic polygonal shapes and cell-cell contact inhibition at confluency, and they expressed VE-Cadherin at their junctions. E. Expression of EC-specific marker genes including Flk1, Tie2, Eng, and Edn – along with Alk1 and Smad1– was examined by RT-PCR analysis on clonally expanded 2f/1f-pECs (from 5 cells per well). Amongst several isolated clones, clone #28 showed the EC morphology and the high Alk1 and Edn expression levels and was therefore chosen for further study. β-actin was used for the loading control. E. F. After 2 days of 4TM (1µM) treatment, cells were cultured on normal ECM devoid of 4TM. Primers that can detect both 2f and 1f alleles (3F, 3R, and 6R) amplified both 2f and 1f bands from the parental cells but only the 1f band from 4TM-treated cells. The 1f/1f cell population was maintained for 7 days without additional 4TM treatment. G. Genomic Southern analysis shows efficient genotype conversion from 2f/1f to 1f/1f by 2 days of 4TM treatment. H, I. semi quantitative (H) and quantitative RT-PCR (I) analyses reveal an undetectable level of Alk1 mRNA level in the TM-treated Alk1 2f/1f ECs. Negative control indicates no reverse transcription reaction. Error bars are SDs (** p
Figure Legend Snippet: Establishment of R26 CreER/+ ; Alk1 2f/1f pulmonary endothelial cell line, and effective deletion of the Alk1 gene by 4TM treatment. A. Schematic diagram of the Alk1 wild-type, Alk1 2loxP (2f), and Alk1 1loxP (1f) alleles. Exons and loxP sequences are denoted by boxes and arrowheads, respectively. Locations of primer pairs detecting specific regions containing loxP sequences for the genomic PCR analysis and the probe recognizing the 5′ region of the Alk1 gene which was used for the genomic Southern blot analysis are indicated. Note that exon 5 encodes the transmembrane domain (indicated by asterisk) of the Alk1 gene. B. After two rounds of FACS with DioAcLDL, about 98% of the sorted immortalized cells were positive for DioAcLDL. C and D. The sorted cells exhibited EC-characteristic polygonal shapes and cell-cell contact inhibition at confluency, and they expressed VE-Cadherin at their junctions. E. Expression of EC-specific marker genes including Flk1, Tie2, Eng, and Edn – along with Alk1 and Smad1– was examined by RT-PCR analysis on clonally expanded 2f/1f-pECs (from 5 cells per well). Amongst several isolated clones, clone #28 showed the EC morphology and the high Alk1 and Edn expression levels and was therefore chosen for further study. β-actin was used for the loading control. E. F. After 2 days of 4TM (1µM) treatment, cells were cultured on normal ECM devoid of 4TM. Primers that can detect both 2f and 1f alleles (3F, 3R, and 6R) amplified both 2f and 1f bands from the parental cells but only the 1f band from 4TM-treated cells. The 1f/1f cell population was maintained for 7 days without additional 4TM treatment. G. Genomic Southern analysis shows efficient genotype conversion from 2f/1f to 1f/1f by 2 days of 4TM treatment. H, I. semi quantitative (H) and quantitative RT-PCR (I) analyses reveal an undetectable level of Alk1 mRNA level in the TM-treated Alk1 2f/1f ECs. Negative control indicates no reverse transcription reaction. Error bars are SDs (** p

Techniques Used: Polymerase Chain Reaction, Southern Blot, FACS, Inhibition, Expressing, Marker, Reverse Transcription Polymerase Chain Reaction, Isolation, Clone Assay, Cell Culture, Amplification, Quantitative RT-PCR, Negative Control

Alk1 -deficient ECs form a more persistent and intricate tube-like network and are resistant to BMP-9. The same number (1×10 5 ) of 2f/1f- and 1f/1f-ECs suspended in chemically defined growth factor- and serum-free ECM containing bFGF (50 ng/ml) and various concentrations of BMP-9 (0, 1, 5, and 20 ng/ml) were seeded onto the Matrigel in 24-well plates. A, B. Representative images of the tube-like network of 2f/1f ( A ) and 1f/1f ( B ) pECs at 3, 6, 9, 12, 24, and 48 hrs after plating. C, D. Histogram showing the total tubular lengths of 2f/1f ( C ) and 1f/1f ( D ) ECs. Tubular lengths tended to be lower at all time points for 5 and 20 ng/ml of BMP-9 treated 2f/1f-ECs, which reached a statistically significant level at 24 and 48 hours (C), whereas no such a trend was observed in 1f/1f-ECs (D). *P
Figure Legend Snippet: Alk1 -deficient ECs form a more persistent and intricate tube-like network and are resistant to BMP-9. The same number (1×10 5 ) of 2f/1f- and 1f/1f-ECs suspended in chemically defined growth factor- and serum-free ECM containing bFGF (50 ng/ml) and various concentrations of BMP-9 (0, 1, 5, and 20 ng/ml) were seeded onto the Matrigel in 24-well plates. A, B. Representative images of the tube-like network of 2f/1f ( A ) and 1f/1f ( B ) pECs at 3, 6, 9, 12, 24, and 48 hrs after plating. C, D. Histogram showing the total tubular lengths of 2f/1f ( C ) and 1f/1f ( D ) ECs. Tubular lengths tended to be lower at all time points for 5 and 20 ng/ml of BMP-9 treated 2f/1f-ECs, which reached a statistically significant level at 24 and 48 hours (C), whereas no such a trend was observed in 1f/1f-ECs (D). *P

Techniques Used:

34) Product Images from "Pyoderma gangrenosum after trauma in a dog"

Article Title: Pyoderma gangrenosum after trauma in a dog

Journal: The Journal of Veterinary Medical Science

doi: 10.1292/jvms.15-0724

Relative transcription levels of cytokine mRNA in lesional skin before treatment (Before), skin after treatment (After) and normal skin of healthy dogs (Healthy, n=5). Transcription levels of tumor necrosis factor (TNF)-α and interleukin (IL)-1β, IL-8 and IL-17A were higher in lesional skin before treatment than in skin after treatment or normal skin of healthy dogs. N.D.: not detected.
Figure Legend Snippet: Relative transcription levels of cytokine mRNA in lesional skin before treatment (Before), skin after treatment (After) and normal skin of healthy dogs (Healthy, n=5). Transcription levels of tumor necrosis factor (TNF)-α and interleukin (IL)-1β, IL-8 and IL-17A were higher in lesional skin before treatment than in skin after treatment or normal skin of healthy dogs. N.D.: not detected.

Techniques Used:

35) Product Images from "Identification of Differentially Expressed Non-coding RNA in Porcine Alveolar Macrophages from Tongcheng and Large White Pigs Responded to PRRSV"

Article Title: Identification of Differentially Expressed Non-coding RNA in Porcine Alveolar Macrophages from Tongcheng and Large White Pigs Responded to PRRSV

Journal: Scientific Reports

doi: 10.1038/s41598-018-33891-0

Log 2 (FC) obtained from stem-loop qRT-PCR and small RNA-seq data. x -axis is the name of selected DEmiRNA and y -axis is the value of log 2 (FC).
Figure Legend Snippet: Log 2 (FC) obtained from stem-loop qRT-PCR and small RNA-seq data. x -axis is the name of selected DEmiRNA and y -axis is the value of log 2 (FC).

Techniques Used: Quantitative RT-PCR, RNA Sequencing Assay

miR-199a-3p could inhibit the expression of CD151. Endogenous CD151 mRNA ( a ) and protein ( b ) expression under the condition of miR-199a-3p overexpression (NC, negative control; MUT, seed region mutated type; WT, wild type) was detected by qRT-PCR and Western blot, respectively; ( c ) Relative expression of CD151 normalized to GAPDH. Every four bands were a replicate, and this experiment was repeated for three times. Student’s t -test was used to compare the differences between miR-199-3p-MUT/WT and NC, and one star (*) represented p
Figure Legend Snippet: miR-199a-3p could inhibit the expression of CD151. Endogenous CD151 mRNA ( a ) and protein ( b ) expression under the condition of miR-199a-3p overexpression (NC, negative control; MUT, seed region mutated type; WT, wild type) was detected by qRT-PCR and Western blot, respectively; ( c ) Relative expression of CD151 normalized to GAPDH. Every four bands were a replicate, and this experiment was repeated for three times. Student’s t -test was used to compare the differences between miR-199-3p-MUT/WT and NC, and one star (*) represented p

Techniques Used: Expressing, Over Expression, Negative Control, Quantitative RT-PCR, Western Blot

( a ) Log2 (FC) obtained from qRT-PCR and RNA-seq data. X-axis is the name of selected DElincRNA and Y-axis is the value of log2 (FC); ( b ) Tissue/cell expression analysis of five lincRNAs. MLN represents for mesenteric lymph nodes and ILN represents for inguinal lymph nodes.
Figure Legend Snippet: ( a ) Log2 (FC) obtained from qRT-PCR and RNA-seq data. X-axis is the name of selected DElincRNA and Y-axis is the value of log2 (FC); ( b ) Tissue/cell expression analysis of five lincRNAs. MLN represents for mesenteric lymph nodes and ILN represents for inguinal lymph nodes.

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

36) Product Images from "Elevated 14,15- epoxyeicosatrienoic acid by increasing of cytochrome P450 2C8, 2C9 and 2J2 and decreasing of soluble epoxide hydrolase associated with aggressiveness of human breast cancer"

Article Title: Elevated 14,15- epoxyeicosatrienoic acid by increasing of cytochrome P450 2C8, 2C9 and 2J2 and decreasing of soluble epoxide hydrolase associated with aggressiveness of human breast cancer

Journal: BMC Cancer

doi: 10.1186/1471-2407-14-841

Blockage of cancer-derived EETs by siRNA targeting CYP or overexpression of sEH inhibits proliferation and migration of breast cancer cells. RT-PCR analysis of mRNA expression of CYP2C8, 2C9, and 2J2, and sEH in MDA-MB-231 cells treated with siRNA (A) or adenovirus-sEH (B) for 24 hr. (C) MTT assay of cell proliferation, expressed as the normalized mean OD 490 . (D) Transwell assay for cell migration. The number of migrated cells was measured by counting 5 randomly chosen fields under a microscope. (E) Representative transwell assay of cells stained with crystal violet (200X). Data are mean ± SD from 3 independent experiments each performed in triplicate (*P
Figure Legend Snippet: Blockage of cancer-derived EETs by siRNA targeting CYP or overexpression of sEH inhibits proliferation and migration of breast cancer cells. RT-PCR analysis of mRNA expression of CYP2C8, 2C9, and 2J2, and sEH in MDA-MB-231 cells treated with siRNA (A) or adenovirus-sEH (B) for 24 hr. (C) MTT assay of cell proliferation, expressed as the normalized mean OD 490 . (D) Transwell assay for cell migration. The number of migrated cells was measured by counting 5 randomly chosen fields under a microscope. (E) Representative transwell assay of cells stained with crystal violet (200X). Data are mean ± SD from 3 independent experiments each performed in triplicate (*P

Techniques Used: Derivative Assay, Over Expression, Migration, Reverse Transcription Polymerase Chain Reaction, Expressing, Multiple Displacement Amplification, MTT Assay, Transwell Assay, Microscopy, Staining

Expression and distribution of CYP2C8, 2C9, and 2J2, and sEH in breast cancer. qRT-PCR analysis of mRNA expression (A-D) and immunohistochemistry (E) of CYP2C8, 2C9, and 2J2, and sEH in breast cancer and paired adjacent noncancerous tissue. Human hepatocellular carcinoma tissues were used as a positive control. The red staining was by AEC and the nuclei were lightly stained with haematoxylin (original magnification 200X).
Figure Legend Snippet: Expression and distribution of CYP2C8, 2C9, and 2J2, and sEH in breast cancer. qRT-PCR analysis of mRNA expression (A-D) and immunohistochemistry (E) of CYP2C8, 2C9, and 2J2, and sEH in breast cancer and paired adjacent noncancerous tissue. Human hepatocellular carcinoma tissues were used as a positive control. The red staining was by AEC and the nuclei were lightly stained with haematoxylin (original magnification 200X).

Techniques Used: Expressing, Quantitative RT-PCR, Immunohistochemistry, Positive Control, Staining

37) Product Images from "MiR-34c induces apoptosis and inhibits the viability of M4e cells by targeting BCL2"

Article Title: MiR-34c induces apoptosis and inhibits the viability of M4e cells by targeting BCL2

Journal: Oncology Letters

doi: 10.3892/ol.2017.7640

Effect of miR-34c on BCL2 mRNA and protein expression. Analyses by Reverse transcription-quantitative polymerase chain reaction and western blotting were employed to investigate the (A) mRNA and (B) protein expression level of BCL2, respectively. All data are expressed as the mean ± standard deviation of three independent experiments. The cells were infected with lenti-miR-34c or scramble for 72 h. **P
Figure Legend Snippet: Effect of miR-34c on BCL2 mRNA and protein expression. Analyses by Reverse transcription-quantitative polymerase chain reaction and western blotting were employed to investigate the (A) mRNA and (B) protein expression level of BCL2, respectively. All data are expressed as the mean ± standard deviation of three independent experiments. The cells were infected with lenti-miR-34c or scramble for 72 h. **P

Techniques Used: Expressing, Real-time Polymerase Chain Reaction, Western Blot, Standard Deviation, Infection

38) Product Images from "Circular RNA hsa_circ_0000885 Levels are Increased in Tissue and Serum Samples from Patients with Osteosarcoma"

Article Title: Circular RNA hsa_circ_0000885 Levels are Increased in Tissue and Serum Samples from Patients with Osteosarcoma

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

doi: 10.12659/MSM.914899

Serum hsa_circ_0000885 levels were increased in serum from patients with osteosarcoma. ( A ) Expression levels of hsa_circ_0000885 in 30 patients with osteosarcoma, 27 patients with benign bone tumors, and 25 age-matched and sex-matched healthy controls were analyzed by quantitative reverse transcription polymerase chain reaction (qRT-PCR) and normalized to the GAPDH level. ( B ) Relative expression of hsa_circ_0000885 in 11 patients with osteosarcoma, Enneking stage I or IIA and 19 patients with osteosarcoma, Enneking stage IB or III. ( C ) Relative expression of hsa_circ_0000885 in 20 patients with osteosarcoma without metastasis and 10 patients with osteosarcoma with lung metastasis. ( D ) Relative expression of hsa_circ_0000885 in 30 patients with osteosarcoma before and after chemotherapy. ( E ) Relative expression of hsa_circ_0000885 in 30 patients with osteosarcoma before and after surgery. ( F ) Receiver operating characteristic (ROC) curve shows the diagnostic value of hsa_circ_0000885 in distinguishing between patients with osteosarcoma from healthy individuals. ( G ) ROC curve shows the diagnostic value of hsa_circ_0000885 in distinguishing between patients with osteosarcoma and benign bone tumors.
Figure Legend Snippet: Serum hsa_circ_0000885 levels were increased in serum from patients with osteosarcoma. ( A ) Expression levels of hsa_circ_0000885 in 30 patients with osteosarcoma, 27 patients with benign bone tumors, and 25 age-matched and sex-matched healthy controls were analyzed by quantitative reverse transcription polymerase chain reaction (qRT-PCR) and normalized to the GAPDH level. ( B ) Relative expression of hsa_circ_0000885 in 11 patients with osteosarcoma, Enneking stage I or IIA and 19 patients with osteosarcoma, Enneking stage IB or III. ( C ) Relative expression of hsa_circ_0000885 in 20 patients with osteosarcoma without metastasis and 10 patients with osteosarcoma with lung metastasis. ( D ) Relative expression of hsa_circ_0000885 in 30 patients with osteosarcoma before and after chemotherapy. ( E ) Relative expression of hsa_circ_0000885 in 30 patients with osteosarcoma before and after surgery. ( F ) Receiver operating characteristic (ROC) curve shows the diagnostic value of hsa_circ_0000885 in distinguishing between patients with osteosarcoma from healthy individuals. ( G ) ROC curve shows the diagnostic value of hsa_circ_0000885 in distinguishing between patients with osteosarcoma and benign bone tumors.

Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction, Quantitative RT-PCR, Diagnostic Assay

Increased hsa_circ_0000885 expression was correlated with poor clinical prognosis. ( A ) Disease-free survival (DFS) in patients with high hsa_circ_0000885 expression and low hsa_circ_0000885 expression (p
Figure Legend Snippet: Increased hsa_circ_0000885 expression was correlated with poor clinical prognosis. ( A ) Disease-free survival (DFS) in patients with high hsa_circ_0000885 expression and low hsa_circ_0000885 expression (p

Techniques Used: Expressing

hsa_circ_0000885 expression was upregulated in osteosarcoma tissue. ( A ) The heatmap shows the circRNA expression profiles of osteosarcoma tissues and adjacent non-tumor tissues. Red and green indicate high and low expression, respectively. ( B ) Expression levels of hsa_circ_0000885 in 50 pairs of osteosarcoma tissues and adjacent non-tumor tissues, analyzed by quantitative real-time polymerase chain reaction (qRT-PCR) and normalized to GAPDH. The results were analyzed using the 2 −ΔΔCT method and compared using paired Student’s t-tests. ( C ) Relative hsa_circ_0000885 expression ratios in osteosarcoma tissues versus adjacent non-tumor tissues shown on the logarithmic scale. Data are shown as the means ± standard deviation (SD). *** P
Figure Legend Snippet: hsa_circ_0000885 expression was upregulated in osteosarcoma tissue. ( A ) The heatmap shows the circRNA expression profiles of osteosarcoma tissues and adjacent non-tumor tissues. Red and green indicate high and low expression, respectively. ( B ) Expression levels of hsa_circ_0000885 in 50 pairs of osteosarcoma tissues and adjacent non-tumor tissues, analyzed by quantitative real-time polymerase chain reaction (qRT-PCR) and normalized to GAPDH. The results were analyzed using the 2 −ΔΔCT method and compared using paired Student’s t-tests. ( C ) Relative hsa_circ_0000885 expression ratios in osteosarcoma tissues versus adjacent non-tumor tissues shown on the logarithmic scale. Data are shown as the means ± standard deviation (SD). *** P

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

39) Product Images from "PAX3 is a novel tumor suppressor by regulating the activities of major signaling pathways and transcription factor FOXO3a in thyroid cancer"

Article Title: PAX3 is a novel tumor suppressor by regulating the activities of major signaling pathways and transcription factor FOXO3a in thyroid cancer

Journal: Oncotarget

doi: 10.18632/oncotarget.10753

Inhibition of EMT process and the expression of metastasis-associated genes by PAX3 in thyroid cancer cells To test the effect of PAX3 re-expression on the process of EMT, the expression of E-cadherin, Vimentin and N-cadherin was determined at both mRNA ( A ) and protein ( B ) levels in the indicated cells by using qRT-PCR and western blot assays, respectively. 18S rRNA was used as an endogenous control for qRT-PCR assay. GAPDH was used as loading control. ( C ) Immunofluorescence staining was performed to assess the expression of E-cadherin, Vimentin and N-cadherin proteins in the indicated cells. Red color represents target protein fluorescence and blue color represents Hoechst33342 staining for nuclei. ( D ) qRT-PCR assay was used to test the effect of PAX3 re-expression on mRNA expression of E-cadherin transcription suppressors Twist , Snail and Slug in the indicated cells. ( E ) qRT-PCR assay was also used to evaluate the effect of PAX3 re-expression on expression of metastasis-related genes MMP - 2 , - 9 and - 14 in the indicated cells. 18S rRNA was used as a normalized control. Data were presented as mean ± SD. Statistically significant differences were indicated: * P
Figure Legend Snippet: Inhibition of EMT process and the expression of metastasis-associated genes by PAX3 in thyroid cancer cells To test the effect of PAX3 re-expression on the process of EMT, the expression of E-cadherin, Vimentin and N-cadherin was determined at both mRNA ( A ) and protein ( B ) levels in the indicated cells by using qRT-PCR and western blot assays, respectively. 18S rRNA was used as an endogenous control for qRT-PCR assay. GAPDH was used as loading control. ( C ) Immunofluorescence staining was performed to assess the expression of E-cadherin, Vimentin and N-cadherin proteins in the indicated cells. Red color represents target protein fluorescence and blue color represents Hoechst33342 staining for nuclei. ( D ) qRT-PCR assay was used to test the effect of PAX3 re-expression on mRNA expression of E-cadherin transcription suppressors Twist , Snail and Slug in the indicated cells. ( E ) qRT-PCR assay was also used to evaluate the effect of PAX3 re-expression on expression of metastasis-related genes MMP - 2 , - 9 and - 14 in the indicated cells. 18S rRNA was used as a normalized control. Data were presented as mean ± SD. Statistically significant differences were indicated: * P

Techniques Used: Inhibition, Expressing, Quantitative RT-PCR, Western Blot, Immunofluorescence, Staining, Fluorescence

Inhibition of cell proliferation and colony formation by PAX3 in thyroid cancer cells Ectopic expression of PAX3 mRNA ( A ) and protein ( B ) in thyroid cancer cell lines BCPAP, 8305C and FTC133 was evidenced by qRT-PCR and western blot assays, respectively. 18S rRNA was used as a normalized control for qRT-PCR assay. GAPDH was used as loading control in western blot assay. ( C ) PAX3 re-expression significantly inhibited cell proliferation in thyroid cancer cells. ( D ) The effect of PAX3 re-expression on cell growth was further confirmed by colony formation assay. Left panel shows the representative images of colony formation in cells transfected with pcDNA3.1-PAX3 or empty vector. Quantitative analysis of colony numbers is shown in the right panel. Data were presented as mean ± SD. Statistically significant differences were indicated: * P
Figure Legend Snippet: Inhibition of cell proliferation and colony formation by PAX3 in thyroid cancer cells Ectopic expression of PAX3 mRNA ( A ) and protein ( B ) in thyroid cancer cell lines BCPAP, 8305C and FTC133 was evidenced by qRT-PCR and western blot assays, respectively. 18S rRNA was used as a normalized control for qRT-PCR assay. GAPDH was used as loading control in western blot assay. ( C ) PAX3 re-expression significantly inhibited cell proliferation in thyroid cancer cells. ( D ) The effect of PAX3 re-expression on cell growth was further confirmed by colony formation assay. Left panel shows the representative images of colony formation in cells transfected with pcDNA3.1-PAX3 or empty vector. Quantitative analysis of colony numbers is shown in the right panel. Data were presented as mean ± SD. Statistically significant differences were indicated: * P

Techniques Used: Inhibition, Expressing, Quantitative RT-PCR, Western Blot, Colony Assay, Transfection, Plasmid Preparation

Modulation of the activities of major signaling pathways and FOXO3a by PAX3 in thyroid cancer cells ( A ) The indicated cells were lysed and lysates were subjected to western blot analysis. The antibodies against phospho-Akt (p-Akt), total Akt (t-Akt), phospho-Erk (p-Erk) and total Erk (t-Erk) were used to determine the effect of PAX3 re-expression on the activities of PI3K/Akt and MAPK/Erk pathways. The antibodies against p21, p27, p16, phospho-Rb (p-Rb) and Rb were used to determine the effect of PAX3 re-expression on the regulation of cell cycle. The antibodies against p53, Bax, Bak, Smac, activated caspase-3 and -9 were used to determine the effect of PAX3 re-expression on cell apoptosis. GAPDH was used as loading control. ( B ) Total RNA and protein were extracted from the indicated cells. qRT-PCR assay was performed to determine the effect of PAX3 re-expression on FOXO3a expression (left panel). Western blotting was used to assess the effect of PAX3 re-expression on the levels of FOXO3a protein and phosphorylated FOXO3a (p-FOXO3a) (right panel). 18S rRNA was used as an endogenous control for qRT-PCR assay. GAPDH was used as loading control for western blot analysis. ( C ) Immunofluorescence assay was used to test the effect of PAX3 re-expression on the expression and cellular localization of FOXO3a (upper panel). Colocalization of FOXO3a with Hoechst33342 was quantified in low panel. Green color represents FOXO3a fluorescence and blue color represents Hoechst33342 staining for nuclei. ( D ) qRT-PCR assay was used to test the effect of PAX3 re-expression on the expression of a set of cell cycle- and apoptosis-related genes. Expression levels of these genes were normalized with 18S rRNA levels. Data were presented as mean ± SD. Statistically significant differences were indicated: * P
Figure Legend Snippet: Modulation of the activities of major signaling pathways and FOXO3a by PAX3 in thyroid cancer cells ( A ) The indicated cells were lysed and lysates were subjected to western blot analysis. The antibodies against phospho-Akt (p-Akt), total Akt (t-Akt), phospho-Erk (p-Erk) and total Erk (t-Erk) were used to determine the effect of PAX3 re-expression on the activities of PI3K/Akt and MAPK/Erk pathways. The antibodies against p21, p27, p16, phospho-Rb (p-Rb) and Rb were used to determine the effect of PAX3 re-expression on the regulation of cell cycle. The antibodies against p53, Bax, Bak, Smac, activated caspase-3 and -9 were used to determine the effect of PAX3 re-expression on cell apoptosis. GAPDH was used as loading control. ( B ) Total RNA and protein were extracted from the indicated cells. qRT-PCR assay was performed to determine the effect of PAX3 re-expression on FOXO3a expression (left panel). Western blotting was used to assess the effect of PAX3 re-expression on the levels of FOXO3a protein and phosphorylated FOXO3a (p-FOXO3a) (right panel). 18S rRNA was used as an endogenous control for qRT-PCR assay. GAPDH was used as loading control for western blot analysis. ( C ) Immunofluorescence assay was used to test the effect of PAX3 re-expression on the expression and cellular localization of FOXO3a (upper panel). Colocalization of FOXO3a with Hoechst33342 was quantified in low panel. Green color represents FOXO3a fluorescence and blue color represents Hoechst33342 staining for nuclei. ( D ) qRT-PCR assay was used to test the effect of PAX3 re-expression on the expression of a set of cell cycle- and apoptosis-related genes. Expression levels of these genes were normalized with 18S rRNA levels. Data were presented as mean ± SD. Statistically significant differences were indicated: * P

Techniques Used: Western Blot, Expressing, Quantitative RT-PCR, Immunofluorescence, Fluorescence, Staining

PAX3 inactivation by promoter hypermethylation in primary PTCs and thyroid cancer cell lines ( A ) qRT-PCR assay was performed to evaluate mRNA expression of PAX3 in primary PTCs and their matched non-cancerous thyroid tissues ( n = 17). PAX3 expression was normalized with 18S rRNA levels. Data are shown as Log2 fold change of PAX3 expression in tumor/non-cancerous tissues. ( B ) Western blotting was performed to analyze the protein levels of PAX3 in PTCs (T) and matched non-cancerous tissues (N). GAPDH was used as loading control. ( C ) Promoter methylation of PAX3 in primary PTCs (upper panel) and thyroid cancer cell lines (lower panel) was determined with MSP assay. In vitro methylated DNA was used as positive control for methylated gene (Pos-M), bisulfite-modified normal leukocyte DNA as positive control for unmethylated gene (Pos-U), and H 2 O as blank control to confirm the specificity of MSP. Mk, DNA marker; M, methylated gene; U, unmethylated gene. PTC-1 and -2 present two PTC cases with different methylation status of PAX3 . ( D ) PAX3 expression in thyroid cancer cell lines was determined by conventional RT-PCR. β-actin was run as loading control. ( E ) PAX3 expression was restored after treatment with 5-Aza-dC or SAHA. Its expression was determined by qRT-PCR. 18S rRNA was used as a normalized control. Data were presented as mean ± SD. Statistically significant differences were indicated: * P
Figure Legend Snippet: PAX3 inactivation by promoter hypermethylation in primary PTCs and thyroid cancer cell lines ( A ) qRT-PCR assay was performed to evaluate mRNA expression of PAX3 in primary PTCs and their matched non-cancerous thyroid tissues ( n = 17). PAX3 expression was normalized with 18S rRNA levels. Data are shown as Log2 fold change of PAX3 expression in tumor/non-cancerous tissues. ( B ) Western blotting was performed to analyze the protein levels of PAX3 in PTCs (T) and matched non-cancerous tissues (N). GAPDH was used as loading control. ( C ) Promoter methylation of PAX3 in primary PTCs (upper panel) and thyroid cancer cell lines (lower panel) was determined with MSP assay. In vitro methylated DNA was used as positive control for methylated gene (Pos-M), bisulfite-modified normal leukocyte DNA as positive control for unmethylated gene (Pos-U), and H 2 O as blank control to confirm the specificity of MSP. Mk, DNA marker; M, methylated gene; U, unmethylated gene. PTC-1 and -2 present two PTC cases with different methylation status of PAX3 . ( D ) PAX3 expression in thyroid cancer cell lines was determined by conventional RT-PCR. β-actin was run as loading control. ( E ) PAX3 expression was restored after treatment with 5-Aza-dC or SAHA. Its expression was determined by qRT-PCR. 18S rRNA was used as a normalized control. Data were presented as mean ± SD. Statistically significant differences were indicated: * P

Techniques Used: Quantitative RT-PCR, Expressing, Western Blot, Methylation, MSP Assay, In Vitro, Positive Control, Modification, Marker, Reverse Transcription Polymerase Chain Reaction

40) Product Images from "Suppression of autophagy by mycophenolic acid contributes to inhibition of HCV replication in human hepatoma cells"

Article Title: Suppression of autophagy by mycophenolic acid contributes to inhibition of HCV replication in human hepatoma cells

Journal: Scientific Reports

doi: 10.1038/srep44039

The effects of MPA treatment on LC3 and p62 levels in Huh7 cells, tunicamycin(Tu)-treated, or HCV-infected Huh 7 cells in the presence of Bafilomycin A1 (Baf A1). After 2 h pre-incubation with the autophagy inhibitor Baf A1 (5 nM), ( a – c ) Huh7 cells were treated or untreated with MPA (5 μg/mL) for 24 h; ( d – f ) Huh7 cells treated with tunicamycin (5 μg/mL) were simultaneously treated or untreated with MPA (5 μg/mL) for 24 h; ( g – i ) Huh 7 cells were infected with HCV JFH-1 at an MOI of 0.1. At day 3 postinfection, the HCV-infected cells were treated or untreated with MPA (5 μg/mL) for 24 h. After MPA treatment, the cellular RNA and proteins were extracted for real time RT PCR and western blot analysis. ( a , d , g ) The levels of intracellular LC3 mRNA in MPA-treated or untreated cells under different conditions, with normalization to corresponding GAPDH mRNA level, are expressed as the fold of control (without MPA treatment under different conditions, which was defined as 1, respectively; in Huh 7 cells, Tu-treated, or HCV-infected cells, the control group is MPA untreated cells, MPA untreated Tu-treated cells, or MPA untreated HCV-infected cells, respectively). ( b , e , h ) Representative western blot images show LC3B-I, LC3B-II, p62 protein levels in MPA-treated or untreated cells under different conditions. ( c , f , i ) Quantitative assessment of LC3B-I, LC3B-II, LC3B-II/LC3B-I, and p62 at protein level in MPA-treated or untreated cells under different conditions. The densitometric intensities of LC3B-I, LC3B-II, p62 and β-actin bands were quantified by image J software. The relative LC3B-I/β-actin, LC3B-II/β-actin, LC3B-II/LC3B-I, and p62/β-actin ratios were calculated and shown as the fold of control (without MPA treatment under different conditions, which was defined as 1, respectively). The data shown in Fig. 3a,c,d,f,g,i, are the mean ± SD of the results of three independent experiments. The p value was calculated by Student’s t -test (* p
Figure Legend Snippet: The effects of MPA treatment on LC3 and p62 levels in Huh7 cells, tunicamycin(Tu)-treated, or HCV-infected Huh 7 cells in the presence of Bafilomycin A1 (Baf A1). After 2 h pre-incubation with the autophagy inhibitor Baf A1 (5 nM), ( a – c ) Huh7 cells were treated or untreated with MPA (5 μg/mL) for 24 h; ( d – f ) Huh7 cells treated with tunicamycin (5 μg/mL) were simultaneously treated or untreated with MPA (5 μg/mL) for 24 h; ( g – i ) Huh 7 cells were infected with HCV JFH-1 at an MOI of 0.1. At day 3 postinfection, the HCV-infected cells were treated or untreated with MPA (5 μg/mL) for 24 h. After MPA treatment, the cellular RNA and proteins were extracted for real time RT PCR and western blot analysis. ( a , d , g ) The levels of intracellular LC3 mRNA in MPA-treated or untreated cells under different conditions, with normalization to corresponding GAPDH mRNA level, are expressed as the fold of control (without MPA treatment under different conditions, which was defined as 1, respectively; in Huh 7 cells, Tu-treated, or HCV-infected cells, the control group is MPA untreated cells, MPA untreated Tu-treated cells, or MPA untreated HCV-infected cells, respectively). ( b , e , h ) Representative western blot images show LC3B-I, LC3B-II, p62 protein levels in MPA-treated or untreated cells under different conditions. ( c , f , i ) Quantitative assessment of LC3B-I, LC3B-II, LC3B-II/LC3B-I, and p62 at protein level in MPA-treated or untreated cells under different conditions. The densitometric intensities of LC3B-I, LC3B-II, p62 and β-actin bands were quantified by image J software. The relative LC3B-I/β-actin, LC3B-II/β-actin, LC3B-II/LC3B-I, and p62/β-actin ratios were calculated and shown as the fold of control (without MPA treatment under different conditions, which was defined as 1, respectively). The data shown in Fig. 3a,c,d,f,g,i, are the mean ± SD of the results of three independent experiments. The p value was calculated by Student’s t -test (* p

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

The effects of MPA treatment on LC3 and p62 levels in Huh7 cells, tunicamycin(Tu)-treated, or HCV-infected Huh 7 cells in the absence of Bafilomycin A1 (Baf A1). ( a – c ) Huh7 cells were treated or untreated with MPA (5 μg/mL) for 24 h; ( d – f) Huh7 cells treated with tunicamycin (5 μg/mL) were simultaneously treated or untreated with MPA (5 μg/mL) for 24 h; ( g – i ) Huh 7 cells were infected with HCV JFH-1 at an MOI of 0.1. At day 3 postinfection, the HCV-infected cells were treated or untreated with MPA (5 μg/mL) for 24 h. After MPA treatment, the cellular RNA and proteins were extracted for real time RT PCR and western blot analysis. ( a , d , g ) The levels of intracellular LC3 mRNA in MPA-treated or untreated cells under different conditions, with normalization to corresponding GAPDH mRNA level, are expressed as the fold of control (without MPA treatment under different conditions, which was defined as 1, respectively; in Huh 7 cells, Tu-treated, or HCV-infected cells, the control group is MPA untreated cells, MPA untreated Tu-treated cells, or MPA untreated HCV-infected cells, respectively). ( b , e , h ) Representative western blot images show LC3B-I, LC3B-II, p62 protein levels in MPA-treated or untreated cells under different conditions. ( c , f , i ) Quantitative assessment of LC3B-I, LC3B-II, LC3B-II/LC3B-I, and p62 at protein level in MPA-treated or untreated cells under different conditions. The densitometric intensities of LC3B-I, LC3B-II, p62 and β-actin bands were quantified by image J software. The relative LC3B-I/β-actin, LC3B-II/β-actin, LC3B-II/LC3B-I, and p62/β-actin ratios were calculated and shown as the fold of control (without MPA treatment under different conditions, which was defined as 1, respectively). The data shown in Fig. 2a,c,d,f,g,i, are the mean ± SD of the results of three independent experiments. The p value was calculated by Student’s t -test (* p
Figure Legend Snippet: The effects of MPA treatment on LC3 and p62 levels in Huh7 cells, tunicamycin(Tu)-treated, or HCV-infected Huh 7 cells in the absence of Bafilomycin A1 (Baf A1). ( a – c ) Huh7 cells were treated or untreated with MPA (5 μg/mL) for 24 h; ( d – f) Huh7 cells treated with tunicamycin (5 μg/mL) were simultaneously treated or untreated with MPA (5 μg/mL) for 24 h; ( g – i ) Huh 7 cells were infected with HCV JFH-1 at an MOI of 0.1. At day 3 postinfection, the HCV-infected cells were treated or untreated with MPA (5 μg/mL) for 24 h. After MPA treatment, the cellular RNA and proteins were extracted for real time RT PCR and western blot analysis. ( a , d , g ) The levels of intracellular LC3 mRNA in MPA-treated or untreated cells under different conditions, with normalization to corresponding GAPDH mRNA level, are expressed as the fold of control (without MPA treatment under different conditions, which was defined as 1, respectively; in Huh 7 cells, Tu-treated, or HCV-infected cells, the control group is MPA untreated cells, MPA untreated Tu-treated cells, or MPA untreated HCV-infected cells, respectively). ( b , e , h ) Representative western blot images show LC3B-I, LC3B-II, p62 protein levels in MPA-treated or untreated cells under different conditions. ( c , f , i ) Quantitative assessment of LC3B-I, LC3B-II, LC3B-II/LC3B-I, and p62 at protein level in MPA-treated or untreated cells under different conditions. The densitometric intensities of LC3B-I, LC3B-II, p62 and β-actin bands were quantified by image J software. The relative LC3B-I/β-actin, LC3B-II/β-actin, LC3B-II/LC3B-I, and p62/β-actin ratios were calculated and shown as the fold of control (without MPA treatment under different conditions, which was defined as 1, respectively). The data shown in Fig. 2a,c,d,f,g,i, are the mean ± SD of the results of three independent experiments. The p value was calculated by Student’s t -test (* p

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

The effects of MPA treatment on the expression of autophagy-related genes (ATGs) in Huh7 cells or HCV-infected cells. ( a – c ) Huh7 cells were treated or untreated with MPA (5 μg/mL) for 24 h; ( d – f) Huh 7 cells were infected with HCV JFH-1 at an MOI of 0.1. At day 3 postinfection, the HCV-infected cells were treated or untreated with MPA (5 μg/mL) for 24 h. After MPA treatment, the cellular RNA and proteins were extracted for real time RT PCR and western blot analysis. ( a , d ) The mRNA levels of ATGs (Beclin1, ATG3, ATG5, ATG7, ATG16L2, EIF4G1, GABARAP, HSP90AA1, and HSPA8) in MPA-treated or untreated cells under different conditions, with normalization to corresponding GAPDH mRNA level, are expressed as the fold of control (without MPA treatment under different conditions, which was defined as 1, respectively; in Huh 7 cells or HCV-infected cells, the control group is MPA untreated cells or MPA untreated HCV-infected cells, respectively). ( b , e ) Representative western blot images show ATG3, ATG5 and ATG7 protein levels in MPA-treated or untreated cells under different conditions. ( c , f ) Quantitative assessment of ATG3, ATG5 and ATG7 at protein level in MPA-treated or untreated cells under different conditions. The densitometric intensities of ATG3, ATG5, ATG7 and β-actin bands were quantified by image J software. The relative ATG3/β-actin, ATG5/β-actin, ATG7/β-actin ratios were calculated and shown as the fold of control (without MPA treatment under different conditions, which was defined as 1, respectively). The data shown in Fig. 5a,c,d,f are the mean ± SD of the results of three independent experiments. The p value was calculated by Student’s t -test (* p
Figure Legend Snippet: The effects of MPA treatment on the expression of autophagy-related genes (ATGs) in Huh7 cells or HCV-infected cells. ( a – c ) Huh7 cells were treated or untreated with MPA (5 μg/mL) for 24 h; ( d – f) Huh 7 cells were infected with HCV JFH-1 at an MOI of 0.1. At day 3 postinfection, the HCV-infected cells were treated or untreated with MPA (5 μg/mL) for 24 h. After MPA treatment, the cellular RNA and proteins were extracted for real time RT PCR and western blot analysis. ( a , d ) The mRNA levels of ATGs (Beclin1, ATG3, ATG5, ATG7, ATG16L2, EIF4G1, GABARAP, HSP90AA1, and HSPA8) in MPA-treated or untreated cells under different conditions, with normalization to corresponding GAPDH mRNA level, are expressed as the fold of control (without MPA treatment under different conditions, which was defined as 1, respectively; in Huh 7 cells or HCV-infected cells, the control group is MPA untreated cells or MPA untreated HCV-infected cells, respectively). ( b , e ) Representative western blot images show ATG3, ATG5 and ATG7 protein levels in MPA-treated or untreated cells under different conditions. ( c , f ) Quantitative assessment of ATG3, ATG5 and ATG7 at protein level in MPA-treated or untreated cells under different conditions. The densitometric intensities of ATG3, ATG5, ATG7 and β-actin bands were quantified by image J software. The relative ATG3/β-actin, ATG5/β-actin, ATG7/β-actin ratios were calculated and shown as the fold of control (without MPA treatment under different conditions, which was defined as 1, respectively). The data shown in Fig. 5a,c,d,f are the mean ± SD of the results of three independent experiments. The p value was calculated by Student’s t -test (* p

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

The effects of overexpression of ATG3, ATG5 or ATG7 on the inhibitory effect of MPA on HCV replication. Huh7 cells were infected with HCV JFH-1 at an MOI of 0.1. At day 3 postinfection, Huh7 cells were transfected with plasmids pCMV-myc-Atg3, pCMV-myc-Atg5 and/or pCMV-myc-Atg7, and the transfection maintained for 48 h. ( a ) Overexpression efficiency of pATG3, pATG5, and pATG7 were determined by western blot. ( b – d ) MPA (5 μg/mL) was added to cultures of plasmid-transfected cells and the treatment was maintained for 48 h. The cellular RNA and proteins were extracted for real-time RT-PCR and western blot analysis. ( b ) The effects of overexpression of ATG3, ATG5, ATG7 on HCV RNA expression. The levels of intracellular HCV RNA in Huh7 cells, with normalization to corresponding GAPDH mRNA level, are expressed as the fold of control (without MPA treatment/plasmid transfection, which was defined as 1). ( c ) A representative western blot image shows HCV core, LC3B-I, LC3B-II, p62 protein levels in Huh7 cells. ( d ) Quantitative assessment of HCV core, LC3B-II, LC3B-II/LC3B-I, p62 at protein level. The densitometric intensities of HCV core, LC3B-I, LC3B-II, p62, β-actin bands were quantified by image J software. The relative HCV core/β-actin, LC3B-II/β-actin, LC3B-II/LC3B-I, and p62/β-actin ratios were calculated and shown as the fold of control (without MPA treatment/plasmid transfection, which was defined as 1). The data shown in Fig. 6b,d are the mean ± SD of the results of three independent experiments. The p value was calculated by Student’s t -test (* p
Figure Legend Snippet: The effects of overexpression of ATG3, ATG5 or ATG7 on the inhibitory effect of MPA on HCV replication. Huh7 cells were infected with HCV JFH-1 at an MOI of 0.1. At day 3 postinfection, Huh7 cells were transfected with plasmids pCMV-myc-Atg3, pCMV-myc-Atg5 and/or pCMV-myc-Atg7, and the transfection maintained for 48 h. ( a ) Overexpression efficiency of pATG3, pATG5, and pATG7 were determined by western blot. ( b – d ) MPA (5 μg/mL) was added to cultures of plasmid-transfected cells and the treatment was maintained for 48 h. The cellular RNA and proteins were extracted for real-time RT-PCR and western blot analysis. ( b ) The effects of overexpression of ATG3, ATG5, ATG7 on HCV RNA expression. The levels of intracellular HCV RNA in Huh7 cells, with normalization to corresponding GAPDH mRNA level, are expressed as the fold of control (without MPA treatment/plasmid transfection, which was defined as 1). ( c ) A representative western blot image shows HCV core, LC3B-I, LC3B-II, p62 protein levels in Huh7 cells. ( d ) Quantitative assessment of HCV core, LC3B-II, LC3B-II/LC3B-I, p62 at protein level. The densitometric intensities of HCV core, LC3B-I, LC3B-II, p62, β-actin bands were quantified by image J software. The relative HCV core/β-actin, LC3B-II/β-actin, LC3B-II/LC3B-I, and p62/β-actin ratios were calculated and shown as the fold of control (without MPA treatment/plasmid transfection, which was defined as 1). The data shown in Fig. 6b,d are the mean ± SD of the results of three independent experiments. The p value was calculated by Student’s t -test (* p

Techniques Used: Over Expression, Infection, Transfection, Western Blot, Plasmid Preparation, Quantitative RT-PCR, RNA Expression, Software

Inhibition of HCV replication by MPA treatment in Huh7 cells. Huh7 cells were infected with HCV JFH-1 at an MOI of 0.1. At day 3 postinfection, the infected cells were treated with MPA at indicated concentrations for 48 h. The cellular/extracellular RNA and cellular proteins were extracted for real-time RT PCR ( a , b ) and western blot analysis ( c , d ). ( a ) The levels of intracellular HCV RNA in MPA-treated or untreated control cells, with normalization to corresponding GAPDH mRNA level, are expressed as the fold of control (without MPA treatment, which was defined as 1). ( b ) The levels of extracellular HCV RNA in culture supernatants of MPA-treated or untreated control cells were measured and expressed as copies/mL. ( c ) The effect of MPA on HCV core expression in JFH-1 infected Huh7 cells. A representative western blot image shows the HCV core protein expression. ( d ) Quantitative assessment of HCV core protein expression in MPA-treated or untreated Huh7 cells. The densitometric intensities of HCV core and β-actin bands were quantified by image J software. The relative HCV core/β-actin ratios were calculated and shown as the fold of control (without MPA treatment, which was defined as 1). The data shown in Fig. 1a,b and d are the mean ± SD of the results of three independent experiments. The p value was calculated by Student’s t -test (* p
Figure Legend Snippet: Inhibition of HCV replication by MPA treatment in Huh7 cells. Huh7 cells were infected with HCV JFH-1 at an MOI of 0.1. At day 3 postinfection, the infected cells were treated with MPA at indicated concentrations for 48 h. The cellular/extracellular RNA and cellular proteins were extracted for real-time RT PCR ( a , b ) and western blot analysis ( c , d ). ( a ) The levels of intracellular HCV RNA in MPA-treated or untreated control cells, with normalization to corresponding GAPDH mRNA level, are expressed as the fold of control (without MPA treatment, which was defined as 1). ( b ) The levels of extracellular HCV RNA in culture supernatants of MPA-treated or untreated control cells were measured and expressed as copies/mL. ( c ) The effect of MPA on HCV core expression in JFH-1 infected Huh7 cells. A representative western blot image shows the HCV core protein expression. ( d ) Quantitative assessment of HCV core protein expression in MPA-treated or untreated Huh7 cells. The densitometric intensities of HCV core and β-actin bands were quantified by image J software. The relative HCV core/β-actin ratios were calculated and shown as the fold of control (without MPA treatment, which was defined as 1). The data shown in Fig. 1a,b and d are the mean ± SD of the results of three independent experiments. The p value was calculated by Student’s t -test (* p

Techniques Used: Inhibition, Infection, Quantitative RT-PCR, Western Blot, Expressing, Software

The effects of silencing expression of ATG3, ATG5 or ATG7 on the inhibitory effect of MPA on HCV replication. Huh7 cells were infected with HCV JFH-1 at an MOI of 0.1. At day 3 postinfection, Huh7 cells were transfected with specific siRNAs against ATG3, ATG5 or ATG7, and the transfection maintained for 48 h. ( a ) Silencing expression efficiency of si-ATG3, si-ATG5, and si-ATG7 were determined by western blot. ( b – d ) MPA (5 μg/mL) was added to cultures of siRNA-transfected cells and the treatment was maintained for 48 h. The cellular RNA and proteins were extracted for real-time RT-PCR and western blot analysis. ( b ) The effects of silencing expression of ATG3, ATG5, ATG7 on HCV RNA expression. The levels of intracellular HCV RNA in Huh7 cells, with normalization to corresponding GAPDH mRNA level, are expressed as the fold of control (without MPA treatment/siRNA transfection, which was defined as 1). ( c ) A representative western blot image shows HCV core, LC3B-I, LC3B-II, p62 protein levels in Huh7 cells. ( d ) Quantitative assessment of HCV core, LC3B-II, LC3B-II/LC3B-I, p62 at protein level. The densitometric intensities of HCV core, LC3B-I, LC3B-II, p62, β-actin bands were quantified by image J software. The relative HCV core/β-actin, LC3B-II/β-actin, LC3B-II/LC3B-I, and p62/β-actin ratios were calculated and shown as the fold of control (without MPA treatment/siRNA transfection, which was defined as 1). The data shown in Fig. 7b,d are the mean ± SD of the results of three independent experiments. The p value was calculated by Student’s t -test (* p
Figure Legend Snippet: The effects of silencing expression of ATG3, ATG5 or ATG7 on the inhibitory effect of MPA on HCV replication. Huh7 cells were infected with HCV JFH-1 at an MOI of 0.1. At day 3 postinfection, Huh7 cells were transfected with specific siRNAs against ATG3, ATG5 or ATG7, and the transfection maintained for 48 h. ( a ) Silencing expression efficiency of si-ATG3, si-ATG5, and si-ATG7 were determined by western blot. ( b – d ) MPA (5 μg/mL) was added to cultures of siRNA-transfected cells and the treatment was maintained for 48 h. The cellular RNA and proteins were extracted for real-time RT-PCR and western blot analysis. ( b ) The effects of silencing expression of ATG3, ATG5, ATG7 on HCV RNA expression. The levels of intracellular HCV RNA in Huh7 cells, with normalization to corresponding GAPDH mRNA level, are expressed as the fold of control (without MPA treatment/siRNA transfection, which was defined as 1). ( c ) A representative western blot image shows HCV core, LC3B-I, LC3B-II, p62 protein levels in Huh7 cells. ( d ) Quantitative assessment of HCV core, LC3B-II, LC3B-II/LC3B-I, p62 at protein level. The densitometric intensities of HCV core, LC3B-I, LC3B-II, p62, β-actin bands were quantified by image J software. The relative HCV core/β-actin, LC3B-II/β-actin, LC3B-II/LC3B-I, and p62/β-actin ratios were calculated and shown as the fold of control (without MPA treatment/siRNA transfection, which was defined as 1). The data shown in Fig. 7b,d are the mean ± SD of the results of three independent experiments. The p value was calculated by Student’s t -test (* p

Techniques Used: Expressing, Infection, Transfection, Western Blot, Quantitative RT-PCR, RNA Expression, Software

41) Product Images from "Induction of GD3/α1-adrenergic receptor/transglutaminase 2-mediated erythroid differentiation in chronic myelogenous leukemic K562 cells"

Article Title: Induction of GD3/α1-adrenergic receptor/transglutaminase 2-mediated erythroid differentiation in chronic myelogenous leukemic K562 cells

Journal: Oncotarget

doi: 10.18632/oncotarget.20080

GD3 synthase expression and expression of several erythroid differentiation marker genes induced by α1-AR/TG2-mediated signaling K562 cells were transfected with GD3 synthase (GD3S) and TG2 cDNAs. Without α1-AR antagonist prazosin, cells were treated with α2-AR and β-AR-specific antagonists such as rauwolscine and propranolol in the presence of 10 μM epinephrine, classified as +Epi+Raw+Prop. Then, 1 μg of total RNA isolated from K562 cells was subjected to RT-PCR using primers specifically designed for megakaryotic or several erythroid lineage marker genes, as described in Experimental Procedures. β-Actin mRNA expression indicated that equal amounts of mRNA were used for RT-PCR in each lane. ( A ) RT-PCR of GD3 expression induced by α1-AR/TG2-mediated signaling. ( B ) Induction of several differentiation marker genes by α1-AR/TG2-mediated signaling. ( C ) Cells were incubated with 10 μM epinephrine for 2 days and benzidine staining was performed as described in the ‘Materials and Methods’ section. Data are representative of three experiments (means ± SD). * P
Figure Legend Snippet: GD3 synthase expression and expression of several erythroid differentiation marker genes induced by α1-AR/TG2-mediated signaling K562 cells were transfected with GD3 synthase (GD3S) and TG2 cDNAs. Without α1-AR antagonist prazosin, cells were treated with α2-AR and β-AR-specific antagonists such as rauwolscine and propranolol in the presence of 10 μM epinephrine, classified as +Epi+Raw+Prop. Then, 1 μg of total RNA isolated from K562 cells was subjected to RT-PCR using primers specifically designed for megakaryotic or several erythroid lineage marker genes, as described in Experimental Procedures. β-Actin mRNA expression indicated that equal amounts of mRNA were used for RT-PCR in each lane. ( A ) RT-PCR of GD3 expression induced by α1-AR/TG2-mediated signaling. ( B ) Induction of several differentiation marker genes by α1-AR/TG2-mediated signaling. ( C ) Cells were incubated with 10 μM epinephrine for 2 days and benzidine staining was performed as described in the ‘Materials and Methods’ section. Data are representative of three experiments (means ± SD). * P

Techniques Used: Expressing, Marker, Transfection, Isolation, Reverse Transcription Polymerase Chain Reaction, Incubation, Staining

Increase in membrane recruitment of TG2 in response to epinephrine First, 50 μg of membrane proteins were isolated and subjected to 10% SDS-PAGE, as described in Experimental Procedures. TG2 expression levels were determined by Western blot analysis. RT-PCR analysis of GD3 synthase mRNA. Total RNAs were isolated from K562 cells after 0, 0.5, 1, 2, 3, or 4 days of treatment with 10 μM epinephrine. Then 1 μg of total RNA from each cell was subjected to RT-PCR. β-Actin indicates that equal amounts of RNA were loaded in each lane. Data are representative of three experiments (means ± SD). * P
Figure Legend Snippet: Increase in membrane recruitment of TG2 in response to epinephrine First, 50 μg of membrane proteins were isolated and subjected to 10% SDS-PAGE, as described in Experimental Procedures. TG2 expression levels were determined by Western blot analysis. RT-PCR analysis of GD3 synthase mRNA. Total RNAs were isolated from K562 cells after 0, 0.5, 1, 2, 3, or 4 days of treatment with 10 μM epinephrine. Then 1 μg of total RNA from each cell was subjected to RT-PCR. β-Actin indicates that equal amounts of RNA were loaded in each lane. Data are representative of three experiments (means ± SD). * P

Techniques Used: Isolation, SDS Page, Expressing, Western Blot, Reverse Transcription Polymerase Chain Reaction

42) Product Images from "Altered expression of microRNA-98 in IL-1β-induced cartilage degradation and its role in chondrocyte apoptosis"

Article Title: Altered expression of microRNA-98 in IL-1β-induced cartilage degradation and its role in chondrocyte apoptosis

Journal: Molecular Medicine Reports

doi: 10.3892/mmr.2017.7028

Bcl-2 was identified as a target gene of miR-98. Cells were transfected with 50 nM miR-98 mimic or control mimic for 24 h. (A) Reverse transcription-quantitative polymerase chain and western blotting were performed to evaluate the expression levels of miR-98 and Bcl-2. (B) Sequence alignment (miRBase sequence database; http://www.mirbase.org ) of miR-98 with 3′-UTR of Bcl-2. Luciferase reporter activity was significantly decreased following transfection with the miR-98 mimic compared with in the control group (lower panel) (n=6/group). *P
Figure Legend Snippet: Bcl-2 was identified as a target gene of miR-98. Cells were transfected with 50 nM miR-98 mimic or control mimic for 24 h. (A) Reverse transcription-quantitative polymerase chain and western blotting were performed to evaluate the expression levels of miR-98 and Bcl-2. (B) Sequence alignment (miRBase sequence database; http://www.mirbase.org ) of miR-98 with 3′-UTR of Bcl-2. Luciferase reporter activity was significantly decreased following transfection with the miR-98 mimic compared with in the control group (lower panel) (n=6/group). *P

Techniques Used: Transfection, Western Blot, Expressing, Sequencing, Luciferase, Activity Assay

Expression levels of miR-98 and apoptotic factors in IL-1β-induced chondrocytes. Cells were cultured with IL-1β (10 ng/ml) or control medium for 12, 24 and 48 h. (A) miR-98, Fas, caspase-3, caspase-8, Bax and Bcl-2 mRNA expression levels were determined by reverse transcription-quantitative polymerase chain reaction using U6 and GAPDH as internal controls. (B) Bcl-2 protein levels were determined by western blotting. Each data point was normalized to the control. Data are presented as the mean ± standard error from six independent experiments. *P
Figure Legend Snippet: Expression levels of miR-98 and apoptotic factors in IL-1β-induced chondrocytes. Cells were cultured with IL-1β (10 ng/ml) or control medium for 12, 24 and 48 h. (A) miR-98, Fas, caspase-3, caspase-8, Bax and Bcl-2 mRNA expression levels were determined by reverse transcription-quantitative polymerase chain reaction using U6 and GAPDH as internal controls. (B) Bcl-2 protein levels were determined by western blotting. Each data point was normalized to the control. Data are presented as the mean ± standard error from six independent experiments. *P

Techniques Used: Expressing, Cell Culture, Real-time Polymerase Chain Reaction, Western Blot

Analysis of the role of miR-98 in the expression of Bcl-2 in IL-1β-treated chondrocytes. (A) Reverse transcription-quantitative polymerase chain reaction analysis of the mRNA expression of Bcl-2. (B) Western blot analysis of the protein levels of Bcl-2. miR-98 inhibitor significantly alleviated IL-1β-induced downregulation of Bcl-2 at the mRNA and protein level (n=6/group). *P
Figure Legend Snippet: Analysis of the role of miR-98 in the expression of Bcl-2 in IL-1β-treated chondrocytes. (A) Reverse transcription-quantitative polymerase chain reaction analysis of the mRNA expression of Bcl-2. (B) Western blot analysis of the protein levels of Bcl-2. miR-98 inhibitor significantly alleviated IL-1β-induced downregulation of Bcl-2 at the mRNA and protein level (n=6/group). *P

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

43) Product Images from "Pyruvate kinase M2 phosphorylates H2AX and promotes genomic instability in human tumor cells"

Article Title: Pyruvate kinase M2 phosphorylates H2AX and promotes genomic instability in human tumor cells

Journal: Oncotarget

doi: 10.18632/oncotarget.22621

PKM2 promotes cell growth under DNA damage condition Expression of empty vector (EV), wild type (WT) PKM2 or PKM2-Del 515-520 mutant in PKM2-depleted MCF7 cells followed by treatment with (B) or without (A) etoposide. Then the cell proliferations were measured by CCK-8 assay. ** p
Figure Legend Snippet: PKM2 promotes cell growth under DNA damage condition Expression of empty vector (EV), wild type (WT) PKM2 or PKM2-Del 515-520 mutant in PKM2-depleted MCF7 cells followed by treatment with (B) or without (A) etoposide. Then the cell proliferations were measured by CCK-8 assay. ** p

Techniques Used: Expressing, Plasmid Preparation, Mutagenesis, CCK-8 Assay

PKM2 expression increases chromosomal aberrations following DDR Three representative metaphase spreads of MCF7 cells reconstitutively expressing empty vector (EV), PKM2-WT or PKM2-Del 515-520 mutant either untreated or 24hr following etoposide treatment. FISH-mediated whole chromosomal staining was used to identify aberrations of chromosome 5. Bottom panel shows the quantification of total chromosome aberrations/metaphase (30 metaphases scored/group). Data are mean ± SD of triplicates in an independent experiment, which were repeated at least for three times with the same results. ** p
Figure Legend Snippet: PKM2 expression increases chromosomal aberrations following DDR Three representative metaphase spreads of MCF7 cells reconstitutively expressing empty vector (EV), PKM2-WT or PKM2-Del 515-520 mutant either untreated or 24hr following etoposide treatment. FISH-mediated whole chromosomal staining was used to identify aberrations of chromosome 5. Bottom panel shows the quantification of total chromosome aberrations/metaphase (30 metaphases scored/group). Data are mean ± SD of triplicates in an independent experiment, which were repeated at least for three times with the same results. ** p

Techniques Used: Expressing, Plasmid Preparation, Mutagenesis, Fluorescence In Situ Hybridization, Staining

Knockdown of PKM2 reduces chromosomal aberrations following DDR (A, C, E) Three representative metaphase spreads of MCF7 cells infected with NC or shPKM2#1 either untreated or 24hr following etoposide treatment. Fluorescence in situ hybridization (FISH)-mediated whole chromosomal staining was used to identify aberrations of chromosome 2 (A), 3 (C) and 5 (E). (B, D, F) Quantification of total chromosome aberrations/metaphase (30 metaphases scored/group). Data are mean ± SD of triplicates in an independent experiment, which were repeated at least for three times with the same results.
Figure Legend Snippet: Knockdown of PKM2 reduces chromosomal aberrations following DDR (A, C, E) Three representative metaphase spreads of MCF7 cells infected with NC or shPKM2#1 either untreated or 24hr following etoposide treatment. Fluorescence in situ hybridization (FISH)-mediated whole chromosomal staining was used to identify aberrations of chromosome 2 (A), 3 (C) and 5 (E). (B, D, F) Quantification of total chromosome aberrations/metaphase (30 metaphases scored/group). Data are mean ± SD of triplicates in an independent experiment, which were repeated at least for three times with the same results.

Techniques Used: Infection, Fluorescence, In Situ Hybridization, Fluorescence In Situ Hybridization, Staining

PKM2 promotes DDR (A-C) MCF7 cells infected with shPKM2#1 or NC were exposed to etoposide (A and B) or UV (C) for hours as indicated, followed by immunoblots for the indicated proteins (A and C) or immunofluorescent analyses (B) with DAPI co-staining. The protein bands on the gels were quantified by densitometry and shown on the bottom panels (A and C). * p
Figure Legend Snippet: PKM2 promotes DDR (A-C) MCF7 cells infected with shPKM2#1 or NC were exposed to etoposide (A and B) or UV (C) for hours as indicated, followed by immunoblots for the indicated proteins (A and C) or immunofluorescent analyses (B) with DAPI co-staining. The protein bands on the gels were quantified by densitometry and shown on the bottom panels (A and C). * p

Techniques Used: Infection, Western Blot, Staining

PKM2 phosphorylates H2AX at Ser139 in vitro (A) Purified recombinant PKM2-WT or PKM2-R399E mutant was incubated with recombinant GST-H2AX in the presence of PEP or ATP. H2AX, γ-H2AX and PKM2 were detected by immunoblotting. (B) The in vitro kinase reactions were performed by incubating recombinant PKM2 or ATM with WT GST-H2AX or GST-H2AX-S139A mutant in the presence of PEP (for PKM2) or ATP (for ATM). (C) PKM2 (0.25μM) was incubated with varied purified H2AX in the presence of excess PEP at room temperature for 2min. The reaction mixtures were then analyzed by Western blot using indicated antibodies (upper panel). The phosphorylated H2AX protein bands were quantified by densitometry and the kinetic constants were determined by fitting the data with Michaelis-Menten equation using GraphPad Prism. (D) Phosphorylation of GST-H2AX by recombinant PKM2 in the presence of FBP, ADP or PEP was detected by immunoblotting. (E, F) The in vitro kinase assay was carried out by mixing WT PKM2 or its mutants as indicated with GST-H2AX. The protein bands on the gels were quantified by densitometry and shown on the bottom panel (D, F). (G, H) Phosphorylation of recombinant H2AX by ATM or PKM2 in the presence of escalating concentration of PKM2 (G) or ATM (H) was analyzed by in vitro kinase assay. Immunoblots were performed for indicated proteins. All these experiments were repeated at least for three times with the same results.
Figure Legend Snippet: PKM2 phosphorylates H2AX at Ser139 in vitro (A) Purified recombinant PKM2-WT or PKM2-R399E mutant was incubated with recombinant GST-H2AX in the presence of PEP or ATP. H2AX, γ-H2AX and PKM2 were detected by immunoblotting. (B) The in vitro kinase reactions were performed by incubating recombinant PKM2 or ATM with WT GST-H2AX or GST-H2AX-S139A mutant in the presence of PEP (for PKM2) or ATP (for ATM). (C) PKM2 (0.25μM) was incubated with varied purified H2AX in the presence of excess PEP at room temperature for 2min. The reaction mixtures were then analyzed by Western blot using indicated antibodies (upper panel). The phosphorylated H2AX protein bands were quantified by densitometry and the kinetic constants were determined by fitting the data with Michaelis-Menten equation using GraphPad Prism. (D) Phosphorylation of GST-H2AX by recombinant PKM2 in the presence of FBP, ADP or PEP was detected by immunoblotting. (E, F) The in vitro kinase assay was carried out by mixing WT PKM2 or its mutants as indicated with GST-H2AX. The protein bands on the gels were quantified by densitometry and shown on the bottom panel (D, F). (G, H) Phosphorylation of recombinant H2AX by ATM or PKM2 in the presence of escalating concentration of PKM2 (G) or ATM (H) was analyzed by in vitro kinase assay. Immunoblots were performed for indicated proteins. All these experiments were repeated at least for three times with the same results.

Techniques Used: In Vitro, Purification, Recombinant, Mutagenesis, Incubation, Western Blot, Kinase Assay, Concentration Assay

Knockdown of PKM2 decreased γ-H2AX level in xenografts MCF7 cells infected with NC or shPKM2#1 were injected into the left or right side of the mammary fat pad of nude mice and grown for three weeks. Afterwards, a dose of 0.1mg/kg of etoposide was administrated intraperitoneally for 7 consecutive days. Two weeks later, the fresh tumors from the mice were collected, homogenated and subjected to western blot analysis with the indicated antibodies.
Figure Legend Snippet: Knockdown of PKM2 decreased γ-H2AX level in xenografts MCF7 cells infected with NC or shPKM2#1 were injected into the left or right side of the mammary fat pad of nude mice and grown for three weeks. Afterwards, a dose of 0.1mg/kg of etoposide was administrated intraperitoneally for 7 consecutive days. Two weeks later, the fresh tumors from the mice were collected, homogenated and subjected to western blot analysis with the indicated antibodies.

Techniques Used: Infection, Injection, Mouse Assay, Western Blot

PKM2 increased chemical- and UV-damaged DNA (A) MCF7 cells infected with shPKM2#1 or NC were exposed to etoposide or UV for indicated hours, followed by detection of DNA damage through alkaline comet assay. (B) Images were analysed with an ImageJ software plugin Comet Assay from Microscopy Services Laboratory ( https://www.med.unc.edu/microscopy ) and levels of DNA damage were shown.
Figure Legend Snippet: PKM2 increased chemical- and UV-damaged DNA (A) MCF7 cells infected with shPKM2#1 or NC were exposed to etoposide or UV for indicated hours, followed by detection of DNA damage through alkaline comet assay. (B) Images were analysed with an ImageJ software plugin Comet Assay from Microscopy Services Laboratory ( https://www.med.unc.edu/microscopy ) and levels of DNA damage were shown.

Techniques Used: Infection, Alkaline Single Cell Gel Electrophoresis, Software, Single Cell Gel Electrophoresis, Microscopy

Nuclear PKM2 interacts with H2AX upon DNA damage (A) Cytosolic and nuclear extracts were prepared from MCF7 cells treated with or without etoposide, followed by western blot analysis for the indicated proteins. LaminB was a nuclear marker, and β-actin acted as a cytosol marker. (B) Heat map of gene ontology (GO) for DNA damage signaling pathway based upon the identified PKM2-interacting proteins from LC-MS/MS. GO analysis (DAVID 6.7) was applied to the specific PKM2-associated complex under etoposide treatment and untreatment for molecular function and biological process enrichment [ 27 ]. The colors in the map represent the quantitative value (normalized total spectra) according to the Scaffold_4.3.3. Color code ranges from 0 to 10. (C) Co-IP analysis of the interaction between endogenous PKM2 and H2AX or γ-H2AX using antibodies against PKM2 in MCF7 cells treated with or without etoposide for 1 hour. IP using rabbit IgG was a negative control. Input, 10% whole cell lysate. (D) GST pull-down analysis of H2AX with PKM2 using purified PKM2 and GST-tagged H2AX fusion protein. (E) Co-IP analysis of the interaction between endogenous PKM2 and γ-H2AX using antibodies against γ-H2AXin MCF7 cells treated with etoposide. (F) Immunofluorescent staining of endogenous PKM2 and γ-H2AX in MCF7 cells treated with or without etoposide for 1 hour. The bottom panel showed the amplified images for cells lined with white line. All these experiments were repeated at least for three times with the same results.
Figure Legend Snippet: Nuclear PKM2 interacts with H2AX upon DNA damage (A) Cytosolic and nuclear extracts were prepared from MCF7 cells treated with or without etoposide, followed by western blot analysis for the indicated proteins. LaminB was a nuclear marker, and β-actin acted as a cytosol marker. (B) Heat map of gene ontology (GO) for DNA damage signaling pathway based upon the identified PKM2-interacting proteins from LC-MS/MS. GO analysis (DAVID 6.7) was applied to the specific PKM2-associated complex under etoposide treatment and untreatment for molecular function and biological process enrichment [ 27 ]. The colors in the map represent the quantitative value (normalized total spectra) according to the Scaffold_4.3.3. Color code ranges from 0 to 10. (C) Co-IP analysis of the interaction between endogenous PKM2 and H2AX or γ-H2AX using antibodies against PKM2 in MCF7 cells treated with or without etoposide for 1 hour. IP using rabbit IgG was a negative control. Input, 10% whole cell lysate. (D) GST pull-down analysis of H2AX with PKM2 using purified PKM2 and GST-tagged H2AX fusion protein. (E) Co-IP analysis of the interaction between endogenous PKM2 and γ-H2AX using antibodies against γ-H2AXin MCF7 cells treated with etoposide. (F) Immunofluorescent staining of endogenous PKM2 and γ-H2AX in MCF7 cells treated with or without etoposide for 1 hour. The bottom panel showed the amplified images for cells lined with white line. All these experiments were repeated at least for three times with the same results.

Techniques Used: Western Blot, Marker, Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry, Co-Immunoprecipitation Assay, Negative Control, Purification, Staining, Amplification

44) Product Images from "miR-26a inhibits atherosclerosis progression by targeting TRPC3"

Article Title: miR-26a inhibits atherosclerosis progression by targeting TRPC3

Journal: Cell & Bioscience

doi: 10.1186/s13578-018-0203-9

TRPC3 was a target of miR-26a in HAECs. a Schematic of the interaction sites of wild-type or mutated TRPC3-3′UTR with miR-26a. b Luciferase activity was measured by luciferase reporter assay in HAECs after cotransfection with WT-TRPC3 or MUT-TRPC3 and miR-26a or miR-NC. c The protein level of TRPC3 in HAECs treated with miR-26a, anti-miR-26a, or corresponding controls were determined by western blot. * P
Figure Legend Snippet: TRPC3 was a target of miR-26a in HAECs. a Schematic of the interaction sites of wild-type or mutated TRPC3-3′UTR with miR-26a. b Luciferase activity was measured by luciferase reporter assay in HAECs after cotransfection with WT-TRPC3 or MUT-TRPC3 and miR-26a or miR-NC. c The protein level of TRPC3 in HAECs treated with miR-26a, anti-miR-26a, or corresponding controls were determined by western blot. * P

Techniques Used: Luciferase, Activity Assay, Reporter Assay, Cotransfection, Western Blot

miR-26a overexpression promoted cell viability and inhibited apoptosis in ox-LDL-treated HAECs by targeting TRPC3. HAECs transfected with miR-26a, or together with TRPC3 were stimulated with ox-LDL for 24 h, followed by western blot analysis of TRPC3 protein level ( a ), MTT assay of cell viability ( b ), and TUNEL assay of apoptosis ( c ). HAECs introduced with anti-miR-26a, or combined with si-TRPC3 were treated with ox-LDL for 24 h, followed by western blot analysis of TRPC3 protein level ( d ), MTT assay of cell viability (E) and TUNEL assay of apoptosis ( f ). * P
Figure Legend Snippet: miR-26a overexpression promoted cell viability and inhibited apoptosis in ox-LDL-treated HAECs by targeting TRPC3. HAECs transfected with miR-26a, or together with TRPC3 were stimulated with ox-LDL for 24 h, followed by western blot analysis of TRPC3 protein level ( a ), MTT assay of cell viability ( b ), and TUNEL assay of apoptosis ( c ). HAECs introduced with anti-miR-26a, or combined with si-TRPC3 were treated with ox-LDL for 24 h, followed by western blot analysis of TRPC3 protein level ( d ), MTT assay of cell viability (E) and TUNEL assay of apoptosis ( f ). * P

Techniques Used: Over Expression, Transfection, Western Blot, MTT Assay, TUNEL Assay

miR-26a overexpression inhibited activation of the nuclear factor-kappa B (NF-κB) pathway by downregulating TRPC3 in ox-LDL-treated HAECs. a HAECs were transfected with miR-26a, miR-NC, miR-26a + Empty, or miR-26a + TRPC3, followed by treatment with 50 nM ox-LDL for 24 h. Then, the protein levels of p65, p-p65, IκBα and p-IκBα in the treated HAECs were detected by western blot. b HAECs were transfected with anti-miR-26a, anti-miR-NC, anti-miR-26a + si-NC, or anti-miR-26a + si-TRPC3, followed by treatment with 50 nM ox-LDL for 24 h. Then, the protein levels of p65, p-p65, IκBα and p-IκBα in the treated HAECs were detected by western blot
Figure Legend Snippet: miR-26a overexpression inhibited activation of the nuclear factor-kappa B (NF-κB) pathway by downregulating TRPC3 in ox-LDL-treated HAECs. a HAECs were transfected with miR-26a, miR-NC, miR-26a + Empty, or miR-26a + TRPC3, followed by treatment with 50 nM ox-LDL for 24 h. Then, the protein levels of p65, p-p65, IκBα and p-IκBα in the treated HAECs were detected by western blot. b HAECs were transfected with anti-miR-26a, anti-miR-NC, anti-miR-26a + si-NC, or anti-miR-26a + si-TRPC3, followed by treatment with 50 nM ox-LDL for 24 h. Then, the protein levels of p65, p-p65, IκBα and p-IκBα in the treated HAECs were detected by western blot

Techniques Used: Over Expression, Activation Assay, Transfection, Western Blot

Effects of miR-26a on hyperlipidemia in HFD-treated apoE −/− mice. a RT-qPCR analysis of miR-26a expression in apoE −/− mice fed with HFD or normal diet. b The body weight of atherosclerosis mice in control, AG, AG-NC, AN, and AN-NC groups. The serum concentrations of TC ( c ), TG ( d ), HDL-C ( e ), and LDL-C ( f ) in atherosclerosis mice in control, AG, AG-NC, AN, and AN-NC groups. AG: miR-26a agomir, AG-NC: miR-26a agomir negative control, AN: miR-26a antagomir, AN-NC: miR-26a antagomir negative control, * P
Figure Legend Snippet: Effects of miR-26a on hyperlipidemia in HFD-treated apoE −/− mice. a RT-qPCR analysis of miR-26a expression in apoE −/− mice fed with HFD or normal diet. b The body weight of atherosclerosis mice in control, AG, AG-NC, AN, and AN-NC groups. The serum concentrations of TC ( c ), TG ( d ), HDL-C ( e ), and LDL-C ( f ) in atherosclerosis mice in control, AG, AG-NC, AN, and AN-NC groups. AG: miR-26a agomir, AG-NC: miR-26a agomir negative control, AN: miR-26a antagomir, AN-NC: miR-26a antagomir negative control, * P

Techniques Used: Mouse Assay, Quantitative RT-PCR, Expressing, Negative Control

Effects of miR-26a on atherosclerotic lesion in ApoE − / − mice. a Representative Oil-red-O staining of aorta in control, AG, AG-NC, AN, and AN-NC groups are shown. Atherosclerotic lesion area indicates the level of atherogenesis. b Characterization of aortic sinus atherosclerotic lesion area by HE staining in control, AG, AG-NC, AN, and AN-NC groups. * P
Figure Legend Snippet: Effects of miR-26a on atherosclerotic lesion in ApoE − / − mice. a Representative Oil-red-O staining of aorta in control, AG, AG-NC, AN, and AN-NC groups are shown. Atherosclerotic lesion area indicates the level of atherogenesis. b Characterization of aortic sinus atherosclerotic lesion area by HE staining in control, AG, AG-NC, AN, and AN-NC groups. * P

Techniques Used: Mouse Assay, Staining

miR-26a overexpression attenuated the development of atherosclerosis in ox-LDL-treated HAECs. HAECs were transfected with miR-26a or miR-NC, followed by treatment with 50 nM ox-LDL for 24 h. a RT-qPCR analysis of miR-26a expression in treated HAECs. The concentrations of TNF-α ( b ), IL-1β ( c ), IL-6 ( d ), and MCP-1 ( e ) in the supernatant of treated HAECs were measured by ELISA assay. f Cell viability of treated HAECs was estimated by MTT assay. g Apoptosis of treated HAECs was assessed by TUNEL assay. * P
Figure Legend Snippet: miR-26a overexpression attenuated the development of atherosclerosis in ox-LDL-treated HAECs. HAECs were transfected with miR-26a or miR-NC, followed by treatment with 50 nM ox-LDL for 24 h. a RT-qPCR analysis of miR-26a expression in treated HAECs. The concentrations of TNF-α ( b ), IL-1β ( c ), IL-6 ( d ), and MCP-1 ( e ) in the supernatant of treated HAECs were measured by ELISA assay. f Cell viability of treated HAECs was estimated by MTT assay. g Apoptosis of treated HAECs was assessed by TUNEL assay. * P

Techniques Used: Over Expression, Transfection, Quantitative RT-PCR, Expressing, Enzyme-linked Immunosorbent Assay, MTT Assay, TUNEL Assay

Effects of miR-26a on the production of inflammatory factors in HFD-treated apoE −/− mice. ELISA assay of concentrations of TNF-α ( a ), IL-1β ( b ), IL-6 ( c ), and MCP-1 ( d ) in the plasma of HFD-treated apoE −/− mice from control, AG, AG-NC, AN, and AN-NC groups. * P
Figure Legend Snippet: Effects of miR-26a on the production of inflammatory factors in HFD-treated apoE −/− mice. ELISA assay of concentrations of TNF-α ( a ), IL-1β ( b ), IL-6 ( c ), and MCP-1 ( d ) in the plasma of HFD-treated apoE −/− mice from control, AG, AG-NC, AN, and AN-NC groups. * P

Techniques Used: Mouse Assay, Enzyme-linked Immunosorbent Assay

45) Product Images from "BC-Box Motif-Mediated Neuronal Differentiation of Somatic Stem Cells"

Article Title: BC-Box Motif-Mediated Neuronal Differentiation of Somatic Stem Cells

Journal: International Journal of Molecular Sciences

doi: 10.3390/ijms19020466

( A ) Isothemal titration caloritetry (ITC) thermograms of elongin BC with various kinds of peptides. Results of ITC experiments by titrating elongin BC with VHL peptides are shown. The panels represent raw ITC data. Sequences of peptides used are shown in the corresponding thermograms. ( B ) Structure of elongin BC and VHL complex (PDB ID 4WQO). Elongin B, elongin C, and VHL are colored green, pink, and cyan, respectively. N-terminal residues of the BC-box of VHL are highlighted as cyan sticks and some relevant residues of elongin C are shown by pink sticks. Blue, red, and yellow represent nitrogen, oxygen, and sulfur atoms, respectively.
Figure Legend Snippet: ( A ) Isothemal titration caloritetry (ITC) thermograms of elongin BC with various kinds of peptides. Results of ITC experiments by titrating elongin BC with VHL peptides are shown. The panels represent raw ITC data. Sequences of peptides used are shown in the corresponding thermograms. ( B ) Structure of elongin BC and VHL complex (PDB ID 4WQO). Elongin B, elongin C, and VHL are colored green, pink, and cyan, respectively. N-terminal residues of the BC-box of VHL are highlighted as cyan sticks and some relevant residues of elongin C are shown by pink sticks. Blue, red, and yellow represent nitrogen, oxygen, and sulfur atoms, respectively.

Techniques Used: Titration

( A ) Ten divided sequences of von Hippel-Lindau tumor suppressor protein (pVHL) and the relation to the st ructure of pVHL. The elongin BC binding site exists in α-domain; and the HIF-1α binding site, in β-domain; ( B ) Immunoblotting study with anti-Neurofilament-H (anti-NFH) antibody. The most potent expression of NFH was obtained with sequence G [VHL(155–171)]; ( C ) Immunocytochemical study with anti-NFH antibody. The greatest rate of immunoreactive cells for NFH was found at the “G” sequence (VHL(155–171)); ( D ) Rates of cells having neurites for the 10 divided sequences. The “G” sequence (VHL(155–171)) shows the greatest rate of cells having neurites. Phase-contrast microphotographs showed cells for “G” sequence (VHL(155–171)) and cells for “A” sequence [VHL(1–13)]; ( E ) The cells for “A” sequence (VHL(1–13)) and the cells for TAT showed no morphological change, as with non-treated cells ( left ), whereas the cells for “G” sequence [VHL(155–171)] assumed a neuron-like morphology ( right ). Scale bar = 20 μm.
Figure Legend Snippet: ( A ) Ten divided sequences of von Hippel-Lindau tumor suppressor protein (pVHL) and the relation to the st ructure of pVHL. The elongin BC binding site exists in α-domain; and the HIF-1α binding site, in β-domain; ( B ) Immunoblotting study with anti-Neurofilament-H (anti-NFH) antibody. The most potent expression of NFH was obtained with sequence G [VHL(155–171)]; ( C ) Immunocytochemical study with anti-NFH antibody. The greatest rate of immunoreactive cells for NFH was found at the “G” sequence (VHL(155–171)); ( D ) Rates of cells having neurites for the 10 divided sequences. The “G” sequence (VHL(155–171)) shows the greatest rate of cells having neurites. Phase-contrast microphotographs showed cells for “G” sequence (VHL(155–171)) and cells for “A” sequence [VHL(1–13)]; ( E ) The cells for “A” sequence (VHL(1–13)) and the cells for TAT showed no morphological change, as with non-treated cells ( left ), whereas the cells for “G” sequence [VHL(155–171)] assumed a neuron-like morphology ( right ). Scale bar = 20 μm.

Techniques Used: Binding Assay, Expressing, Sequencing

46) Product Images from "Gbvdr6, a Gene Encoding a Receptor-Like Protein of Cotton (Gossypium barbadense), Confers Resistance to Verticillium Wilt in Arabidopsis and Upland Cotton"

Article Title: Gbvdr6, a Gene Encoding a Receptor-Like Protein of Cotton (Gossypium barbadense), Confers Resistance to Verticillium Wilt in Arabidopsis and Upland Cotton

Journal: Frontiers in Plant Science

doi: 10.3389/fpls.2017.02272

Upregulation of pathogenesis-related genes in the Gbvdr6 over-expressed Arabidopsis . The expression levels of marker genes PR1, PR2 , and PR5 in SA signaling, PR3, PDF1.2 , and ERF1 in ET/JA signaling, and GST2 in ET signaling were measured by real-time reverse-transcription PCR with β-tubulin gene as the internal control. Data were the means ± SE of three biological replicates. Duncan's multiple range test was carried out within genes, and different letters in the graphs indicate significant differences between treatments ( P
Figure Legend Snippet: Upregulation of pathogenesis-related genes in the Gbvdr6 over-expressed Arabidopsis . The expression levels of marker genes PR1, PR2 , and PR5 in SA signaling, PR3, PDF1.2 , and ERF1 in ET/JA signaling, and GST2 in ET signaling were measured by real-time reverse-transcription PCR with β-tubulin gene as the internal control. Data were the means ± SE of three biological replicates. Duncan's multiple range test was carried out within genes, and different letters in the graphs indicate significant differences between treatments ( P

Techniques Used: Expressing, Marker, Polymerase Chain Reaction

Hydrogen peroxide and callose accumulation in the Gbvdr6 over-expressed cotton and WT plant in response to V. dahliae . Roots from the transgenic and the WT at 5 days post-inoculation with V. dahliae were stained with 3,3′-diaminobenzidinetetrahydrochloride (DAB), and photos were taken under a fluorescence microscope with bright light. Scale bar = 100 μm (upper) . Callose accumulation (lower) . Roots from the transgenic and the WT at 5 days post-inoculation with V. dahliae were stained with aniline blue, and photos were taken under a fluorescence microscope with UV light. Scale bar = 100 μm.
Figure Legend Snippet: Hydrogen peroxide and callose accumulation in the Gbvdr6 over-expressed cotton and WT plant in response to V. dahliae . Roots from the transgenic and the WT at 5 days post-inoculation with V. dahliae were stained with 3,3′-diaminobenzidinetetrahydrochloride (DAB), and photos were taken under a fluorescence microscope with bright light. Scale bar = 100 μm (upper) . Callose accumulation (lower) . Roots from the transgenic and the WT at 5 days post-inoculation with V. dahliae were stained with aniline blue, and photos were taken under a fluorescence microscope with UV light. Scale bar = 100 μm.

Techniques Used: Transgenic Assay, Staining, Fluorescence, Microscopy

Gbvdr6 over-expressed cotton enhanced resistance to V. dahliae . (A) Varied expressional levels of Gbvdr6 in the transformed plant s. Gbvdr6 relative expressional levels of the T3 generation were measured by qRT-PCR and calculated in relation to the wild type plants according to the ΔΔCt method with the UBQ14 gene as the internal control. L1, L4, L5, L9, and L12 are the transgenic cotton lines. Different letters on the bars designate statistically significant differences ( P
Figure Legend Snippet: Gbvdr6 over-expressed cotton enhanced resistance to V. dahliae . (A) Varied expressional levels of Gbvdr6 in the transformed plant s. Gbvdr6 relative expressional levels of the T3 generation were measured by qRT-PCR and calculated in relation to the wild type plants according to the ΔΔCt method with the UBQ14 gene as the internal control. L1, L4, L5, L9, and L12 are the transgenic cotton lines. Different letters on the bars designate statistically significant differences ( P

Techniques Used: Transformation Assay, Quantitative RT-PCR, Transgenic Assay

Expression pattern analysis of the Gbvdr6 gene. (A) The transcript levels of Gbvdr6 in different tissues of Hai7124. Values were expressed as fold changes of transcript levels in the different tissues with respect to that of leaves with the 2 −ΔΔCT Method. Error bars represented SE of three biological replicates. Duncan's multiple range test was conducted, and the different letters in graphs indicate significant differences between treatments ( P
Figure Legend Snippet: Expression pattern analysis of the Gbvdr6 gene. (A) The transcript levels of Gbvdr6 in different tissues of Hai7124. Values were expressed as fold changes of transcript levels in the different tissues with respect to that of leaves with the 2 −ΔΔCT Method. Error bars represented SE of three biological replicates. Duncan's multiple range test was conducted, and the different letters in graphs indicate significant differences between treatments ( P

Techniques Used: Expressing

Subcellular localization of Gbvdr6 in epidermal cells of N. tabacum leaves. The Gbvdr6-GFP fusion was transiently co-expressed with the plasma membrane marker mCherry. The images were taken under a confocal microscope at 48 h after agro-infiltration. GFP: fluorescence of Gbvdr6-GFP fusion, mCherry: fluorescence of the plasma membrane marker mCherry, DIC, differential interference contrast; merged, a merged image. Scale bar = 60 μm.
Figure Legend Snippet: Subcellular localization of Gbvdr6 in epidermal cells of N. tabacum leaves. The Gbvdr6-GFP fusion was transiently co-expressed with the plasma membrane marker mCherry. The images were taken under a confocal microscope at 48 h after agro-infiltration. GFP: fluorescence of Gbvdr6-GFP fusion, mCherry: fluorescence of the plasma membrane marker mCherry, DIC, differential interference contrast; merged, a merged image. Scale bar = 60 μm.

Techniques Used: Marker, Microscopy, Fluorescence

Gbvdr6 over-expressed Arabidopsis enhanced resistance to V. dahliae . (A) The semi-quantitative RT-PCR of Gbvdr6 over-expressed Arabidopsis . The β-tubulin was the internal control, and L1-L6 are the transgenic Arabidopsis lines. (B) The numbers of healthy, stunted and dead Arabidopsis plants were scored and statistically analyzed. Thirty plantlets were tested in each line. The experiment was conducted twice with similar results. The chi-squared test is used to determine whether there is a significant difference. The asterisk indicated above the columns means ** P
Figure Legend Snippet: Gbvdr6 over-expressed Arabidopsis enhanced resistance to V. dahliae . (A) The semi-quantitative RT-PCR of Gbvdr6 over-expressed Arabidopsis . The β-tubulin was the internal control, and L1-L6 are the transgenic Arabidopsis lines. (B) The numbers of healthy, stunted and dead Arabidopsis plants were scored and statistically analyzed. Thirty plantlets were tested in each line. The experiment was conducted twice with similar results. The chi-squared test is used to determine whether there is a significant difference. The asterisk indicated above the columns means ** P

Techniques Used: Quantitative RT-PCR, Transgenic Assay

Gbvdr6 over-expressed Arabidopsis is more insensitive to MeJA compared with the wild type. (A) The phenotype of Gbvdr6 over-expressed Arabidopsis ( Gbvdr6 -OE) and wild type after MeJA treatment. The WT and Gbvdr 6-OE are the seedlings grown on the plate without MeJA, and the WT-MeJA and Gbvdr6 -OE-MeJA indicates the seedling on the plate with 20 um MeJA. The photos were taken at 30 days after sowing. (B) Assay of seed germination rate of Gbvdr6 over-expressed Arabidopsis ( Gbvdr6 -OE) and wild type in the presence of exogenous MeJA. Germination rates of the seeds were analyzed at the indicated time points. The data represent means ± SD of three independent replicates with at least 50 seeds counted per replicate. (C) Assay of root length of Gbvdr6 over-expressed Arabidopsis ( Gbvdr6 -OE) and wild type in the presence of exogenous MeJA at 30 days after sowing. Significant difference between different lines is indicated by different letters ( P
Figure Legend Snippet: Gbvdr6 over-expressed Arabidopsis is more insensitive to MeJA compared with the wild type. (A) The phenotype of Gbvdr6 over-expressed Arabidopsis ( Gbvdr6 -OE) and wild type after MeJA treatment. The WT and Gbvdr 6-OE are the seedlings grown on the plate without MeJA, and the WT-MeJA and Gbvdr6 -OE-MeJA indicates the seedling on the plate with 20 um MeJA. The photos were taken at 30 days after sowing. (B) Assay of seed germination rate of Gbvdr6 over-expressed Arabidopsis ( Gbvdr6 -OE) and wild type in the presence of exogenous MeJA. Germination rates of the seeds were analyzed at the indicated time points. The data represent means ± SD of three independent replicates with at least 50 seeds counted per replicate. (C) Assay of root length of Gbvdr6 over-expressed Arabidopsis ( Gbvdr6 -OE) and wild type in the presence of exogenous MeJA at 30 days after sowing. Significant difference between different lines is indicated by different letters ( P

Techniques Used:

Phylogenetic and structural analysis of Gbvdr6 gene. (A) Phylogenetic relationship of Gbvdr6 with other Ve-like proteins by MEGA6 software with the neighbor-joining (NJ) algorithm under 1,000 replicates of bootstrap. The numbers on the internal nodes are the percentage bootstrap support values. (B) Schematic diagram of Gbvdr6 protein domain architecture showing signal peptide (SP) at N-terminus, followed by extracellular leucine-rich repeat (eLRRs), transmembrane (TM) domain and cytoplasmic domain (CD) at C-terminus. The numbers indicate the domain regions. (C) Schematic diagram of physical locations of Gbvdr6, Gbvdr5 , and GbVe1 and the SSR markers flanking the known Verticillium wilt-resistant QTLs in the chromosomes of tetraploid cotton. The numbers in brackets indicate the physical positions in the chromosome. NAU2741 and CGR5056a are the SSR markers that flank Verticillium wilt resistance QTLs qVW-A1-1 and qVW-C15-4 in the A01(c1) and D01(c15) chromosomes, respectively.
Figure Legend Snippet: Phylogenetic and structural analysis of Gbvdr6 gene. (A) Phylogenetic relationship of Gbvdr6 with other Ve-like proteins by MEGA6 software with the neighbor-joining (NJ) algorithm under 1,000 replicates of bootstrap. The numbers on the internal nodes are the percentage bootstrap support values. (B) Schematic diagram of Gbvdr6 protein domain architecture showing signal peptide (SP) at N-terminus, followed by extracellular leucine-rich repeat (eLRRs), transmembrane (TM) domain and cytoplasmic domain (CD) at C-terminus. The numbers indicate the domain regions. (C) Schematic diagram of physical locations of Gbvdr6, Gbvdr5 , and GbVe1 and the SSR markers flanking the known Verticillium wilt-resistant QTLs in the chromosomes of tetraploid cotton. The numbers in brackets indicate the physical positions in the chromosome. NAU2741 and CGR5056a are the SSR markers that flank Verticillium wilt resistance QTLs qVW-A1-1 and qVW-C15-4 in the A01(c1) and D01(c15) chromosomes, respectively.

Techniques Used: Software

47) Product Images from "The Effect of MCM3AP-AS1/miR-211/KLF5/AGGF1 Axis Regulating Glioblastoma Angiogenesis"

Article Title: The Effect of MCM3AP-AS1/miR-211/KLF5/AGGF1 Axis Regulating Glioblastoma Angiogenesis

Journal: Frontiers in Molecular Neuroscience

doi: 10.3389/fnmol.2017.00437

Knockdown of MCM3AP-AS1 inhibited GBM angiogenesis, KLF5 and AGGF1 expression via increasing miR-211 expression. (A) Quantitative real-time PCR analysis for miR-211 expression negatively correlative with MCM3AP-AS1 in GECs. Data are presented as mean ± SD. ( n = 5, each group), ∗ P
Figure Legend Snippet: Knockdown of MCM3AP-AS1 inhibited GBM angiogenesis, KLF5 and AGGF1 expression via increasing miR-211 expression. (A) Quantitative real-time PCR analysis for miR-211 expression negatively correlative with MCM3AP-AS1 in GECs. Data are presented as mean ± SD. ( n = 5, each group), ∗ P

Techniques Used: Expressing, Real-time Polymerase Chain Reaction

The schematic cartoon of the mechanism of MCM3AP-AS1/miR-211/KLF5/AGGF1 axis in GBM angiogenesis.
Figure Legend Snippet: The schematic cartoon of the mechanism of MCM3AP-AS1/miR-211/KLF5/AGGF1 axis in GBM angiogenesis.

Techniques Used:

MCM3AP-AS1 expression in glioma-associated endothelial cells (GECs) and inhibition of MCM3AP-AS1 knockdown on glioblastoma (GBM) angiogenesis, KLF5, and AGGF1 expression. (A) Relative MCM3AP-AS1 expression in ECs and GECs by quantitative real-time PCR. Data are presented as mean ± SD. ( n = 5, each group), ∗ P
Figure Legend Snippet: MCM3AP-AS1 expression in glioma-associated endothelial cells (GECs) and inhibition of MCM3AP-AS1 knockdown on glioblastoma (GBM) angiogenesis, KLF5, and AGGF1 expression. (A) Relative MCM3AP-AS1 expression in ECs and GECs by quantitative real-time PCR. Data are presented as mean ± SD. ( n = 5, each group), ∗ P

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

MCM3AP-AS1 knockdown combined with miR-211 overexpression inhibited GBM angiogenesis. (A) Matrigel plug assay was used to measure GBM angiogenesis. (B) The amount of hemoglobin was measured. Data are presented as mean ± SD. ( n = 5, each group), ∗ P
Figure Legend Snippet: MCM3AP-AS1 knockdown combined with miR-211 overexpression inhibited GBM angiogenesis. (A) Matrigel plug assay was used to measure GBM angiogenesis. (B) The amount of hemoglobin was measured. Data are presented as mean ± SD. ( n = 5, each group), ∗ P

Techniques Used: Over Expression, Matrigel Assay

48) Product Images from "miR-185 inhibits non-small cell lung cancer cell proliferation and invasion through targeting of SOX9 and regulation of Wnt signaling"

Article Title: miR-185 inhibits non-small cell lung cancer cell proliferation and invasion through targeting of SOX9 and regulation of Wnt signaling

Journal: Molecular Medicine Reports

doi: 10.3892/mmr.2017.8050

Downstream Wnt signaling of SOX9 is involved in the miR-185 regulation of NSCLC cell proliferation, invasion and migration. A549 and SK-MES-1 cells were transfected with miR-185 mimics or miR-185 inhibitor; the protein levels of SOX9, β-catenin and c-Myc in A549 and SK-MES-1 cells were determined using (A) western blotting and (B) densitometric analysis. The data are presented as the mean ± standard deviation of three independent experiments. *P
Figure Legend Snippet: Downstream Wnt signaling of SOX9 is involved in the miR-185 regulation of NSCLC cell proliferation, invasion and migration. A549 and SK-MES-1 cells were transfected with miR-185 mimics or miR-185 inhibitor; the protein levels of SOX9, β-catenin and c-Myc in A549 and SK-MES-1 cells were determined using (A) western blotting and (B) densitometric analysis. The data are presented as the mean ± standard deviation of three independent experiments. *P

Techniques Used: Migration, Transfection, Western Blot, Standard Deviation

49) Product Images from "Isolation and Role of PmRGL2 in GA-mediated Floral Bud Dormancy Release in Japanese Apricot (Prunus mume Siebold et Zucc.)"

Article Title: Isolation and Role of PmRGL2 in GA-mediated Floral Bud Dormancy Release in Japanese Apricot (Prunus mume Siebold et Zucc.)

Journal: Frontiers in Plant Science

doi: 10.3389/fpls.2018.00027

GA 4 levels in transgenic and wild-type poplar as measured by LC-MS/MS. Small letters over a column indicate significant differences between the WT and transgenic lines at P ≤ 0.05 (Duncan’s multiple range test). Data are the mean ± SD ( n = 3).
Figure Legend Snippet: GA 4 levels in transgenic and wild-type poplar as measured by LC-MS/MS. Small letters over a column indicate significant differences between the WT and transgenic lines at P ≤ 0.05 (Duncan’s multiple range test). Data are the mean ± SD ( n = 3).

Techniques Used: Transgenic Assay, Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry

Confirmation of PmRGL2 in three, independent lines of transgenic poplar (T 1 , T 2 , T 3 ), and its absence in wild-type poplar lines. WT, wild-type.
Figure Legend Snippet: Confirmation of PmRGL2 in three, independent lines of transgenic poplar (T 1 , T 2 , T 3 ), and its absence in wild-type poplar lines. WT, wild-type.

Techniques Used: Transgenic Assay

Phenotypic characterization of transgenic poplar plants constitutively expressing PmRGL2 in comparison with non-transgenic, wild-type, poplar plants. A, B, C are different transgenic lines. WT, wild-type. Plant height (measured from soil line to tip of flag leaf): WT: 90 cm, T 1 : 88 cm, T 2 : 81 cm, T 3 : 78 cm. Wild-type on the left and transgenic lines on the right in each figure. In the four pictures of each group, photos were taken after 0, 52, 70, and 83 days after entering into dormancy. The WT is on the left in each picture (A–F) , and the transgenic tree is on the right. T 1 (A,D) , T 2 (B,E) , T 3 (C,G) . (G) , close-up of buds in WT (left) and transgenic poplar (right) during dormancy release.
Figure Legend Snippet: Phenotypic characterization of transgenic poplar plants constitutively expressing PmRGL2 in comparison with non-transgenic, wild-type, poplar plants. A, B, C are different transgenic lines. WT, wild-type. Plant height (measured from soil line to tip of flag leaf): WT: 90 cm, T 1 : 88 cm, T 2 : 81 cm, T 3 : 78 cm. Wild-type on the left and transgenic lines on the right in each figure. In the four pictures of each group, photos were taken after 0, 52, 70, and 83 days after entering into dormancy. The WT is on the left in each picture (A–F) , and the transgenic tree is on the right. T 1 (A,D) , T 2 (B,E) , T 3 (C,G) . (G) , close-up of buds in WT (left) and transgenic poplar (right) during dormancy release.

Techniques Used: Transgenic Assay, Expressing

50) Product Images from "LincRNA-ROR promotes metastasis and invasion of esophageal squamous cell carcinoma by regulating miR-145/FSCN1"

Article Title: LincRNA-ROR promotes metastasis and invasion of esophageal squamous cell carcinoma by regulating miR-145/FSCN1

Journal: OncoTargets and therapy

doi: 10.2147/OTT.S157638

The anti-AGO2 RIP followed by qRT-PCR confirmed that ROR could bind to miR-145 (*** p
Figure Legend Snippet: The anti-AGO2 RIP followed by qRT-PCR confirmed that ROR could bind to miR-145 (*** p

Techniques Used: Quantitative RT-PCR

51) Product Images from "Decreased expression of microRNA-17 and microRNA-20b promotes breast cancer resistance to taxol therapy by upregulation of NCOA3"

Article Title: Decreased expression of microRNA-17 and microRNA-20b promotes breast cancer resistance to taxol therapy by upregulation of NCOA3

Journal: Cell Death & Disease

doi: 10.1038/cddis.2016.367

Identification of NCOA3 as a direct target of miR-17 and miR-20b. ( a ) The predicted miR-17 and miR-20b target sites in the 3′-UTR of NCOA3 mRNA and their mutated version. ( b and c ) Luciferase activity assays in 231/Tax1 and 293T cells showed that miR-17 and miR-20b inhibited the expression of NCOA3 . 231/Tax1 and 293T cells were co-transfected with pGL3 vector containing the wild-type or mutated 3′-UTR of NCOA3 , or pGL3-control vector, along with miR-17 or miR-20b mimics and NC. After 48 h, luciferase activity was detected. Data were normalized to luciferase activity in the corresponding cells transfected with NC and are represented as the mean±S.D. of three replicates. ( d - g ) Protein and mRNA levels of NCOA3 were downregulated by miR-17 or miR-20b mimics in breast cancer cells. MCF-7/Tax1 ( d and e ) and 231/Tax1 ( f and g ) cells were transfected with miR-17 or miR-20b mimics and NC, respectively. (A) Western blot was performed to detect the protein expression of NCOA3. Actin was used as a loading control. Data were from three independent experiments. (B) RT-PCR was performed to detect the mRNA expression of NCOA3. Actin was used as control. Data are mean±S.D. from three independent experiments. *** P
Figure Legend Snippet: Identification of NCOA3 as a direct target of miR-17 and miR-20b. ( a ) The predicted miR-17 and miR-20b target sites in the 3′-UTR of NCOA3 mRNA and their mutated version. ( b and c ) Luciferase activity assays in 231/Tax1 and 293T cells showed that miR-17 and miR-20b inhibited the expression of NCOA3 . 231/Tax1 and 293T cells were co-transfected with pGL3 vector containing the wild-type or mutated 3′-UTR of NCOA3 , or pGL3-control vector, along with miR-17 or miR-20b mimics and NC. After 48 h, luciferase activity was detected. Data were normalized to luciferase activity in the corresponding cells transfected with NC and are represented as the mean±S.D. of three replicates. ( d - g ) Protein and mRNA levels of NCOA3 were downregulated by miR-17 or miR-20b mimics in breast cancer cells. MCF-7/Tax1 ( d and e ) and 231/Tax1 ( f and g ) cells were transfected with miR-17 or miR-20b mimics and NC, respectively. (A) Western blot was performed to detect the protein expression of NCOA3. Actin was used as a loading control. Data were from three independent experiments. (B) RT-PCR was performed to detect the mRNA expression of NCOA3. Actin was used as control. Data are mean±S.D. from three independent experiments. *** P

Techniques Used: Luciferase, Activity Assay, Expressing, Transfection, Plasmid Preparation, Western Blot, Reverse Transcription Polymerase Chain Reaction

MiR-17 and miR-20b are negatively correlated with NCOA3 mRNA levels in breast cancer. Relative expression of NCOA3 along with miR-17 and miR-20b was determined by RT-PCR in 22 taxol-sensitive and 33 taxol-resistant breast cancer tissues from patients with taxol treatment. ( a - d ) Relative expression of NCOA3 along with miR-17 and miR-20b were determined by RT-PCR in 22 taxol-sensitive breast cancer tissues ( a and c ) and 33 taxol-resistant breast cancer tissues ( b and d ). For NCOA3, β -Actin was used as an internal control; For miR-17 and miR-20b, U6 was used as an internal control. Their expression correlations were analyzed by correlation coefficient and t -test
Figure Legend Snippet: MiR-17 and miR-20b are negatively correlated with NCOA3 mRNA levels in breast cancer. Relative expression of NCOA3 along with miR-17 and miR-20b was determined by RT-PCR in 22 taxol-sensitive and 33 taxol-resistant breast cancer tissues from patients with taxol treatment. ( a - d ) Relative expression of NCOA3 along with miR-17 and miR-20b were determined by RT-PCR in 22 taxol-sensitive breast cancer tissues ( a and c ) and 33 taxol-resistant breast cancer tissues ( b and d ). For NCOA3, β -Actin was used as an internal control; For miR-17 and miR-20b, U6 was used as an internal control. Their expression correlations were analyzed by correlation coefficient and t -test

Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction

MiR-17 and miR-20b are predicted to target NCOA3 and decreased in taxol-resistant breast cancer. ( a ) Flowchart for the selection of the miR-17 and miR-20b. ( b ) RT-PCR was performed to detect the expression of miR-17 and miR-20b in MCF-7, 231, MCF-7/Tax1, MCF-7/Tax2, 231/Tax1 and 231/Tax2 cells. U6 was used as an internal control. Data represent mean±S.D. *** P
Figure Legend Snippet: MiR-17 and miR-20b are predicted to target NCOA3 and decreased in taxol-resistant breast cancer. ( a ) Flowchart for the selection of the miR-17 and miR-20b. ( b ) RT-PCR was performed to detect the expression of miR-17 and miR-20b in MCF-7, 231, MCF-7/Tax1, MCF-7/Tax2, 231/Tax1 and 231/Tax2 cells. U6 was used as an internal control. Data represent mean±S.D. *** P

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

MiR-17 and miR-20b decrease resistance of breast tumor to taxol in xenograft tumor models. MCF-7/Tax1 and 231/Tax1 cells stably expressing miR-17 or miR-20b mimics or NC mimics were injected into nude mice. Nude mice were administered with taxol (20 mg/kg) as indicated at each time point. ( a and b ) Tumor volume was measured once per three days by using calipers (as indicated at each time point) for 30–36 days. ( c and d ) Average body weight changes were measured over the course of the study. Data are shown as mean±S.D. ( n =6 per group). *** P
Figure Legend Snippet: MiR-17 and miR-20b decrease resistance of breast tumor to taxol in xenograft tumor models. MCF-7/Tax1 and 231/Tax1 cells stably expressing miR-17 or miR-20b mimics or NC mimics were injected into nude mice. Nude mice were administered with taxol (20 mg/kg) as indicated at each time point. ( a and b ) Tumor volume was measured once per three days by using calipers (as indicated at each time point) for 30–36 days. ( c and d ) Average body weight changes were measured over the course of the study. Data are shown as mean±S.D. ( n =6 per group). *** P

Techniques Used: Stable Transfection, Expressing, Injection, Mouse Assay

Both miR-17 and miR-20b enhances taxol-induced apoptosis in breast cancer cells. ( a and b ) (a) 231/Tax1 and MCF-7/Tax1 were transfected with miR-17 mimics ( a ) or miR-20b mimics ( b ) and NC for 48 h, respectively. (B) MCF-7 and 231 cells were transfected with miR-17 inhibitors ( a ) or miR-20b inhibitors ( b ) and NC for 48 h, respectively. RT-PCR was performed to detect the expression of miR-17 or miR-20b. ( c and d ) MCF-7/Tax1 (A) and 231/Tax1 (B) were transfected with miR-17 or miR-20b mimics. ( e and f ) MCF-7 (A) and 231 (B) were transfected with miR-17 or miR-20b inhibitors. After 8 h, cells were treated with indicated dose of taxol (Tax) for additional 48 h. MTT assay was performed to examine cell viability ( c and e ). Cell apoptosis was assessed by Annexin-V-FITC/PI staining assay by flow cytometry ( d and f ). Columns, means of three determinations; bars, S.D. * P
Figure Legend Snippet: Both miR-17 and miR-20b enhances taxol-induced apoptosis in breast cancer cells. ( a and b ) (a) 231/Tax1 and MCF-7/Tax1 were transfected with miR-17 mimics ( a ) or miR-20b mimics ( b ) and NC for 48 h, respectively. (B) MCF-7 and 231 cells were transfected with miR-17 inhibitors ( a ) or miR-20b inhibitors ( b ) and NC for 48 h, respectively. RT-PCR was performed to detect the expression of miR-17 or miR-20b. ( c and d ) MCF-7/Tax1 (A) and 231/Tax1 (B) were transfected with miR-17 or miR-20b mimics. ( e and f ) MCF-7 (A) and 231 (B) were transfected with miR-17 or miR-20b inhibitors. After 8 h, cells were treated with indicated dose of taxol (Tax) for additional 48 h. MTT assay was performed to examine cell viability ( c and e ). Cell apoptosis was assessed by Annexin-V-FITC/PI staining assay by flow cytometry ( d and f ). Columns, means of three determinations; bars, S.D. * P

Techniques Used: Transfection, Reverse Transcription Polymerase Chain Reaction, Expressing, MTT Assay, Staining, Flow Cytometry, Cytometry

Overexpression of NCOA3 reverses reduction of cell viability and induction of apoptosis by miR-17 and miR-20b in taxol-treated breast cancer cells. ( a - d ) MCF-7/Tax1 ( a and c ) and 231/Tax1 ( b and d ) cells were co-transfected with NC and miR-17 or miR-20b mimics along with control (CTR) or NCOA3 vectors. After 8 h, cells were treated with indicated dose of taxol (Tax) for additional 48 h. ( a and b ) MTT assay was performed to examine cell viability. ( c and d ) Cell apoptosis was assessed by Annexin-V-FITC/PI staining assay by flow cytometry. Columns, means of three determinations; bars, S.D.; ** P
Figure Legend Snippet: Overexpression of NCOA3 reverses reduction of cell viability and induction of apoptosis by miR-17 and miR-20b in taxol-treated breast cancer cells. ( a - d ) MCF-7/Tax1 ( a and c ) and 231/Tax1 ( b and d ) cells were co-transfected with NC and miR-17 or miR-20b mimics along with control (CTR) or NCOA3 vectors. After 8 h, cells were treated with indicated dose of taxol (Tax) for additional 48 h. ( a and b ) MTT assay was performed to examine cell viability. ( c and d ) Cell apoptosis was assessed by Annexin-V-FITC/PI staining assay by flow cytometry. Columns, means of three determinations; bars, S.D.; ** P

Techniques Used: Over Expression, Transfection, MTT Assay, Staining, Flow Cytometry, Cytometry

52) Product Images from "JS-K, a nitric oxide pro-drug, regulates growth and apoptosis through the ubiquitin-proteasome pathway in prostate cancer cells"

Article Title: JS-K, a nitric oxide pro-drug, regulates growth and apoptosis through the ubiquitin-proteasome pathway in prostate cancer cells

Journal: BMC Cancer

doi: 10.1186/s12885-017-3351-0

C4-2 ( a ) and LNCaP ( b ) cells were incubated for three periods (3, 6 and 9 h) with 5 μM JS-K. RT-PCR was performed to access the influence of JS-K on transcription of specific AR target genes ( PSA , NKX3.1 , PMEPA1 and SLC45A3 ). Each assay was performed in triplicate and the expression levels of mRNAs were expressed as 2 -ΔΔCT ; c western blotting was performed to detect the influence of JS-K on PSA in C4-2 and LNCaP cells incubated for three periods (3, 6 and 9 h) with 5 μM JS-K. Results are mean ± SD of three different experiments. Single asterisks (*) indicate a significant difference ( P
Figure Legend Snippet: C4-2 ( a ) and LNCaP ( b ) cells were incubated for three periods (3, 6 and 9 h) with 5 μM JS-K. RT-PCR was performed to access the influence of JS-K on transcription of specific AR target genes ( PSA , NKX3.1 , PMEPA1 and SLC45A3 ). Each assay was performed in triplicate and the expression levels of mRNAs were expressed as 2 -ΔΔCT ; c western blotting was performed to detect the influence of JS-K on PSA in C4-2 and LNCaP cells incubated for three periods (3, 6 and 9 h) with 5 μM JS-K. Results are mean ± SD of three different experiments. Single asterisks (*) indicate a significant difference ( P

Techniques Used: Incubation, Reverse Transcription Polymerase Chain Reaction, Expressing, Western Blot

53) Product Images from "Isolation and Characterization of Encephalomyocarditis Virus from Dogs in China"

Article Title: Isolation and Characterization of Encephalomyocarditis Virus from Dogs in China

Journal: Scientific Reports

doi: 10.1038/s41598-017-00435-x

EMCV-C15 loads in the organs of artificially challenged dogs according to real-time RT-PCR. Viral loads in various organs are shown in the graph. Eight 25-to-30-day-old dogs were obtained from a commercial breeding herd that was free of CPV, CDV, CPIV, CHV, ICHV and EMCV. Dogs were randomly allocated into two groups. Dogs in group A (n = 5) were inoculated with 1.0 mL (10 5 TCID 50 ) of EMCV-C15. Dogs in group B (n = 3) were injected with 1.0 mL of DMEM as a control treatment. At 35 DPI, all dogs were euthanized and subjected to analysis of viral loads/virus distribution of EMCV-C15. Numbers 1, 2, 3, 4 and 5 in the legend correspond to the five dogs inoculated with EMCV-C15. Data from group B (uninfected group) are not visible in the graph because there was no viral load in animals from that group. Error bars represent the standard error of the mean of three replicates.
Figure Legend Snippet: EMCV-C15 loads in the organs of artificially challenged dogs according to real-time RT-PCR. Viral loads in various organs are shown in the graph. Eight 25-to-30-day-old dogs were obtained from a commercial breeding herd that was free of CPV, CDV, CPIV, CHV, ICHV and EMCV. Dogs were randomly allocated into two groups. Dogs in group A (n = 5) were inoculated with 1.0 mL (10 5 TCID 50 ) of EMCV-C15. Dogs in group B (n = 3) were injected with 1.0 mL of DMEM as a control treatment. At 35 DPI, all dogs were euthanized and subjected to analysis of viral loads/virus distribution of EMCV-C15. Numbers 1, 2, 3, 4 and 5 in the legend correspond to the five dogs inoculated with EMCV-C15. Data from group B (uninfected group) are not visible in the graph because there was no viral load in animals from that group. Error bars represent the standard error of the mean of three replicates.

Techniques Used: Quantitative RT-PCR, Injection

54) Product Images from "The Preferential Infection of Astrocytes by Enterovirus 71 Plays a Key Role in the Viral Neurogenic Pathogenesis"

Article Title: The Preferential Infection of Astrocytes by Enterovirus 71 Plays a Key Role in the Viral Neurogenic Pathogenesis

Journal: Frontiers in Cellular and Infection Microbiology

doi: 10.3389/fcimb.2016.00192

Effect of astrocyte engulfment on EV71 infection in astrocytes . Antibodies against EV71-specific receptors failed to inhibit EV71 infection in human (A) and macaque astrocytes (B). (C) Detection of complement C3 mRNA expression in infected human astrocytes. NC: negative control. (D) Incubation of complement and virus facilitates viral replication in human astrocytes, with a peak at 12–36 h. The viral loads of the infected astrocytes and relative expression of complement mRNA were measured using TaqMan-based qRT-PCR. β-actin was used as the house keeping gene.
Figure Legend Snippet: Effect of astrocyte engulfment on EV71 infection in astrocytes . Antibodies against EV71-specific receptors failed to inhibit EV71 infection in human (A) and macaque astrocytes (B). (C) Detection of complement C3 mRNA expression in infected human astrocytes. NC: negative control. (D) Incubation of complement and virus facilitates viral replication in human astrocytes, with a peak at 12–36 h. The viral loads of the infected astrocytes and relative expression of complement mRNA were measured using TaqMan-based qRT-PCR. β-actin was used as the house keeping gene.

Techniques Used: Infection, Expressing, Negative Control, Incubation, Quantitative RT-PCR

Histopathological characteristics of CNS tissue from EV71-infected rhesus macaques and human patients . Typical pathological changes in the brain stem include the following: (A,D) inflammatory cells aggregated near vascular tissue; (B,E) neuron degeneration and glial cell proliferation and glial nodule formation; (C,F) EV71 viral antigen expression in CNS tissues from EV71-infected rhesus macaque and human patients. Monoamine (G) and cytokine (H) detection in brain stem homogenates, and monoamine (I) detection in the peripheral blood from infected rhesus macaques. NA, noradrenalin; AD, adrenalin; DOP, dopamine; NC, negative control. * p ≤ 0.05 compared with the corresponding control group.
Figure Legend Snippet: Histopathological characteristics of CNS tissue from EV71-infected rhesus macaques and human patients . Typical pathological changes in the brain stem include the following: (A,D) inflammatory cells aggregated near vascular tissue; (B,E) neuron degeneration and glial cell proliferation and glial nodule formation; (C,F) EV71 viral antigen expression in CNS tissues from EV71-infected rhesus macaque and human patients. Monoamine (G) and cytokine (H) detection in brain stem homogenates, and monoamine (I) detection in the peripheral blood from infected rhesus macaques. NA, noradrenalin; AD, adrenalin; DOP, dopamine; NC, negative control. * p ≤ 0.05 compared with the corresponding control group.

Techniques Used: Infection, Expressing, Negative Control

EV71 was detected in neurons and astrocytes in CNS tissues from infected rhesus macaques and human patients . The green fluorescent signal of EV71 was detected in neurons (A) and astrocytes (B) labeled with red fluorescence by an anti-neuron antibody and an anti-GAFP antibody, respectively. (B) Immunofluorescence confocal microscopy observations of neurons (C) or astrocytes (D) and EV71 antigen in CNS tissues from infected human patients. (E) The percentages of antigen-positive neurons and astrocytes in the brain stem (from 20 sections from 4 animals and 30 sections from 6 patients).
Figure Legend Snippet: EV71 was detected in neurons and astrocytes in CNS tissues from infected rhesus macaques and human patients . The green fluorescent signal of EV71 was detected in neurons (A) and astrocytes (B) labeled with red fluorescence by an anti-neuron antibody and an anti-GAFP antibody, respectively. (B) Immunofluorescence confocal microscopy observations of neurons (C) or astrocytes (D) and EV71 antigen in CNS tissues from infected human patients. (E) The percentages of antigen-positive neurons and astrocytes in the brain stem (from 20 sections from 4 animals and 30 sections from 6 patients).

Techniques Used: Infection, Labeling, Fluorescence, Immunofluorescence, Confocal Microscopy

Detection of EV71 replication in cultured neurons and astrocytes in vitro . Proliferation of the virus in cultured human neurons (A) and astrocytes (B) was measured based on virus titration and the viral load from 6 to 60 h p.i. Immunofluorescence microscopy observations of the EV71 antigen in cultured EV71-infected neurons (C) images are shown at 100 × magnification) and astrocytes (D) images are shown at 400 × magnification.). Monoamine release by neurons (E) and astrocytes (F) from 6 to 60 h p.i. Cytokines released by astrocytes (G) between 6 and 60 h p.i. The cells were infected with EV71 (MOI = 0.05). NA, noradrenalin; AD, adrenalin; DOP, dopamine; NC, negative control. * p ≤ 0.05, compared with the corresponding control group. (H) A characteristic lattice-like arrangement of viral particles was observed in human astrocytes induced by EV71 infection as observed by TEM. Astrocytes were infected with EV71 (MOI = 0.1), and samples were collected at 24 h p.i. (I) Presence of negative-stranded RNA genomes in the cells is shown (black arrow). M, marker; AS, astrocyte.
Figure Legend Snippet: Detection of EV71 replication in cultured neurons and astrocytes in vitro . Proliferation of the virus in cultured human neurons (A) and astrocytes (B) was measured based on virus titration and the viral load from 6 to 60 h p.i. Immunofluorescence microscopy observations of the EV71 antigen in cultured EV71-infected neurons (C) images are shown at 100 × magnification) and astrocytes (D) images are shown at 400 × magnification.). Monoamine release by neurons (E) and astrocytes (F) from 6 to 60 h p.i. Cytokines released by astrocytes (G) between 6 and 60 h p.i. The cells were infected with EV71 (MOI = 0.05). NA, noradrenalin; AD, adrenalin; DOP, dopamine; NC, negative control. * p ≤ 0.05, compared with the corresponding control group. (H) A characteristic lattice-like arrangement of viral particles was observed in human astrocytes induced by EV71 infection as observed by TEM. Astrocytes were infected with EV71 (MOI = 0.1), and samples were collected at 24 h p.i. (I) Presence of negative-stranded RNA genomes in the cells is shown (black arrow). M, marker; AS, astrocyte.

Techniques Used: Cell Culture, In Vitro, Titration, Immunofluorescence, Microscopy, Infection, Negative Control, Transmission Electron Microscopy, Marker

Modulated IL-6 release by infected astrocytes induces adrenalin secretion . Monoamine release by neurons from the brain stem (A) or KMB 17 cells (B) co-cultured with EV71-infected astrocytes from the brain stem. (C) Adrenalin release by neurons from the brain stem treated with IL-6 (15 ng/ml). (D) Monoamine release by neurons from the cerebrum co-cultured with EV71-infected astrocytes from the brain stem. Astrocytes were infected with EV71 (MOI = 0.05). The cultured cell supernatant samples were collected at different hours p.i. * p ≤ 0.05, compared with the corresponding control group. NA, noradrenalin; AD, adrenalin; DOP, dopamine.
Figure Legend Snippet: Modulated IL-6 release by infected astrocytes induces adrenalin secretion . Monoamine release by neurons from the brain stem (A) or KMB 17 cells (B) co-cultured with EV71-infected astrocytes from the brain stem. (C) Adrenalin release by neurons from the brain stem treated with IL-6 (15 ng/ml). (D) Monoamine release by neurons from the cerebrum co-cultured with EV71-infected astrocytes from the brain stem. Astrocytes were infected with EV71 (MOI = 0.05). The cultured cell supernatant samples were collected at different hours p.i. * p ≤ 0.05, compared with the corresponding control group. NA, noradrenalin; AD, adrenalin; DOP, dopamine.

Techniques Used: Infection, Cell Culture

Dynamic distribution of EV71 in the CNS of infected rhesus macaques . The green fluorescent signal and EV71 antigen were aggregated around the blood vessels on day 3 (A,C) and distributed in the tissues of the brain stem on day 7 after EGFP-EV71 infection (B,D) . The green fluorescent signal was detected and the EV71 antigens were expressed (red rectangle) in olivary nuclei (white rectangle) on day 7 after EGFP-EV71 infection via the respiratory tract (E,G) and cerebral injection (F,H) . The images are shown at 100 × magnification in e and f and were combined automatically using Leica AF Multi-Dimensional Acquisition software.
Figure Legend Snippet: Dynamic distribution of EV71 in the CNS of infected rhesus macaques . The green fluorescent signal and EV71 antigen were aggregated around the blood vessels on day 3 (A,C) and distributed in the tissues of the brain stem on day 7 after EGFP-EV71 infection (B,D) . The green fluorescent signal was detected and the EV71 antigens were expressed (red rectangle) in olivary nuclei (white rectangle) on day 7 after EGFP-EV71 infection via the respiratory tract (E,G) and cerebral injection (F,H) . The images are shown at 100 × magnification in e and f and were combined automatically using Leica AF Multi-Dimensional Acquisition software.

Techniques Used: Infection, Injection, Software

55) Product Images from "Epithelial ovarian cancer-secreted exosomal miR-222-3p induces polarization of tumor-associated macrophages"

Article Title: Epithelial ovarian cancer-secreted exosomal miR-222-3p induces polarization of tumor-associated macrophages

Journal: Oncotarget

doi: 10.18632/oncotarget.9246

MiR-222-3p promotes M2 phenotype polarization of macrophages in vitro and vivo , which can enhance growth and metastasis of EOC cells Macrophages were transfected with the miR-222-3p mimic and compared with a negative control. ( A ) After 24 hours, qRT-PCR was applied using primers for CD206 and Arg-1. ( B ) After 48 hours, the expression of M2 surface marker CD206 was detected by western blot. ( C ) Cytokine levels in the media of macrophages. ( D ) Proliferation and migration capacity of the human EOC cell line Skov3 which incubated with the supernatants of macrophages treated with miR-222-3p or miR-negative control was performed using MTS and Transwell assay. ( E ) Images of appearance of tumour deposits at necropsy of miR-222-3p group compared with miR-NC group in vivo . ( F ) Representative images of CD31, LYVE-1 and CD206 immunostaining are shown in tumor tissues of miR-222-3p group or miR-NC group; scale bar = 50 μm. CD31 positive ‘hotspots’, LYVE-1 positive ‘hotspots’ and CD206-positive cells were calculated as number of positive cells. Data are shown as mean ± SEM, n = 3 independent experiments; * p
Figure Legend Snippet: MiR-222-3p promotes M2 phenotype polarization of macrophages in vitro and vivo , which can enhance growth and metastasis of EOC cells Macrophages were transfected with the miR-222-3p mimic and compared with a negative control. ( A ) After 24 hours, qRT-PCR was applied using primers for CD206 and Arg-1. ( B ) After 48 hours, the expression of M2 surface marker CD206 was detected by western blot. ( C ) Cytokine levels in the media of macrophages. ( D ) Proliferation and migration capacity of the human EOC cell line Skov3 which incubated with the supernatants of macrophages treated with miR-222-3p or miR-negative control was performed using MTS and Transwell assay. ( E ) Images of appearance of tumour deposits at necropsy of miR-222-3p group compared with miR-NC group in vivo . ( F ) Representative images of CD31, LYVE-1 and CD206 immunostaining are shown in tumor tissues of miR-222-3p group or miR-NC group; scale bar = 50 μm. CD31 positive ‘hotspots’, LYVE-1 positive ‘hotspots’ and CD206-positive cells were calculated as number of positive cells. Data are shown as mean ± SEM, n = 3 independent experiments; * p

Techniques Used: In Vitro, Transfection, Negative Control, Quantitative RT-PCR, Expressing, Marker, Western Blot, Migration, Incubation, Transwell Assay, In Vivo, Immunostaining

Exosomes derived EOC cells activate macrophages to a TAM-like phenotype in vitro and vivo , which can promote the progression of EOC Macrophages were treated with EOC-derived exosomes (100 ug/ml) or control (PBS). ( A ) After 48 hours, qRT-PCR was applied using primers for CD206, Arg-1, or MCP-1. ( B ) After 96 hours, the expression of M2 surface marker CD206 was detected by western blot. ( C ) Cytokine levels in the media of macrophages. ( D ) Proliferation and migration capacity of Skov-3 human ovarian cancer cells which incubated with the supernatants of macrophages treated with the exosomes or control (PBS) was performed using MTS and Transwell assay. Cell viability was determined by OD measurement and migrated cells were counted. ( E ) Photographs of appearance of tumor deposits at necropsy of the injected mice. ( F ) Representative IHC examples of CD31, LYVE-1 and CD206 staining are shown in tumors of Skov3-exo group and compared with control group; scale bar = 50 μm. CD31 positive ‘hotspots’, LYVE-1 positive ‘hotspots’ and CD206-positive cells were calculated as number of positive cells. Data are shown as mean ± SEM, n = 3 independent experiments; * p
Figure Legend Snippet: Exosomes derived EOC cells activate macrophages to a TAM-like phenotype in vitro and vivo , which can promote the progression of EOC Macrophages were treated with EOC-derived exosomes (100 ug/ml) or control (PBS). ( A ) After 48 hours, qRT-PCR was applied using primers for CD206, Arg-1, or MCP-1. ( B ) After 96 hours, the expression of M2 surface marker CD206 was detected by western blot. ( C ) Cytokine levels in the media of macrophages. ( D ) Proliferation and migration capacity of Skov-3 human ovarian cancer cells which incubated with the supernatants of macrophages treated with the exosomes or control (PBS) was performed using MTS and Transwell assay. Cell viability was determined by OD measurement and migrated cells were counted. ( E ) Photographs of appearance of tumor deposits at necropsy of the injected mice. ( F ) Representative IHC examples of CD31, LYVE-1 and CD206 staining are shown in tumors of Skov3-exo group and compared with control group; scale bar = 50 μm. CD31 positive ‘hotspots’, LYVE-1 positive ‘hotspots’ and CD206-positive cells were calculated as number of positive cells. Data are shown as mean ± SEM, n = 3 independent experiments; * p

Techniques Used: Derivative Assay, In Vitro, Quantitative RT-PCR, Expressing, Marker, Western Blot, Migration, Incubation, Transwell Assay, Injection, Mouse Assay, Immunohistochemistry, Staining

Regulation of SOCS3 expression and the related signaling pathway by EOC-derived exosomes Macrophages were treated with EOC-derived exosomes (100 ug/ml) or control (PBS). ( A ) Expression of SOCS3 gene in macrophages that were stimulated with EOC-derived exosomes or control (PBS), as performed by qRT-PCR. ( B ) A representative immunoblot of SOCS3, phosphorylated (p-) STAT3 and total STAT3 in macrophages which were treated with EOC-derived exosomes or control (PBS) is displayed along with ( C ) quantitative data by densitometry. ( D ) Macrophages were treated with phosphor-STAT3 inhibitor stattic prior to stimulate with Skov3-derived exosomes. Expression of M2 surface marker CD206 was detected by western blot. Data are shown as mean ± SEM, n = 3 independent experiments.* p
Figure Legend Snippet: Regulation of SOCS3 expression and the related signaling pathway by EOC-derived exosomes Macrophages were treated with EOC-derived exosomes (100 ug/ml) or control (PBS). ( A ) Expression of SOCS3 gene in macrophages that were stimulated with EOC-derived exosomes or control (PBS), as performed by qRT-PCR. ( B ) A representative immunoblot of SOCS3, phosphorylated (p-) STAT3 and total STAT3 in macrophages which were treated with EOC-derived exosomes or control (PBS) is displayed along with ( C ) quantitative data by densitometry. ( D ) Macrophages were treated with phosphor-STAT3 inhibitor stattic prior to stimulate with Skov3-derived exosomes. Expression of M2 surface marker CD206 was detected by western blot. Data are shown as mean ± SEM, n = 3 independent experiments.* p

Techniques Used: Expressing, Derivative Assay, Quantitative RT-PCR, Marker, Western Blot

EOC patients have higher level of miR-222-3p in serum-derived exosomes than healthy individuals Besides, miR-222-3p is richer in EOC-derived exosomes and can be transferred to macrophages via the exosomes. ( A ) MicroRNAs were abstracted in serum-derived exosomes from 6 EOC patients and from 6 healthy individuals. MiR-222-3p expression was detected by qRT-PCR. Data are shown as median ± range, n = 6 independent experiments. ** p
Figure Legend Snippet: EOC patients have higher level of miR-222-3p in serum-derived exosomes than healthy individuals Besides, miR-222-3p is richer in EOC-derived exosomes and can be transferred to macrophages via the exosomes. ( A ) MicroRNAs were abstracted in serum-derived exosomes from 6 EOC patients and from 6 healthy individuals. MiR-222-3p expression was detected by qRT-PCR. Data are shown as median ± range, n = 6 independent experiments. ** p

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

56) Product Images from "A noncoding RNA containing a SINE-B1 motif associates with meiotic metaphase chromatin and has an indispensable function during spermatogenesis"

Article Title: A noncoding RNA containing a SINE-B1 motif associates with meiotic metaphase chromatin and has an indispensable function during spermatogenesis

Journal: PLoS ONE

doi: 10.1371/journal.pone.0179585

QRT-PCR analyses of the subcellular localization of the RNAs in the testicular cells. Testicular cells were prepared from the testes of mice 22 days after birth; this stage approximately corresponds to the time of the highest level of R53 expression ( Fig 5B ). (A) Gel electrophoreses of PCR products using primer pairs for SCP3 (19 cycles), BC1 (22 cycles), Xist (35 cycles), β-actin (18 cycles), R53 RNA (35 cycles, R53-S2 for the primer detecting a R53-B1F-containing sequence downstream of that of R53-S1) and genotyping of the Jmjd1C locus (genome, 35 cycles) in the cytoplasmic extract (Cyt), nuclear soluble (Nuc) and chromatin-bound (Chr) fractions are shown. The genotyping PCR (qJ1C used as the primer) to detect a sequence in the third intron of wild-type Jmjd1C gene was performed to demonstrate that there was almost no contamination of the genomic DNA in any fraction. All the primer pairs used are listed in S1 Table . (B) Reverse-transcription was performed using total RNA prepared from the chromatin-bound fraction and oligo-dT primer with (RT+) or without reverse transcriptase (RT-). As in (A), PCR was performed using 2 sets of primers for R53 RNA (35 cycles) to testify that the PCR products were derived from RT-dependent cDNA. The PCR specificity for R53 transcript was confirmed by sequencing analyses of the PCR products. (C) The amounts of each transcript in the three subcellular fractions were quantitatively analyzed. The values indicated are the relative levels of each transcript against total values of the three subcellular fractions (set as 100%). The error bars indicate the SEM (n = 6).
Figure Legend Snippet: QRT-PCR analyses of the subcellular localization of the RNAs in the testicular cells. Testicular cells were prepared from the testes of mice 22 days after birth; this stage approximately corresponds to the time of the highest level of R53 expression ( Fig 5B ). (A) Gel electrophoreses of PCR products using primer pairs for SCP3 (19 cycles), BC1 (22 cycles), Xist (35 cycles), β-actin (18 cycles), R53 RNA (35 cycles, R53-S2 for the primer detecting a R53-B1F-containing sequence downstream of that of R53-S1) and genotyping of the Jmjd1C locus (genome, 35 cycles) in the cytoplasmic extract (Cyt), nuclear soluble (Nuc) and chromatin-bound (Chr) fractions are shown. The genotyping PCR (qJ1C used as the primer) to detect a sequence in the third intron of wild-type Jmjd1C gene was performed to demonstrate that there was almost no contamination of the genomic DNA in any fraction. All the primer pairs used are listed in S1 Table . (B) Reverse-transcription was performed using total RNA prepared from the chromatin-bound fraction and oligo-dT primer with (RT+) or without reverse transcriptase (RT-). As in (A), PCR was performed using 2 sets of primers for R53 RNA (35 cycles) to testify that the PCR products were derived from RT-dependent cDNA. The PCR specificity for R53 transcript was confirmed by sequencing analyses of the PCR products. (C) The amounts of each transcript in the three subcellular fractions were quantitatively analyzed. The values indicated are the relative levels of each transcript against total values of the three subcellular fractions (set as 100%). The error bars indicate the SEM (n = 6).

Techniques Used: Quantitative RT-PCR, Mouse Assay, Expressing, Polymerase Chain Reaction, Sequencing, Derivative Assay

57) Product Images from "Integrative analyses of transcriptome sequencing identify novel functional lncRNAs in esophageal squamous cell carcinoma"

Article Title: Integrative analyses of transcriptome sequencing identify novel functional lncRNAs in esophageal squamous cell carcinoma

Journal: Oncogenesis

doi: 10.1038/oncsis.2017.1

Gene expression profile analysis after lncRNA625 knockdown. ( a ) Gene expression profile analysis performed after lncRNA625 knockdown in cells stably transfected with either shlncRNA625 or scrambled shRNA (shscramble). ( b ) qRT–PCR of a representative panel of genes in scrambled and silncRNA625 (error bars are s.d., n =6). ( c ) PCGs downregulated by lncRNA625 knockdown and significantly upregulated in RNA-seq samples. ( d ) PCGs upregulated and downregulated, following lncRNA625 silencing, in RNA-seq samples. Genes boxed in red are literature-evidenced cancer-related genes. Genes with an asterisk are literature-evidenced ESCC-related genes.
Figure Legend Snippet: Gene expression profile analysis after lncRNA625 knockdown. ( a ) Gene expression profile analysis performed after lncRNA625 knockdown in cells stably transfected with either shlncRNA625 or scrambled shRNA (shscramble). ( b ) qRT–PCR of a representative panel of genes in scrambled and silncRNA625 (error bars are s.d., n =6). ( c ) PCGs downregulated by lncRNA625 knockdown and significantly upregulated in RNA-seq samples. ( d ) PCGs upregulated and downregulated, following lncRNA625 silencing, in RNA-seq samples. Genes boxed in red are literature-evidenced cancer-related genes. Genes with an asterisk are literature-evidenced ESCC-related genes.

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

LncRNA625 interacts with EP300 to regulate downstream target genes. ( a ) LncRNA625 is located in the cytoplasm and nucleus of tumor tissue. Sense or antisense probe for lncRNA625 FISH were synthesized by in vitro transcription of T7 RNA polymerase, and 3 μm serial slides of ESCC tissues were hybridized with sense or antisense probes conjugated with biotin. Subsequently, the biotin signal was determined with Cy3-conjugated streptavidine. DAPI staining for was for nuclei, and haematoxylin and eosin staining was for tumor histomorphology. Scale bar: 40 ×. ( b ) Cytoplasmic and nuclear RNAs were isolated from KYSE510 cells, and lncRNA625 was detected by real-time RT–PCR. Levels of U6 snRNA (nuclear control transcript) and GAPDH (cytoplasmic control transcript) were detected by real-time RT–PCR. Values are mean±s.e. ( c ) Venn diagram showing the overlap between ESCC-related histone modification proteins and lncRNA625-related transcription regulatory proteins. ( d ) Comparison of 202 differentially expressed genes following silencing lncRNA625 in KYSE150 cells vs. a compendium of UCSC-published EP300 occupancy profiles in diverse cell types. ( e ) LncRNA625 interacts with EP300. RNA immunoprecipitation assays for EP300 were performed and RNA was extracted with 1 ml TRIzol, and lncRNA625 was detected by real-time RT-PCR in both KYSE150 and KYSE510 ESCC cells. IgG and SP1 were used as negative controls in the experiment. ( f ) Gene expression profile analysis was performed after either lncRNA625 or EP300 knockdown in KYSE 150 cells. Genes with |log 2 FC| > log 2 1.5, after lncRNA625 knockdown, are displayed in the heat map. ( g ) GSEA plot showing that genes regulated by lncRNA625 were enriched in the expression profile after knocking down EP300. In particular, those genes downregulated after knocking down EP300 received a high enrichment score. ( h ) qRT–PCR of a representative panel of genes in scrambled, silncRNA625 and siEP300 cells (error bars are s.d., n =6). Genes boxed in red are literature-evidenced cancer-related genes. Genes with an asterisk are literature-evidenced ESCC-related genes.
Figure Legend Snippet: LncRNA625 interacts with EP300 to regulate downstream target genes. ( a ) LncRNA625 is located in the cytoplasm and nucleus of tumor tissue. Sense or antisense probe for lncRNA625 FISH were synthesized by in vitro transcription of T7 RNA polymerase, and 3 μm serial slides of ESCC tissues were hybridized with sense or antisense probes conjugated with biotin. Subsequently, the biotin signal was determined with Cy3-conjugated streptavidine. DAPI staining for was for nuclei, and haematoxylin and eosin staining was for tumor histomorphology. Scale bar: 40 ×. ( b ) Cytoplasmic and nuclear RNAs were isolated from KYSE510 cells, and lncRNA625 was detected by real-time RT–PCR. Levels of U6 snRNA (nuclear control transcript) and GAPDH (cytoplasmic control transcript) were detected by real-time RT–PCR. Values are mean±s.e. ( c ) Venn diagram showing the overlap between ESCC-related histone modification proteins and lncRNA625-related transcription regulatory proteins. ( d ) Comparison of 202 differentially expressed genes following silencing lncRNA625 in KYSE150 cells vs. a compendium of UCSC-published EP300 occupancy profiles in diverse cell types. ( e ) LncRNA625 interacts with EP300. RNA immunoprecipitation assays for EP300 were performed and RNA was extracted with 1 ml TRIzol, and lncRNA625 was detected by real-time RT-PCR in both KYSE150 and KYSE510 ESCC cells. IgG and SP1 were used as negative controls in the experiment. ( f ) Gene expression profile analysis was performed after either lncRNA625 or EP300 knockdown in KYSE 150 cells. Genes with |log 2 FC| > log 2 1.5, after lncRNA625 knockdown, are displayed in the heat map. ( g ) GSEA plot showing that genes regulated by lncRNA625 were enriched in the expression profile after knocking down EP300. In particular, those genes downregulated after knocking down EP300 received a high enrichment score. ( h ) qRT–PCR of a representative panel of genes in scrambled, silncRNA625 and siEP300 cells (error bars are s.d., n =6). Genes boxed in red are literature-evidenced cancer-related genes. Genes with an asterisk are literature-evidenced ESCC-related genes.

Techniques Used: Fluorescence In Situ Hybridization, Synthesized, In Vitro, Staining, Isolation, Quantitative RT-PCR, Modification, Immunoprecipitation, Expressing

Identification of functional lncRNAs in ESCC via downstream target PCGs. ( a ) The extended lncRNA-PCG co-expression network. The network is displayed using Cytoscape software according to the ‘spring' layout. Node size is proportional to the degree of the node. Node color reflects differential expression level. Nodes with a zigzag border line are differential PCGs after lncRNA625 knockdown in the network. Interesting lncRNAs and PCGs are enlarged or labeled using their names. For example, nodes with a blue label are genes regulated by lncRNA625. Circles with dashed lines in the network represent subnetwork modules with many highly upregulated lncRNAs and PCGs. ( b ) An active subnetwork module in the lncRNA-PCG co-expression network. ( c ) PCGs in the lncRNA-PCG co-expression network are annotated to Gene Ontology to identify their functions. ( d ) Schematic overview of URW-LPE. The top figure represents the data stream of URW-LPE, and the bottom figure represents the running process of the random walk that is the core step of URW-LPE. ( e ) Fold change and URWScore value of each lncRNA in the lncRNA-PCG co-expression network. Known disease lncRNAs and ESCC PCGs are labeled. ( f ) Scatter plots of the relative expression levels of qRT–PCR of lncRNAs in an additional 120 paired ESCC patient samples. Note that because a lncRNA may not be identified by qRT–PCR in the corresponding sample, the number of actual samples with expression level for each lncRNA was slightly less than the total number of samples. Comparisons of the relative expression between tumor (T) and non-tumor (N) were performed using a paired t -test. A P -value
Figure Legend Snippet: Identification of functional lncRNAs in ESCC via downstream target PCGs. ( a ) The extended lncRNA-PCG co-expression network. The network is displayed using Cytoscape software according to the ‘spring' layout. Node size is proportional to the degree of the node. Node color reflects differential expression level. Nodes with a zigzag border line are differential PCGs after lncRNA625 knockdown in the network. Interesting lncRNAs and PCGs are enlarged or labeled using their names. For example, nodes with a blue label are genes regulated by lncRNA625. Circles with dashed lines in the network represent subnetwork modules with many highly upregulated lncRNAs and PCGs. ( b ) An active subnetwork module in the lncRNA-PCG co-expression network. ( c ) PCGs in the lncRNA-PCG co-expression network are annotated to Gene Ontology to identify their functions. ( d ) Schematic overview of URW-LPE. The top figure represents the data stream of URW-LPE, and the bottom figure represents the running process of the random walk that is the core step of URW-LPE. ( e ) Fold change and URWScore value of each lncRNA in the lncRNA-PCG co-expression network. Known disease lncRNAs and ESCC PCGs are labeled. ( f ) Scatter plots of the relative expression levels of qRT–PCR of lncRNAs in an additional 120 paired ESCC patient samples. Note that because a lncRNA may not be identified by qRT–PCR in the corresponding sample, the number of actual samples with expression level for each lncRNA was slightly less than the total number of samples. Comparisons of the relative expression between tumor (T) and non-tumor (N) were performed using a paired t -test. A P -value

Techniques Used: Functional Assay, Expressing, Software, Labeling, Quantitative RT-PCR

LncRNA625 modulates cancer cell proliferation, invasion and migration via affecting downstream target PCGs. ( a ) Read distributions of the RNA-seq gene model. ( b ) LncRNA625 expression in various human ESCC cells. ( c ) Colony formation of stably transfected KYSE150 and KYSE510 stained with haematoxylin solution after incubation for 15 days. ( d ) Invasion and migration of KYSE150 and KYSE510 cells stably transfected with shRNA against either lncRNA625 or with a scrambled RNA. ( e ) Migration of SHEEC cells detected at 48 h following transfection with either lncRNA625 expression vector or control vector, and detection of lncRNA625 levels by real-time RT-PCR. Values are mean±s.e.m. ( f ) LncRNA625 downregulation inhibited the proliferation of esophageal cancer cells. 1 × 10 6 KYSE150-shlncRNA625 or shscramble cells were subcutaneously inoculated in the right flank of each BALB/c mouse (nu/nu) ( n =9) and after one week, tumor volumes were measured every two days according to the formula: V=ab 2 /2 (‘a' represents the length of tumor tissue and ‘b' represents the width of tumor tissue). The average weight of tumors was determined after mice were euthanized by CO 2 inhalation.
Figure Legend Snippet: LncRNA625 modulates cancer cell proliferation, invasion and migration via affecting downstream target PCGs. ( a ) Read distributions of the RNA-seq gene model. ( b ) LncRNA625 expression in various human ESCC cells. ( c ) Colony formation of stably transfected KYSE150 and KYSE510 stained with haematoxylin solution after incubation for 15 days. ( d ) Invasion and migration of KYSE150 and KYSE510 cells stably transfected with shRNA against either lncRNA625 or with a scrambled RNA. ( e ) Migration of SHEEC cells detected at 48 h following transfection with either lncRNA625 expression vector or control vector, and detection of lncRNA625 levels by real-time RT-PCR. Values are mean±s.e.m. ( f ) LncRNA625 downregulation inhibited the proliferation of esophageal cancer cells. 1 × 10 6 KYSE150-shlncRNA625 or shscramble cells were subcutaneously inoculated in the right flank of each BALB/c mouse (nu/nu) ( n =9) and after one week, tumor volumes were measured every two days according to the formula: V=ab 2 /2 (‘a' represents the length of tumor tissue and ‘b' represents the width of tumor tissue). The average weight of tumors was determined after mice were euthanized by CO 2 inhalation.

Techniques Used: Migration, RNA Sequencing Assay, Expressing, Stable Transfection, Transfection, Staining, Incubation, shRNA, Plasmid Preparation, Quantitative RT-PCR, Mouse Assay

Kaplan–Meier curves of ESCC patients with either higher or lower expression of lncRNA625 and downstream target PCGs. ( a ) Kaplan–Meier survival curves of patients with ESCC classified into high- and low-risk groups based on their lncRNA625 signature. Expression level and survival information were obtained in 118 cases from 120 ESCC patient samples. For patients with invasive depth 3/4 (T3/T4), lymph node metastasis and stage III, a stratified analysis was done. ( b ) Kaplan–Meier survival based on the lncRNA625 signature for the cancer lncRNA profiles in TCGA. ( c ) Kaplan–Meier survival curves of ESCC patients classified into high- and low-risk groups based on two lncRNA625 downstream target PCG signatures. Expression level and patient information were obtained from TCGA. Red and blue indicates higher and lower expression, respectively.
Figure Legend Snippet: Kaplan–Meier curves of ESCC patients with either higher or lower expression of lncRNA625 and downstream target PCGs. ( a ) Kaplan–Meier survival curves of patients with ESCC classified into high- and low-risk groups based on their lncRNA625 signature. Expression level and survival information were obtained in 118 cases from 120 ESCC patient samples. For patients with invasive depth 3/4 (T3/T4), lymph node metastasis and stage III, a stratified analysis was done. ( b ) Kaplan–Meier survival based on the lncRNA625 signature for the cancer lncRNA profiles in TCGA. ( c ) Kaplan–Meier survival curves of ESCC patients classified into high- and low-risk groups based on two lncRNA625 downstream target PCG signatures. Expression level and patient information were obtained from TCGA. Red and blue indicates higher and lower expression, respectively.

Techniques Used: Expressing

58) Product Images from "ATM participates in the regulation of viability and cell cycle via ellipticine in bladder cancer"

Article Title: ATM participates in the regulation of viability and cell cycle via ellipticine in bladder cancer

Journal: Molecular Medicine Reports

doi: 10.3892/mmr.2017.6141

Cytotoxicity effects of ellipticine in T-24 bladder cancer cells. Cell proliferation and viability were determined by a Cell Counting Kit-8 assay in untreated (control) cells or cells treated with either DMSO vehicle control (DMSO) or various concentrations of ellipticine (1–16 µM). The data are presented as the mean ± standard deviation. *P
Figure Legend Snippet: Cytotoxicity effects of ellipticine in T-24 bladder cancer cells. Cell proliferation and viability were determined by a Cell Counting Kit-8 assay in untreated (control) cells or cells treated with either DMSO vehicle control (DMSO) or various concentrations of ellipticine (1–16 µM). The data are presented as the mean ± standard deviation. *P

Techniques Used: Cell Counting, Standard Deviation

Cell cycle analysis of T-24 bladder cancer cells treated with ellipticine. (A) Cells were treated with different concentrations of ellipticine for 24 h and then stained with propidium iodide. The DNA content was analyzed by flow cytometry. G1, S and G2/M indicate the respective cell cycle phase. (B) Quantification of cell cycle analysis from three independent experiments. The data are presented as mean ± standard deviation. *P
Figure Legend Snippet: Cell cycle analysis of T-24 bladder cancer cells treated with ellipticine. (A) Cells were treated with different concentrations of ellipticine for 24 h and then stained with propidium iodide. The DNA content was analyzed by flow cytometry. G1, S and G2/M indicate the respective cell cycle phase. (B) Quantification of cell cycle analysis from three independent experiments. The data are presented as mean ± standard deviation. *P

Techniques Used: Cell Cycle Assay, Staining, Flow Cytometry, Cytometry, Standard Deviation

mRNA and protein expression analysis in T-24 bladder cancer cells treated with various concentrations of ellipticine for 24 h. The data are presented as mean ± standard deviation. (A) ATM mRNA expression was assessed in untreated (control) and ellipticine-treated cells. Results are plotted as relative expression to the control cells. (B) Western blot analysis of untreated (control) and ellipticine-treated cells. A representative blot is shown from three independent experiments with similar results. (C) Quantification of protein expression from densitometric analysis relative to the GAPDH control from three independent experiments. *P
Figure Legend Snippet: mRNA and protein expression analysis in T-24 bladder cancer cells treated with various concentrations of ellipticine for 24 h. The data are presented as mean ± standard deviation. (A) ATM mRNA expression was assessed in untreated (control) and ellipticine-treated cells. Results are plotted as relative expression to the control cells. (B) Western blot analysis of untreated (control) and ellipticine-treated cells. A representative blot is shown from three independent experiments with similar results. (C) Quantification of protein expression from densitometric analysis relative to the GAPDH control from three independent experiments. *P

Techniques Used: Expressing, Standard Deviation, Western Blot

Effect of ellipticine on T-24 cell migration. Cells treated with 0–0.8 µM ellipticine were allowed to migrate through uncoated Transwell membranes for 24 h, then were fixed, stained, and counted. (A) Representative images of ellipticine treated/untreated migrated cells on the bottom of the Transwells. Scale bars, 100 µm. (B) Quantification of cell migration analysis from three independent experiments. The data are presented as mean ± standard deviation. *P
Figure Legend Snippet: Effect of ellipticine on T-24 cell migration. Cells treated with 0–0.8 µM ellipticine were allowed to migrate through uncoated Transwell membranes for 24 h, then were fixed, stained, and counted. (A) Representative images of ellipticine treated/untreated migrated cells on the bottom of the Transwells. Scale bars, 100 µm. (B) Quantification of cell migration analysis from three independent experiments. The data are presented as mean ± standard deviation. *P

Techniques Used: Migration, Staining, Standard Deviation

59) Product Images from "ATM participates in the regulation of viability and cell cycle via ellipticine in bladder cancer"

Article Title: ATM participates in the regulation of viability and cell cycle via ellipticine in bladder cancer

Journal: Molecular Medicine Reports

doi: 10.3892/mmr.2017.6141

Cytotoxicity effects of ellipticine in T-24 bladder cancer cells. Cell proliferation and viability were determined by a Cell Counting Kit-8 assay in untreated (control) cells or cells treated with either DMSO vehicle control (DMSO) or various concentrations of ellipticine (1–16 µM). The data are presented as the mean ± standard deviation. *P
Figure Legend Snippet: Cytotoxicity effects of ellipticine in T-24 bladder cancer cells. Cell proliferation and viability were determined by a Cell Counting Kit-8 assay in untreated (control) cells or cells treated with either DMSO vehicle control (DMSO) or various concentrations of ellipticine (1–16 µM). The data are presented as the mean ± standard deviation. *P

Techniques Used: Cell Counting, Standard Deviation

60) Product Images from "Therapeutic Effect and Location of GFP-Labeled Placental Mesenchymal Stem Cells on Hepatic Fibrosis in Rats"

Article Title: Therapeutic Effect and Location of GFP-Labeled Placental Mesenchymal Stem Cells on Hepatic Fibrosis in Rats

Journal: Stem Cells International

doi: 10.1155/2017/1798260

Relative mRNA expression levels of α -SMA and TGF- β 1 in the different experimental groups. Quantitative real-time polymerase chain reaction (qRT-PCR) results from n = 6 rats from each group at each time point. The tests were performed in triplicate. The data are presented as the mean ± SD (error bars) and were statistically analyzed using Student's t -test. ∗∗ p
Figure Legend Snippet: Relative mRNA expression levels of α -SMA and TGF- β 1 in the different experimental groups. Quantitative real-time polymerase chain reaction (qRT-PCR) results from n = 6 rats from each group at each time point. The tests were performed in triplicate. The data are presented as the mean ± SD (error bars) and were statistically analyzed using Student's t -test. ∗∗ p

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

Immunohistochemistry results of α -SMA and TGF- β 1 in the different experimental groups. Light-brown coloration in cells means the positive staining results. Immunohistochemical staining for α -SMA and TGF- β 1 (20x), with a young rat liver as a negative control (NC), fibrotic animals (group II), fibrotic animals transplanted with hPMSCs (group II) sacrificed at one week, four weeks, eight weeks, or twelve weeks after transplantation (1 w, 4 w, 8 w, or 12 w). Scale bars: 100 μ m.
Figure Legend Snippet: Immunohistochemistry results of α -SMA and TGF- β 1 in the different experimental groups. Light-brown coloration in cells means the positive staining results. Immunohistochemical staining for α -SMA and TGF- β 1 (20x), with a young rat liver as a negative control (NC), fibrotic animals (group II), fibrotic animals transplanted with hPMSCs (group II) sacrificed at one week, four weeks, eight weeks, or twelve weeks after transplantation (1 w, 4 w, 8 w, or 12 w). Scale bars: 100 μ m.

Techniques Used: Immunohistochemistry, Staining, Negative Control, Transplantation Assay

61) Product Images from "Increased Set1 binding at the promoter induces aberrant epigenetic alterations and up-regulates cyclic adenosine 5'-monophosphate response element modulator alpha in systemic lupus erythematosus"

Article Title: Increased Set1 binding at the promoter induces aberrant epigenetic alterations and up-regulates cyclic adenosine 5'-monophosphate response element modulator alpha in systemic lupus erythematosus

Journal: Clinical Epigenetics

doi: 10.1186/s13148-016-0294-2

H3K4me3 enrichment at the CREMα promoter in SLE and control CD4 + T cells. a Relative H3K4me3 enrichment within the CREMα promoter in SLE and healthy CD4 + T cells was assessed by ChIP and real-time PCR. Results were normalized to input DNA (total chromatin). b Positive correlation between the levels of H3K4me3 and CREMα mRNA in SLE CD4 + T cells. All reactions were run in triplicate
Figure Legend Snippet: H3K4me3 enrichment at the CREMα promoter in SLE and control CD4 + T cells. a Relative H3K4me3 enrichment within the CREMα promoter in SLE and healthy CD4 + T cells was assessed by ChIP and real-time PCR. Results were normalized to input DNA (total chromatin). b Positive correlation between the levels of H3K4me3 and CREMα mRNA in SLE CD4 + T cells. All reactions were run in triplicate

Techniques Used: Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction

Set1 and MLL1 binding at the CREMα promoter in SLE and control CD4 + T cells. a , b Relative levels of Set1 ( a ) and MLL1 ( b ) binding within the CREMα promoter region in SLE and healthy CD4 + T cells were analyzed by ChIP and real-time PCR. Results were normalized to input DNA (total chromatin). c Positive correlation between Set1 promoter binding and H3K4me3 level in SLE CD4 + T cells. d Positive correlation between Set1 promoter binding and CREMα mRNA level in SLE CD4 + T cells. All experiments were repeated three times
Figure Legend Snippet: Set1 and MLL1 binding at the CREMα promoter in SLE and control CD4 + T cells. a , b Relative levels of Set1 ( a ) and MLL1 ( b ) binding within the CREMα promoter region in SLE and healthy CD4 + T cells were analyzed by ChIP and real-time PCR. Results were normalized to input DNA (total chromatin). c Positive correlation between Set1 promoter binding and H3K4me3 level in SLE CD4 + T cells. d Positive correlation between Set1 promoter binding and CREMα mRNA level in SLE CD4 + T cells. All experiments were repeated three times

Techniques Used: Binding Assay, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction

62) Product Images from "Soluble fibrinogen‐like protein 2 ameliorates acute rejection of liver transplantation in rat via inducing Kupffer cells M2 polarization, et al. Soluble fibrinogen‐like protein 2 ameliorates acute rejection of liver transplantation in rat via inducing Kupffer cells M2 polarization"

Article Title: Soluble fibrinogen‐like protein 2 ameliorates acute rejection of liver transplantation in rat via inducing Kupffer cells M2 polarization, et al. Soluble fibrinogen‐like protein 2 ameliorates acute rejection of liver transplantation in rat via inducing Kupffer cells M2 polarization

Journal: Cancer Medicine

doi: 10.1002/cam4.1528

sFGL2 over‐expression in recipients alleviates AR of rat OLT (Lewis to BN). A,B,The mRNA and serum levels of sFGL2 in liver tissues from BN rats (control group), AAV‐FGL2‐treated BN rats or AAV‐null‐treated BN rats were examined using qRT‐PCR or ELISA, respectively. C, H E staining of allografts on day 7 after OLT. (D) RAI scores were performed to grade AR according to Banff schema. E, Kaplan‐Meier curve was used to perform the survival of recipients. F, Serum levels of ALT, AST, and TBIL on day 7 after OLT. G, The mRNA levels of IL‐12, TNF‐α, IL‐10, TGF‐β and Arg‐1 in KCs isolated from allografts on day 7 after OLT were detected using qRT‐PCR. H, The protein level of Arg‐1 in allografts was measured using Western blot. *** P
Figure Legend Snippet: sFGL2 over‐expression in recipients alleviates AR of rat OLT (Lewis to BN). A,B,The mRNA and serum levels of sFGL2 in liver tissues from BN rats (control group), AAV‐FGL2‐treated BN rats or AAV‐null‐treated BN rats were examined using qRT‐PCR or ELISA, respectively. C, H E staining of allografts on day 7 after OLT. (D) RAI scores were performed to grade AR according to Banff schema. E, Kaplan‐Meier curve was used to perform the survival of recipients. F, Serum levels of ALT, AST, and TBIL on day 7 after OLT. G, The mRNA levels of IL‐12, TNF‐α, IL‐10, TGF‐β and Arg‐1 in KCs isolated from allografts on day 7 after OLT were detected using qRT‐PCR. H, The protein level of Arg‐1 in allografts was measured using Western blot. *** P

Techniques Used: Over Expression, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Staining, AST Assay, Isolation, Western Blot

Increased sFGL2 and the frequency of M2 KCs in tolerance group after rat OLT. A, The mRNA levels of FGL2 in liver tissues from BN rats (control group), and that in allografts from recipients in AR or tolerance group were examined by qRT‐PCR. B, Serum levels of sFGL2 from BN rats (control group), recipients in AR or tolerance group were measured by ELISA. C, The F4/80 + CD206 + M2 KCs from BN rats (control group), recipients in AR or tolerance groups were analyzed using flow cytometry. D, Pearson correlation analysis of the frequencies of F4/80 + CD206 + M2 KCs with mRNA and serum levels of sFGL2 in tolerance group. * P
Figure Legend Snippet: Increased sFGL2 and the frequency of M2 KCs in tolerance group after rat OLT. A, The mRNA levels of FGL2 in liver tissues from BN rats (control group), and that in allografts from recipients in AR or tolerance group were examined by qRT‐PCR. B, Serum levels of sFGL2 from BN rats (control group), recipients in AR or tolerance group were measured by ELISA. C, The F4/80 + CD206 + M2 KCs from BN rats (control group), recipients in AR or tolerance groups were analyzed using flow cytometry. D, Pearson correlation analysis of the frequencies of F4/80 + CD206 + M2 KCs with mRNA and serum levels of sFGL2 in tolerance group. * P

Techniques Used: Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Cytometry

63) Product Images from "The Serine/Threonine-Protein Phosphatase 1 From Haemonchus contortus Is Actively Involved in Suppressive Regulatory Roles on Immune Functions of Goat Peripheral Blood Mononuclear Cells"

Article Title: The Serine/Threonine-Protein Phosphatase 1 From Haemonchus contortus Is Actively Involved in Suppressive Regulatory Roles on Immune Functions of Goat Peripheral Blood Mononuclear Cells

Journal: Frontiers in Immunology

doi: 10.3389/fimmu.2018.01627

Localization of serine/threonine-protein phosphatase 1 protein within parasite structure. Immunohistochemically STP-1 protein localizes inner and outer structure of Haemonchus contortus male and female adult worms. The red color indicates target protein stained with Cy3 and blue dots are localization of nuclei stained with DAPI. No red fluorescence was detected in control panel. Scale-bars: 100 µm.
Figure Legend Snippet: Localization of serine/threonine-protein phosphatase 1 protein within parasite structure. Immunohistochemically STP-1 protein localizes inner and outer structure of Haemonchus contortus male and female adult worms. The red color indicates target protein stained with Cy3 and blue dots are localization of nuclei stained with DAPI. No red fluorescence was detected in control panel. Scale-bars: 100 µm.

Techniques Used: Staining, Fluorescence

64) Product Images from "Mutant Runx2 regulates amelogenesis and osteogenesis through a miR-185-5p-Dlx2 axis"

Article Title: Mutant Runx2 regulates amelogenesis and osteogenesis through a miR-185-5p-Dlx2 axis

Journal: Cell Death & Disease

doi: 10.1038/s41419-017-0078-4

iR-185-5p inhibits Dlx2 expression m a Schematic of the miR-185-5p putative target site in mouse Dlx2 3′ UTR. b Expression level of Dlx2 during the amelogenic differentiation of LS8 cells. c Expression change of Dlx2 during the osteogenic differentiation of MC3T3-E1 cells. d Knockdown of Dlx2 in LS8 cells treated with RA/DEX for 48 h inhibited the expression of Mmp20 and Klk4 . e Knockdown Dlx2 in MC3T3-E1 cells at day 0 of osteogenic induction had no obvious effect on the expression of Alp . f Expression of Alp on day 7 of osteogenic induction was inhibited by si- Dlx2 in MC3T3-E1 cells. g Putative miR-185-5p-binding sequence in the 3′ UTR of Dlx2 mRNA. Mutations were generated in the Dlx2 sequence in the complementary site for the seed region of miR-185-5p as indicated. h Normalized luciferase activity from Dlx2 3′ UTR reporter vector containing a wild-type or mutant miR-185-5p. i and j Relative Dlx2 mRNA and protein expression, determined by RT-PCR and western blot, respectively, in LS8 cells or MC3T3-E1 cells transfected with 50 nM miR-NC, miR-185-5p mimic, or miR-185-5p inhibitor. RNA and protein were harvested 2 days after transfection. Cells transfected with control miR-NC were designated as negative control. Data are representative images or expressed as mean ± S.E. of each group of cells from three separate experiments, and the values of the control cells are designated as 1. * p
Figure Legend Snippet: iR-185-5p inhibits Dlx2 expression m a Schematic of the miR-185-5p putative target site in mouse Dlx2 3′ UTR. b Expression level of Dlx2 during the amelogenic differentiation of LS8 cells. c Expression change of Dlx2 during the osteogenic differentiation of MC3T3-E1 cells. d Knockdown of Dlx2 in LS8 cells treated with RA/DEX for 48 h inhibited the expression of Mmp20 and Klk4 . e Knockdown Dlx2 in MC3T3-E1 cells at day 0 of osteogenic induction had no obvious effect on the expression of Alp . f Expression of Alp on day 7 of osteogenic induction was inhibited by si- Dlx2 in MC3T3-E1 cells. g Putative miR-185-5p-binding sequence in the 3′ UTR of Dlx2 mRNA. Mutations were generated in the Dlx2 sequence in the complementary site for the seed region of miR-185-5p as indicated. h Normalized luciferase activity from Dlx2 3′ UTR reporter vector containing a wild-type or mutant miR-185-5p. i and j Relative Dlx2 mRNA and protein expression, determined by RT-PCR and western blot, respectively, in LS8 cells or MC3T3-E1 cells transfected with 50 nM miR-NC, miR-185-5p mimic, or miR-185-5p inhibitor. RNA and protein were harvested 2 days after transfection. Cells transfected with control miR-NC were designated as negative control. Data are representative images or expressed as mean ± S.E. of each group of cells from three separate experiments, and the values of the control cells are designated as 1. * p

Techniques Used: Expressing, ALP Assay, Binding Assay, Sequencing, Generated, Luciferase, Activity Assay, Plasmid Preparation, Mutagenesis, Reverse Transcription Polymerase Chain Reaction, Western Blot, Transfection, Negative Control

miR-185-5p inhibits amelogenesis and osteogenesis by suppressing Dlx2 expression a Overexpression of mutant 3′-UTR- Dlx2 in LS8 could abolish the negative effect of miR-185-5p on the expression of Amelx . b-c Overexpression of Dlx2 in MC3T3-E1 alleviated the inhibitory effect caused by miR-185-5p mimics. Alizarin Red S stain was quantified b and protein level of Ocn c was determined after treatment with STEMPRO for 21 days. d, e Inhibition of miR-185-5p could partially rescue the inhibitory effect of mutant Runx2 on expression of Amelx and Alp (1: Runx2 WT + inhibitor NC, 2: Runx2 WT + miR-185-5p inhibitor, 3: Runx2 M1 + inhibitor NC, 4: Runx2 M1 + miR-185-5p inhibitor, 5: Runx2 M2 + inhibitor NC, 6: Runx2 M2 + miR-185-5p inhibitor, 7: Runx2 M3 + inhibitor NC, 8: Runx2 M3 + miR-185-5p inhibitor)
Figure Legend Snippet: miR-185-5p inhibits amelogenesis and osteogenesis by suppressing Dlx2 expression a Overexpression of mutant 3′-UTR- Dlx2 in LS8 could abolish the negative effect of miR-185-5p on the expression of Amelx . b-c Overexpression of Dlx2 in MC3T3-E1 alleviated the inhibitory effect caused by miR-185-5p mimics. Alizarin Red S stain was quantified b and protein level of Ocn c was determined after treatment with STEMPRO for 21 days. d, e Inhibition of miR-185-5p could partially rescue the inhibitory effect of mutant Runx2 on expression of Amelx and Alp (1: Runx2 WT + inhibitor NC, 2: Runx2 WT + miR-185-5p inhibitor, 3: Runx2 M1 + inhibitor NC, 4: Runx2 M1 + miR-185-5p inhibitor, 5: Runx2 M2 + inhibitor NC, 6: Runx2 M2 + miR-185-5p inhibitor, 7: Runx2 M3 + inhibitor NC, 8: Runx2 M3 + miR-185-5p inhibitor)

Techniques Used: Expressing, Over Expression, Mutagenesis, Staining, Inhibition, ALP Assay

65) Product Images from "circ-SHKBP1 Regulates the Angiogenesis of U87 Glioma-Exposed Endothelial Cells through miR-544a/FOXP1 and miR-379/FOXP2 Pathways"

Article Title: circ-SHKBP1 Regulates the Angiogenesis of U87 Glioma-Exposed Endothelial Cells through miR-544a/FOXP1 and miR-379/FOXP2 Pathways

Journal: Molecular Therapy. Nucleic Acids

doi: 10.1016/j.omtn.2017.12.014

circ-SHKBP1 Knockdown Combined with miR-544a/miR-379 Overexpression Suppressed Angiogenesis In Vivo (A) The co-effect of circ-SHKBP1 and miR-544a on the angiogenesis in vivo was evaluated by Matrigel plug assay. (B) The amount of hemoglobin was measured after combination of circ-SHKBP1 and miR-544a. Data represent the means ± SD (n = 5, each group). *p
Figure Legend Snippet: circ-SHKBP1 Knockdown Combined with miR-544a/miR-379 Overexpression Suppressed Angiogenesis In Vivo (A) The co-effect of circ-SHKBP1 and miR-544a on the angiogenesis in vivo was evaluated by Matrigel plug assay. (B) The amount of hemoglobin was measured after combination of circ-SHKBP1 and miR-544a. Data represent the means ± SD (n = 5, each group). *p

Techniques Used: Over Expression, In Vivo, Matrigel Assay

miR-544a/miR-379 Functional Targeted circ-SHKBP1 (A) Cartoon of circ-SHKBP1 arose from SHKBP1 gene by scanning SHKBP1 genomic DNA and circBase (blue, miR-544a binging site; green, miR-379 binging site). (B) The spliced sequence and putative binging site of circ-SHKBP1 (hsa_circ_0000936) are shown with the help of starBase v2.0 (blue, the putative binging site and sequences of miR-544a; green, the putative binging site and sequences of miR-379). (C and D) The putative binding sites between circ-SHKBP1 and miR-544 (C) and miR-379 (D) were predicted, and the relative luciferase activity was expressed as firefly/Renilla luciferase activity. Values are means ± SD (n = 5, each group). **p
Figure Legend Snippet: miR-544a/miR-379 Functional Targeted circ-SHKBP1 (A) Cartoon of circ-SHKBP1 arose from SHKBP1 gene by scanning SHKBP1 genomic DNA and circBase (blue, miR-544a binging site; green, miR-379 binging site). (B) The spliced sequence and putative binging site of circ-SHKBP1 (hsa_circ_0000936) are shown with the help of starBase v2.0 (blue, the putative binging site and sequences of miR-544a; green, the putative binging site and sequences of miR-379). (C and D) The putative binding sites between circ-SHKBP1 and miR-544 (C) and miR-379 (D) were predicted, and the relative luciferase activity was expressed as firefly/Renilla luciferase activity. Values are means ± SD (n = 5, each group). **p

Techniques Used: Functional Assay, Sequencing, Binding Assay, Luciferase, Activity Assay

circ-SHKBP1 Expression in Glioma Microvessels and GECs, and Knockdown of circ-SHKBP1 Inhibited the Angiogenesis of U87 Glioma and the Expression of AGGF1 (A) Normal brain microvessels and glioma microvessels were respectively captured from NBTs and glioma tissues using laser capture microdissection. Relative expressions of circ-SHKBP1 in normal brain microvessels or glioma microvessels were shown. Data represent means ± SD (n = 5, each group). **p
Figure Legend Snippet: circ-SHKBP1 Expression in Glioma Microvessels and GECs, and Knockdown of circ-SHKBP1 Inhibited the Angiogenesis of U87 Glioma and the Expression of AGGF1 (A) Normal brain microvessels and glioma microvessels were respectively captured from NBTs and glioma tissues using laser capture microdissection. Relative expressions of circ-SHKBP1 in normal brain microvessels or glioma microvessels were shown. Data represent means ± SD (n = 5, each group). **p

Techniques Used: Expressing, Laser Capture Microdissection

66) Product Images from "In vitro inhibition of porcine reproductive and respiratory syndrome virus replication by short antisense oligonucleotides with locked nucleic acid modification"

Article Title: In vitro inhibition of porcine reproductive and respiratory syndrome virus replication by short antisense oligonucleotides with locked nucleic acid modification

Journal: BMC Veterinary Research

doi: 10.1186/s12917-018-1432-1

Viral ORF7 level was further reduced by transfection with LNA modified AS-ONs in PAMs. Antisense oligonucleotides (YN8, LNA-YN8-A and LNA-YN8-B) were used in the transfection in PAMs with different concentrations. The RNA ( a ) and protein ( b ) levels of gene ORF7 were more significantly inhibited by the treatments with LNA modified YN8 than by DNA AS-ON YN8 transfection. After virus challenge and transfection with AS-ONs, total RNA was isolated for RT-qPCR analysis and the total protein was extracted for western blot analysis. The ΔΔCt method for relative quantification of gene expression was used to determine viral ORF7 RNA levels. The Y-axis ( a ) shows the relative RNA levels of the ORF7 gene for each treatment after normalization to the non-transfected reference sample (PRRSV only). Transfection with 8 μM YN8 or 4 μM LNA-YN8-A or LNA-YN8-B completely blocked the synthesis of viral protein ( b ).The histogram and blots shown here are representative data from three independent experiments. β-actin was used as internal control in both RT-qPCR and western blot analysis. NS: not significant; *: P
Figure Legend Snippet: Viral ORF7 level was further reduced by transfection with LNA modified AS-ONs in PAMs. Antisense oligonucleotides (YN8, LNA-YN8-A and LNA-YN8-B) were used in the transfection in PAMs with different concentrations. The RNA ( a ) and protein ( b ) levels of gene ORF7 were more significantly inhibited by the treatments with LNA modified YN8 than by DNA AS-ON YN8 transfection. After virus challenge and transfection with AS-ONs, total RNA was isolated for RT-qPCR analysis and the total protein was extracted for western blot analysis. The ΔΔCt method for relative quantification of gene expression was used to determine viral ORF7 RNA levels. The Y-axis ( a ) shows the relative RNA levels of the ORF7 gene for each treatment after normalization to the non-transfected reference sample (PRRSV only). Transfection with 8 μM YN8 or 4 μM LNA-YN8-A or LNA-YN8-B completely blocked the synthesis of viral protein ( b ).The histogram and blots shown here are representative data from three independent experiments. β-actin was used as internal control in both RT-qPCR and western blot analysis. NS: not significant; *: P

Techniques Used: Transfection, Modification, Isolation, Quantitative RT-PCR, Western Blot, Expressing

67) Product Images from "MicroRNA-495-3p inhibits multidrug resistance by modulating autophagy through GRP78/mTOR axis in gastric cancer"

Article Title: MicroRNA-495-3p inhibits multidrug resistance by modulating autophagy through GRP78/mTOR axis in gastric cancer

Journal: Cell Death & Disease

doi: 10.1038/s41419-018-0950-x

Downregulation of GRP78 by miR-495-3p inhibits autophagy in GC MDR cells. a Representative images and b Quantification of LC3 puncta (green) in GC MDR cells transfected with si-GRP78 or si-nc. Scale bars: 50 μM. c Representative images and d Quantification of LC3 puncta (green) in SGC7901/ADR co-transfected with GRP78 3′-UTR-mutant overexpression vector/nc and miR-495-3p/nc. e The expression level of GRP78, LC3BI/II, and P62 in GC MDR cells transfected with si-GRP78 or si-NC were determined by western blotting. f The protein level of GRP78, LC3BI/II, and P62 in SGC7901/ADR co-transfected with GRP78 3′-UTR-mutant overexpression vector/nc and miR-495-3p/nc. All values expressed as mean ± SD, n = 3 for each group. * P
Figure Legend Snippet: Downregulation of GRP78 by miR-495-3p inhibits autophagy in GC MDR cells. a Representative images and b Quantification of LC3 puncta (green) in GC MDR cells transfected with si-GRP78 or si-nc. Scale bars: 50 μM. c Representative images and d Quantification of LC3 puncta (green) in SGC7901/ADR co-transfected with GRP78 3′-UTR-mutant overexpression vector/nc and miR-495-3p/nc. e The expression level of GRP78, LC3BI/II, and P62 in GC MDR cells transfected with si-GRP78 or si-NC were determined by western blotting. f The protein level of GRP78, LC3BI/II, and P62 in SGC7901/ADR co-transfected with GRP78 3′-UTR-mutant overexpression vector/nc and miR-495-3p/nc. All values expressed as mean ± SD, n = 3 for each group. * P

Techniques Used: Transfection, Mutagenesis, Over Expression, Plasmid Preparation, Expressing, Western Blot

miR-495-3p inhibits autophagy by activating mTOR signaling. a Western blot analysis of phosphorylated mTOR (p-mTOR) and its main substrates 4E-BP1 (p-4E-BP1) and S6 (p-S6) in SGC7901 transfected with anti-miR-495-3p and GC MDR cells transfected with miR-495-3p. b The phosphorylation status of mTOR, S6K (p-S6K), and 4E-BP1 (p-4E-BP1) transfected with si-GRP78 and si-NC were measured by western blotting ( c ). Western blotting determines the phosphorylation of mTOR, S6K (p-S6K), and 4E-BP1 (p-4E-BP1) in SGC7901/ADR co-transfected with GRP78 3′-UTR-mutant overexpression vector/nc and miR-495-3p/nc. All values expressed as mean ± SD, n = 3 for each group. * P
Figure Legend Snippet: miR-495-3p inhibits autophagy by activating mTOR signaling. a Western blot analysis of phosphorylated mTOR (p-mTOR) and its main substrates 4E-BP1 (p-4E-BP1) and S6 (p-S6) in SGC7901 transfected with anti-miR-495-3p and GC MDR cells transfected with miR-495-3p. b The phosphorylation status of mTOR, S6K (p-S6K), and 4E-BP1 (p-4E-BP1) transfected with si-GRP78 and si-NC were measured by western blotting ( c ). Western blotting determines the phosphorylation of mTOR, S6K (p-S6K), and 4E-BP1 (p-4E-BP1) in SGC7901/ADR co-transfected with GRP78 3′-UTR-mutant overexpression vector/nc and miR-495-3p/nc. All values expressed as mean ± SD, n = 3 for each group. * P

Techniques Used: Western Blot, Transfection, Mutagenesis, Over Expression, Plasmid Preparation

Expression levels of miR-495-3p and GRP78 in GC specimens. a The expression levels of miR-495-3p (upper) and GRP78 (lower) in peri-tumor and primary GC tissues, scale bars: 500 μm (top) and 200 μm (bottom). b miR-495-3p and c GRP78 expression staining score in peri-tumor and primary GC tissues. Kaplan–Meier survival curves of GC patients expressing miR-495-3p ( d ) and GRP78 ( e ) GC patients were ranked based on the staining score and divided into high-expression ( > 4) and low-expression (≤4) groups. * P
Figure Legend Snippet: Expression levels of miR-495-3p and GRP78 in GC specimens. a The expression levels of miR-495-3p (upper) and GRP78 (lower) in peri-tumor and primary GC tissues, scale bars: 500 μm (top) and 200 μm (bottom). b miR-495-3p and c GRP78 expression staining score in peri-tumor and primary GC tissues. Kaplan–Meier survival curves of GC patients expressing miR-495-3p ( d ) and GRP78 ( e ) GC patients were ranked based on the staining score and divided into high-expression ( > 4) and low-expression (≤4) groups. * P

Techniques Used: Expressing, Staining

miR-495-3p is downregulated in GC and inhibits MDR and proliferation of GC MDR cells. a Real-time PCR detected the relative expression level of miR-495-3p in peri-tumor tissue and gastric cancer tissue in 15 paired samples. b The expression level of miR-495-3p in normal gastric epithelial cell line, five gastric cancer cell lines and GC MDR cells. c Four chemotherapeutic drugs (c1 adriamycin; c2 cisplatin; c3 fluorouracil; c4 vincristine) with indicated concentration gradient were added to the medium of SGC7901/ADR transfected with miR-495-3p mimics or nc. Then, cell survival was measured using the MTT assay. d Colony formation assays were performed to evaluate the proliferative functions of GC MDR cells transfected with miR-495-3p mimic or nc. 10 3 cells per well were seeded initially in 6-well plates. e Representative images of tumors 4 weeks after subcutaneous injection of 10 7 SGC7901/ADR transfected with lenti-NC or lenti-miR-495-3p into the right flank of the nude mice. (Scale bar = 1 cm). All experiments were performed in triplicates. All values were expressed as mean ± SD, n = 3 for each group. * P
Figure Legend Snippet: miR-495-3p is downregulated in GC and inhibits MDR and proliferation of GC MDR cells. a Real-time PCR detected the relative expression level of miR-495-3p in peri-tumor tissue and gastric cancer tissue in 15 paired samples. b The expression level of miR-495-3p in normal gastric epithelial cell line, five gastric cancer cell lines and GC MDR cells. c Four chemotherapeutic drugs (c1 adriamycin; c2 cisplatin; c3 fluorouracil; c4 vincristine) with indicated concentration gradient were added to the medium of SGC7901/ADR transfected with miR-495-3p mimics or nc. Then, cell survival was measured using the MTT assay. d Colony formation assays were performed to evaluate the proliferative functions of GC MDR cells transfected with miR-495-3p mimic or nc. 10 3 cells per well were seeded initially in 6-well plates. e Representative images of tumors 4 weeks after subcutaneous injection of 10 7 SGC7901/ADR transfected with lenti-NC or lenti-miR-495-3p into the right flank of the nude mice. (Scale bar = 1 cm). All experiments were performed in triplicates. All values were expressed as mean ± SD, n = 3 for each group. * P

Techniques Used: Real-time Polymerase Chain Reaction, Expressing, Concentration Assay, Transfection, MTT Assay, Injection, Mouse Assay

miR-495-3p inhibits autophagy in GC MDR cells. a Transmission electron micrographs of morphological changes in SGC7901 transfected with miR-495-3p inhibitor or GC MDR cells transfected with miR-495-3p mimic. White arrows represent autophagosomes or autolysosomes. (Scale bar = 500 nm). b Quantitative analysis of the number of autophagic vesicles. c Representative images and d Quantification of LC3 puncta (green) in SGC7901 transfected with miR-495-3p inhibitor or GC MDR cells transfected with miR-495-3p mimics. Scale bars: 50 μM. e Western blot analysis of the protein levels of Beclin-1, and LC3I/II in GC MDR cells transfected with miR-495-3p mimic or nc. All values were expressed as mean ± SD, n = 4 for each group. * P
Figure Legend Snippet: miR-495-3p inhibits autophagy in GC MDR cells. a Transmission electron micrographs of morphological changes in SGC7901 transfected with miR-495-3p inhibitor or GC MDR cells transfected with miR-495-3p mimic. White arrows represent autophagosomes or autolysosomes. (Scale bar = 500 nm). b Quantitative analysis of the number of autophagic vesicles. c Representative images and d Quantification of LC3 puncta (green) in SGC7901 transfected with miR-495-3p inhibitor or GC MDR cells transfected with miR-495-3p mimics. Scale bars: 50 μM. e Western blot analysis of the protein levels of Beclin-1, and LC3I/II in GC MDR cells transfected with miR-495-3p mimic or nc. All values were expressed as mean ± SD, n = 4 for each group. * P

Techniques Used: Transmission Assay, Transfection, Western Blot

miR-495-3p directly targets GRP78 at the post-transcriptional level. a Illustration of the GRP78 3′-UTR-containing reporter constructs. Mutations were generated at two predicted miR-495-3p binding sites located in the GRP78 3′-UTR. b Representative luciferase activity in SGC7901 cells co-transfected with wild-type or mutated reporter plasmids and nc, miR-495-3p mimic. c Western blot showed the changes in GRP78 protein levels after transient transfection of miR-495-3p inhibitor or mimic with indicated concentration gradient as compared to the negative controls (nc or anti-nc, respectively). d Real-time PCR determined the GRP78 level in SGC7901 after transfection with miR-495-3p inhibitor/mimic or anti-nc/nc. NS is no significance. All values were expressed as mean ± SD, n = 3 for each group. * P
Figure Legend Snippet: miR-495-3p directly targets GRP78 at the post-transcriptional level. a Illustration of the GRP78 3′-UTR-containing reporter constructs. Mutations were generated at two predicted miR-495-3p binding sites located in the GRP78 3′-UTR. b Representative luciferase activity in SGC7901 cells co-transfected with wild-type or mutated reporter plasmids and nc, miR-495-3p mimic. c Western blot showed the changes in GRP78 protein levels after transient transfection of miR-495-3p inhibitor or mimic with indicated concentration gradient as compared to the negative controls (nc or anti-nc, respectively). d Real-time PCR determined the GRP78 level in SGC7901 after transfection with miR-495-3p inhibitor/mimic or anti-nc/nc. NS is no significance. All values were expressed as mean ± SD, n = 3 for each group. * P

Techniques Used: Construct, Generated, Binding Assay, Luciferase, Activity Assay, Transfection, Western Blot, Concentration Assay, Real-time Polymerase Chain Reaction

68) Product Images from "Identification of key genes and long non-coding RNAs in celecoxib-treated lung squamous cell carcinoma cell line by RNA-sequencing"

Article Title: Identification of key genes and long non-coding RNAs in celecoxib-treated lung squamous cell carcinoma cell line by RNA-sequencing

Journal: Molecular Medicine Reports

doi: 10.3892/mmr.2018.8656

Expression levels of (A) VEGFA, (B) FN and (C) lncAP000769 1–2:10 were determined in SK-MES-1 cells using reverse transcription-quantitative polymerase chain reaction. *P
Figure Legend Snippet: Expression levels of (A) VEGFA, (B) FN and (C) lncAP000769 1–2:10 were determined in SK-MES-1 cells using reverse transcription-quantitative polymerase chain reaction. *P

Techniques Used: Expressing, Real-time Polymerase Chain Reaction

69) Product Images from "Diagnostic utility of plasma lncRNA small nucleolar RNA host gene 1 in patients with hepatocellular carcinoma"

Article Title: Diagnostic utility of plasma lncRNA small nucleolar RNA host gene 1 in patients with hepatocellular carcinoma

Journal: Molecular Medicine Reports

doi: 10.3892/mmr.2018.9336

lncRNA expressions in HCC tissues. (A) lncRNA expression spectrum clustering map from six paired tumor samples and adjacent normal tissues. Green and red bars indicate downregulation and upregulation, respectively. (B) Relative expression levels of 10 aberrantly expressed lncRNAs were validated by reverse transcription-quantitative polymerase chain reaction. *P
Figure Legend Snippet: lncRNA expressions in HCC tissues. (A) lncRNA expression spectrum clustering map from six paired tumor samples and adjacent normal tissues. Green and red bars indicate downregulation and upregulation, respectively. (B) Relative expression levels of 10 aberrantly expressed lncRNAs were validated by reverse transcription-quantitative polymerase chain reaction. *P

Techniques Used: Expressing, Real-time Polymerase Chain Reaction

Relative expression levels of lncRNA SNHG1 in plasma samples from patients with HCC, patients with HCH and healthy Control individuals. (A-C) Relative lncRNA expression levels were determined by reverse transcription-quantitative polymerase chain reaction for (A) SNHG1, (B) ASLNC12773 and (C) BF896662 in the three groups. (D) A positive correlation was identified between plasma and tissue SNHG1 expression by Pearson correlation analysis. r=0.6210; R 2 =0.3857; P
Figure Legend Snippet: Relative expression levels of lncRNA SNHG1 in plasma samples from patients with HCC, patients with HCH and healthy Control individuals. (A-C) Relative lncRNA expression levels were determined by reverse transcription-quantitative polymerase chain reaction for (A) SNHG1, (B) ASLNC12773 and (C) BF896662 in the three groups. (D) A positive correlation was identified between plasma and tissue SNHG1 expression by Pearson correlation analysis. r=0.6210; R 2 =0.3857; P

Techniques Used: Expressing, Real-time Polymerase Chain Reaction

70) Product Images from "Emodin inhibits pancreatic cancer EMT and invasion by up-regulating microRNA-1271"

Article Title: Emodin inhibits pancreatic cancer EMT and invasion by up-regulating microRNA-1271

Journal: Molecular Medicine Reports

doi: 10.3892/mmr.2018.9304

Effect of Emodin on the level of miR-1271 and EMT marker in SW1990 cells. (A) The expression of miRNA-1271, E-cadherin, ZEB1 and TWIST1 in pancreatic cancer cells were detected by qRT-PCR. (B) The protein content of EMT-related markers E-cadherin, ZEB1 and TWIST1 in pancreatic cancer cells was detected by western-blot assay. (C) Transwell chamber model was used to detect the change of invasion ability of pancreatic cancer cells in each experimental group. Magnification, ×100. *P
Figure Legend Snippet: Effect of Emodin on the level of miR-1271 and EMT marker in SW1990 cells. (A) The expression of miRNA-1271, E-cadherin, ZEB1 and TWIST1 in pancreatic cancer cells were detected by qRT-PCR. (B) The protein content of EMT-related markers E-cadherin, ZEB1 and TWIST1 in pancreatic cancer cells was detected by western-blot assay. (C) Transwell chamber model was used to detect the change of invasion ability of pancreatic cancer cells in each experimental group. Magnification, ×100. *P

Techniques Used: Marker, Expressing, Quantitative RT-PCR, Western Blot

miRNA-1271 expression in different groups in SW1990 cells. 48 h after cell transfection, the expression of miRNA-1271 in pancreatic cancer cells were detected by qRT-PCR. *P
Figure Legend Snippet: miRNA-1271 expression in different groups in SW1990 cells. 48 h after cell transfection, the expression of miRNA-1271 in pancreatic cancer cells were detected by qRT-PCR. *P

Techniques Used: Expressing, Transfection, Quantitative RT-PCR

Effect of miRNA-1271 on EMT in SW1990 cells. (A) The expression of E-cadherin, ZEB1 and TWIST1 in pancreatic cancer cells were detected by qRT-PCR. (B) The protein content of EMT-related markers E-cadherin, ZEB1 and TWIST1 in pancreatic cancer cells was detected by western-blot assay. (C) Transwell chamber model was used to detect the change of invasion ability of pancreatic cancer cells in each experimental group. Magnification, ×100. *P
Figure Legend Snippet: Effect of miRNA-1271 on EMT in SW1990 cells. (A) The expression of E-cadherin, ZEB1 and TWIST1 in pancreatic cancer cells were detected by qRT-PCR. (B) The protein content of EMT-related markers E-cadherin, ZEB1 and TWIST1 in pancreatic cancer cells was detected by western-blot assay. (C) Transwell chamber model was used to detect the change of invasion ability of pancreatic cancer cells in each experimental group. Magnification, ×100. *P

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

Emodin inhibited SW1990 cell proliferation ability. (A) After treatment, cell proliferation ability was detected using MTT assay. (B) The level of miR-1271 in SW1990 cells was detected using qRT-PCR before and after Emodin treatment. *P
Figure Legend Snippet: Emodin inhibited SW1990 cell proliferation ability. (A) After treatment, cell proliferation ability was detected using MTT assay. (B) The level of miR-1271 in SW1990 cells was detected using qRT-PCR before and after Emodin treatment. *P

Techniques Used: MTT Assay, Quantitative RT-PCR

71) Product Images from "Micro RNA‐30c suppresses the pro‐fibrogenic effects of cardiac fibroblasts induced by TGF‐β1 and prevents atrial fibrosis by targeting TGFβ RII, et al. MicroRNA‐30c suppresses the pro‐fibrogenic effects of cardiac fibroblasts induced by TGF‐β1 and prevents atrial fibrosis by targeting TGFβRII"

Article Title: Micro RNA‐30c suppresses the pro‐fibrogenic effects of cardiac fibroblasts induced by TGF‐β1 and prevents atrial fibrosis by targeting TGFβ RII, et al. MicroRNA‐30c suppresses the pro‐fibrogenic effects of cardiac fibroblasts induced by TGF‐β1 and prevents atrial fibrosis by targeting TGFβRII

Journal: Journal of Cellular and Molecular Medicine

doi: 10.1111/jcmm.13548

MiR‐30c targets TGF β RII . (A) Diagram of miR‐30c binding site in TGF β RII 3′ UTR . TGF β RII 3′ UTR mutant was constructed to evaluate miR‐30c binding. (B) After 293T cell cotransfection, luciferase activity was measured using a Dual Luciferase Report Assay Kit. The luciferase activity was significantly decreased in TGF β RII ‐ WT + miR‐30c group compared with that in TGF β RII ‐ WT + miR‐30c NC and TGF β RII ‐mu + miR‐30c. ### P
Figure Legend Snippet: MiR‐30c targets TGF β RII . (A) Diagram of miR‐30c binding site in TGF β RII 3′ UTR . TGF β RII 3′ UTR mutant was constructed to evaluate miR‐30c binding. (B) After 293T cell cotransfection, luciferase activity was measured using a Dual Luciferase Report Assay Kit. The luciferase activity was significantly decreased in TGF β RII ‐ WT + miR‐30c group compared with that in TGF β RII ‐ WT + miR‐30c NC and TGF β RII ‐mu + miR‐30c. ### P

Techniques Used: Binding Assay, Mutagenesis, Construct, Cotransfection, Luciferase, Activity Assay

MiR‐30c was decreased in atrial fibrosis. (A, B) Masson's trichrome staining and quantitative analysis in left atrium of Sham (n = 6) and AAC (n = 7) groups. The blue staining indicates interstitial fibrosis. Interstitial fibrosis was increased in AAC rats. ** P
Figure Legend Snippet: MiR‐30c was decreased in atrial fibrosis. (A, B) Masson's trichrome staining and quantitative analysis in left atrium of Sham (n = 6) and AAC (n = 7) groups. The blue staining indicates interstitial fibrosis. Interstitial fibrosis was increased in AAC rats. ** P

Techniques Used: Staining

MiR‐30c regulates atrial fibrogenesis. TGF ‐β1 decreased miR‐30c expression. Overexpression of miR‐30c in cardiac fibroblasts inhibited TGF β RII expression, thus preventing cell proliferation, differentiation and migration as well as collagen synthesis and atrial fibrogenesis
Figure Legend Snippet: MiR‐30c regulates atrial fibrogenesis. TGF ‐β1 decreased miR‐30c expression. Overexpression of miR‐30c in cardiac fibroblasts inhibited TGF β RII expression, thus preventing cell proliferation, differentiation and migration as well as collagen synthesis and atrial fibrogenesis

Techniques Used: Expressing, Over Expression, Migration

The miR‐30c inhibitor reversed the anti‐fibrotic effects on CF s. (A) CF s were transfected with a miR‐30c inhibitor or miR‐30c inhibitor negative control ( NC ) for 72 hours. The level of miR‐30c was decreased in CF s transfected with miR‐30c inhibitor by qRT ‐ PCR . NC was used as an inhibitor negative control. # P
Figure Legend Snippet: The miR‐30c inhibitor reversed the anti‐fibrotic effects on CF s. (A) CF s were transfected with a miR‐30c inhibitor or miR‐30c inhibitor negative control ( NC ) for 72 hours. The level of miR‐30c was decreased in CF s transfected with miR‐30c inhibitor by qRT ‐ PCR . NC was used as an inhibitor negative control. # P

Techniques Used: Transfection, Negative Control, Quantitative RT-PCR

MiR‐30c exhibited an anti‐fibrotic effect on CF s. (A) CF s were transfected with a miR‐30c mimic or miR‐30c negative control ( NC ) for 72 hours. NC was used as a mimic negative control. The level of miR‐30c was increased in CF s transfected with miR‐30c mimic by qRT ‐ PCR . # P
Figure Legend Snippet: MiR‐30c exhibited an anti‐fibrotic effect on CF s. (A) CF s were transfected with a miR‐30c mimic or miR‐30c negative control ( NC ) for 72 hours. NC was used as a mimic negative control. The level of miR‐30c was increased in CF s transfected with miR‐30c mimic by qRT ‐ PCR . # P

Techniques Used: Transfection, Negative Control, Quantitative RT-PCR

In vivo overexpression of miR‐30c via AAV 9 attenuates atrial fibrosis. (A) After administering AAV 9‐miR‐30c into the inferior vena cava for 8 weeks, the level of miR‐30c in the left atrium was increased by qRT ‐ PCR . ** P
Figure Legend Snippet: In vivo overexpression of miR‐30c via AAV 9 attenuates atrial fibrosis. (A) After administering AAV 9‐miR‐30c into the inferior vena cava for 8 weeks, the level of miR‐30c in the left atrium was increased by qRT ‐ PCR . ** P

Techniques Used: In Vivo, Over Expression, Quantitative RT-PCR

72) Product Images from "miR-1303 regulates BBB permeability and promotes CNS lesions following CA16 infections by directly targeting MMP9"

Article Title: miR-1303 regulates BBB permeability and promotes CNS lesions following CA16 infections by directly targeting MMP9

Journal: Emerging Microbes & Infections

doi: 10.1038/s41426-018-0157-3

MMP9 is a target gene of miR-1303. a Verification of the expression of miR-1303 by qRT-PCR compared to miRNA sequencing. b Detection of the expression of MMP9 induced by CA16 infection using qRT-PCR. c Dual-luciferase reporter analysis verified the targetting relationship between miR-1303 and MMP9. d qRT-PCR and WB analysis of MMP9 expression in miR-1303 transfected cells. Error bars represent the mean ± SEM, and the data are averages from three biological replicates, * P
Figure Legend Snippet: MMP9 is a target gene of miR-1303. a Verification of the expression of miR-1303 by qRT-PCR compared to miRNA sequencing. b Detection of the expression of MMP9 induced by CA16 infection using qRT-PCR. c Dual-luciferase reporter analysis verified the targetting relationship between miR-1303 and MMP9. d qRT-PCR and WB analysis of MMP9 expression in miR-1303 transfected cells. Error bars represent the mean ± SEM, and the data are averages from three biological replicates, * P

Techniques Used: Expressing, Quantitative RT-PCR, Sequencing, Infection, Luciferase, Western Blot, Transfection

73) Product Images from "The Y4-RNA fragment, a potential diagnostic marker, exists in saliva"

Article Title: The Y4-RNA fragment, a potential diagnostic marker, exists in saliva

Journal: Non-coding RNA Research

doi: 10.1016/j.ncrna.2017.07.002

Quantitative RT-PCR. (A) Primer pairs. The sequences of the primers SL-forward_Y4_3 and Y4RNA_R4 for amplification of the 31/32-nt Y4-RNA fragment and the sequences of the primers SL-forward_Y4_3 and Y4RNA_R5 for amplification of the full-length Y4-RNA are presented together with the sequence of Y4-RNA, a middle part of which is omitted. Three nucleotides are added to the 5′ terminus of each primer to stabilize the primer/template hybrid. (B) A representative RT-PCR data for the synthetic 31-nt Y4-RNA fragment (0.001, 0.01, 0.05, and 0.1 fmol) and saliva RNA (1 ng) from the examinees E1, E3, and E4. The right and left halves represent the data for the full-length Y4-RNA and the 31/32-nt fragment, respectively. (C) A standard curve was drawn using the RT-PCR data for the synthetic 31-nt Y4-RNA fragment. (D) Quantitation of the 31/32-nt Y4-RNA fragment in saliva RNA from the examinees E1, E3, and E4. Error bars denote standard deviations (n = 3).
Figure Legend Snippet: Quantitative RT-PCR. (A) Primer pairs. The sequences of the primers SL-forward_Y4_3 and Y4RNA_R4 for amplification of the 31/32-nt Y4-RNA fragment and the sequences of the primers SL-forward_Y4_3 and Y4RNA_R5 for amplification of the full-length Y4-RNA are presented together with the sequence of Y4-RNA, a middle part of which is omitted. Three nucleotides are added to the 5′ terminus of each primer to stabilize the primer/template hybrid. (B) A representative RT-PCR data for the synthetic 31-nt Y4-RNA fragment (0.001, 0.01, 0.05, and 0.1 fmol) and saliva RNA (1 ng) from the examinees E1, E3, and E4. The right and left halves represent the data for the full-length Y4-RNA and the 31/32-nt fragment, respectively. (C) A standard curve was drawn using the RT-PCR data for the synthetic 31-nt Y4-RNA fragment. (D) Quantitation of the 31/32-nt Y4-RNA fragment in saliva RNA from the examinees E1, E3, and E4. Error bars denote standard deviations (n = 3).

Techniques Used: Quantitative RT-PCR, Amplification, Sequencing, Reverse Transcription Polymerase Chain Reaction, Quantitation Assay

74) Product Images from "MiR-29a function as tumor suppressor in cervical cancer by targeting SIRT1 and predict patient prognosis"

Article Title: MiR-29a function as tumor suppressor in cervical cancer by targeting SIRT1 and predict patient prognosis

Journal: OncoTargets and therapy

doi: 10.2147/OTT.S218043

Low expression of miR-29a predicted poor prognosis of cervical cancer. ( A ) miR-29a was downregulated in cervical cancer tissues versus corresponding paracancerous tissues. ( B ) Kaplan–Meier method elucidated low expression of miR-29a predicted poor overall survival. ( C ) The expression of miR-29a was downregulation in HeLa cells than Ect1/E6E7 cells. ( D ) The transfection efficiency of transfecting miR-29a mimic and miR-29a inhibitor in HeLa cells. * P
Figure Legend Snippet: Low expression of miR-29a predicted poor prognosis of cervical cancer. ( A ) miR-29a was downregulated in cervical cancer tissues versus corresponding paracancerous tissues. ( B ) Kaplan–Meier method elucidated low expression of miR-29a predicted poor overall survival. ( C ) The expression of miR-29a was downregulation in HeLa cells than Ect1/E6E7 cells. ( D ) The transfection efficiency of transfecting miR-29a mimic and miR-29a inhibitor in HeLa cells. * P

Techniques Used: Expressing, Transfection

miR-29a regulated the expression of SIRT1 through directly binding to the 3’-UTR of its mRNA. ( A ) TargetScan predicts SIRT1 was a potential target gene of miR-29a. ( B ) The miR-29a mimic inhibited the luciferase activity of cells that transfected wild-type SIRT1 3’-UTR. ( C ) The mRNA level of SIRT1 was inhibited by the miR-29a mimic, while that was enhanced by the miR-29a inhibitor in HeLa cells. ( D ) The protein level of SIRT1 was regulated by miR-29a in HeLa cells. *Compared with NC, P
Figure Legend Snippet: miR-29a regulated the expression of SIRT1 through directly binding to the 3’-UTR of its mRNA. ( A ) TargetScan predicts SIRT1 was a potential target gene of miR-29a. ( B ) The miR-29a mimic inhibited the luciferase activity of cells that transfected wild-type SIRT1 3’-UTR. ( C ) The mRNA level of SIRT1 was inhibited by the miR-29a mimic, while that was enhanced by the miR-29a inhibitor in HeLa cells. ( D ) The protein level of SIRT1 was regulated by miR-29a in HeLa cells. *Compared with NC, P

Techniques Used: Expressing, Binding Assay, Luciferase, Activity Assay, Transfection

miR-29a impaired cell metastasis and EMT of HeLa cells. ( A ) The miR-29a mimic inhibited the migratory capacity, whereas it was inhibited by the miR-29a inhibitor. ( B ) miR-29a regulated cell invasion in HeLa cells. ( C ) The miR-29a mimic suppressed the expression of E-cadherin while improved the expression of N-cadherin. Meanwhile, the miR-29a inhibitor enhanced the expression of E-cadherin whereas suppressed the expression of N-cadherin. *Compared with NC, P
Figure Legend Snippet: miR-29a impaired cell metastasis and EMT of HeLa cells. ( A ) The miR-29a mimic inhibited the migratory capacity, whereas it was inhibited by the miR-29a inhibitor. ( B ) miR-29a regulated cell invasion in HeLa cells. ( C ) The miR-29a mimic suppressed the expression of E-cadherin while improved the expression of N-cadherin. Meanwhile, the miR-29a inhibitor enhanced the expression of E-cadherin whereas suppressed the expression of N-cadherin. *Compared with NC, P

Techniques Used: Expressing

SIRT1 reversed partial functions of miR-29a. ( A ) The transfection efficiency was calculated of re-transfecting pcDNA3.1-SIRT1 plasmid in miR-29a overexpressed HeLa cells. ( B ) The migratory and invasive abilities were increased when re-transfected SIRT1 in miR-29a overexpressed cells. ( C ) SIRT1 could reverse partial functions of miR-29a on the EMT capacity in HeLa cells. * P
Figure Legend Snippet: SIRT1 reversed partial functions of miR-29a. ( A ) The transfection efficiency was calculated of re-transfecting pcDNA3.1-SIRT1 plasmid in miR-29a overexpressed HeLa cells. ( B ) The migratory and invasive abilities were increased when re-transfected SIRT1 in miR-29a overexpressed cells. ( C ) SIRT1 could reverse partial functions of miR-29a on the EMT capacity in HeLa cells. * P

Techniques Used: Transfection, Plasmid Preparation

75) Product Images from "Lentivirus-mediated LIGHT overexpression inhibits human colorectal carcinoma cell growth in vitro and in vivo"

Article Title: Lentivirus-mediated LIGHT overexpression inhibits human colorectal carcinoma cell growth in vitro and in vivo

Journal: Oncology Letters

doi: 10.3892/ol.2013.1505

Effects of overexpressing LIGHT on the mRNA level of LIGHT. (A) Electrophoresis and semiquantitative analysis of PCR products. The mRNA level of LIGHT was higher in the HCT116/LIGHT cells compared with the HCT116/GFP and HCT116 cells. Lanes 1 and 4, HCT116; lanes 2 and 5, HCT116/GFP; lanes 3 and 6, HCT116/LIGHT; M, DL1000 DNA marker. (B) Statistical analysis of the mRNA level of LIGHT relative to that of GAPDH. LIGHT, lymphotoxin-related inducible ligand that competes for glycoprotein D binding to herpesvirus entry mediator on T cells.
Figure Legend Snippet: Effects of overexpressing LIGHT on the mRNA level of LIGHT. (A) Electrophoresis and semiquantitative analysis of PCR products. The mRNA level of LIGHT was higher in the HCT116/LIGHT cells compared with the HCT116/GFP and HCT116 cells. Lanes 1 and 4, HCT116; lanes 2 and 5, HCT116/GFP; lanes 3 and 6, HCT116/LIGHT; M, DL1000 DNA marker. (B) Statistical analysis of the mRNA level of LIGHT relative to that of GAPDH. LIGHT, lymphotoxin-related inducible ligand that competes for glycoprotein D binding to herpesvirus entry mediator on T cells.

Techniques Used: Electrophoresis, Polymerase Chain Reaction, Marker, Binding Assay

Related Articles

Real-time Polymerase Chain Reaction:

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Article Title: Overexpression of Programmed Death Ligands in Naturally Occurring Postweaning Multisystemic Wasting Syndrome
Article Snippet: Residual DNA was removed from the total RNA samples, and first-strand cDNA was synthesized from 1 μg of total RNA with a PrimeScript RT Reagent Kit with gDNA eraser (perfect real-time; Takara, code: RR047A) according to the manufacturer's protocol. .. The reaction was performed at 37°C for 15 min, followed by incubation in an 85°C water bath for 5 s. The synthesized cDNA was cooled at 4°C for several minutes and then stored at −20°C until use in real-time quantitative PCR (RT-qPCR) reactions.

RNA Extraction:

Article Title: Arabidopsis EMB1990 Encoding a Plastid-Targeted YlmG Protein Is Required for Chloroplast Biogenesis and Embryo Development
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Article Title: Functional interaction between TATA and upstream CACGTG elements regulates the temporally specific expression of Otx mRNAs during early embryogenesis of the sea urchin, Hemicentrotus pulcherrimus
Article Snippet: For RT–PCR analysis, total RNA was prepared from collected embryos using the Isogen RNA extraction system (Wako Pure Chemical Industries, Ltd). .. The 800 bp DNA fragment encoding the region from immediately 3′ of the transcriptional initiation site of the HpOtxE promoter to the middle of the open reading frame (ORF) of firefly luciferase was amplified from 0.3 µg of total RNA using the One Step RNA PCR Kit (AMV) (TaKaRa Biomedicals) and the primers TK1682 and TK1686.

Article Title: Overexpression of Programmed Death Ligands in Naturally Occurring Postweaning Multisystemic Wasting Syndrome
Article Snippet: Paragraph title: RNA extraction and cDNA synthesis ... Residual DNA was removed from the total RNA samples, and first-strand cDNA was synthesized from 1 μg of total RNA with a PrimeScript RT Reagent Kit with gDNA eraser (perfect real-time; Takara, code: RR047A) according to the manufacturer's protocol.

Amplification:

Article Title: Circular RNA AKT3 upregulates PIK3R1 to enhance cisplatin resistance in gastric cancer via miR-198 suppression
Article Snippet: For Quantitative real-time PCR (RT-PCR), 500 ng of treated RNA was directly reverse transcribed using Prime Script RT Master Mix (Takara, Japan) and either random or oligo(dT) primers. .. Reverse transcription of miRNA was performed using a New Poly(A) Tailing Kit (ThermoFisher Scientific, China). mRNA was reverse transcribed into cDNA with a PrimeScript RT Master Mix Kit (Takara, RR036A, Japan). cDNA was amplified using Universal SYBR Green Master Mix (4,913,914,001, Roche, Shanghai, China).

Article Title: RNA-seq profiling reveals differentially expressed genes as potential markers for vital reaction in skin contusion: a pilot study
Article Snippet: Quantitative real-time reverse transcriptase polymerase chain reaction (RT-qPCR) Briefly, cDNA copies of total RNA were obtained using PrimerScript RT reagent Kit (TaKaRa, Japan). .. The RT-qPCR conditions, thermal cycler parameters and gene-specific primers used for amplification are listed in Supplemental Table S1.

Article Title: Functional interaction between TATA and upstream CACGTG elements regulates the temporally specific expression of Otx mRNAs during early embryogenesis of the sea urchin, Hemicentrotus pulcherrimus
Article Snippet: .. The 800 bp DNA fragment encoding the region from immediately 3′ of the transcriptional initiation site of the HpOtxE promoter to the middle of the open reading frame (ORF) of firefly luciferase was amplified from 0.3 µg of total RNA using the One Step RNA PCR Kit (AMV) (TaKaRa Biomedicals) and the primers TK1682 and TK1686. .. As a reference, the 190 bp DNA fragment encoding the 3′-untranslated region (3′-UTR) of the ubiquitin gene was amplified using the primer pair TK1591 and TK1592.

Article Title: Chlamydia pneumoniae Infection Promotes Vascular Smooth Muscle Cell Migration through a Toll-Like Receptor 2-Related Signaling Pathway
Article Snippet: cDNA was prepared from RNA using 200 U of Moloney murine leukemia virus reverse transcriptase (TaKaRa, Otsu, Japan) according to a standard protocol. .. For standard PCR, the generated cDNA was amplified by PCR using specific primers: TLR2, 5′-GCTCTCCTGTTGTGCTTCTCCAC-3′ (forward) and 5′-CAGGAGCAGATGAAATGGTTGT-3′ (reverse) ( ); GAPDH, 5′-AGTTCAACGGCACAGTCAAG-3′ (forward) and 5′-TTGTCTGTGGGACACTGCTC-3′ (reverse).

Synthesized:

Article Title: Up-regulation of microRNA-340 promotes osteosarcoma cell apoptosis while suppressing proliferation, migration, and invasion by inactivating the CTNNB1-mediated Notch signaling pathway
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Article Title: Overexpression of Programmed Death Ligands in Naturally Occurring Postweaning Multisystemic Wasting Syndrome
Article Snippet: .. Residual DNA was removed from the total RNA samples, and first-strand cDNA was synthesized from 1 μg of total RNA with a PrimeScript RT Reagent Kit with gDNA eraser (perfect real-time; Takara, code: RR047A) according to the manufacturer's protocol. .. Briefly, residual DNA was removed as follows: 10 μL total volume of 1 μg total RNA, 2 μL 5×gDNA eraser buffer, 1 μL gDNA eraser and RNase-free dH2 O at 42°C for 2 min, and then put on the ice.

Isolation:

Article Title: Arabidopsis EMB1990 Encoding a Plastid-Targeted YlmG Protein Is Required for Chloroplast Biogenesis and Embryo Development
Article Snippet: .. RNA Extraction and RT-PCR Total RNA from different kinds of Arabidopsis tissues was isolated by RNAiso Plus, except the siliques and ovules were extracted using MiniBEST Plant RNA Extraction Kit (TaKaRa, Japan). .. Afterward, RNA was transcribed into cDNA using random hexamer primer with RevertAid RT Reverse Transcription Kit (Fermentas, United States), following the manufacturer’s instructions.

Article Title: Overexpression of Programmed Death Ligands in Naturally Occurring Postweaning Multisystemic Wasting Syndrome
Article Snippet: Total RNA was extracted from freshly isolated PBMCs using TRIzol reagent (Invitrogen) according to the manufacturer's instructions. .. Residual DNA was removed from the total RNA samples, and first-strand cDNA was synthesized from 1 μg of total RNA with a PrimeScript RT Reagent Kit with gDNA eraser (perfect real-time; Takara, code: RR047A) according to the manufacturer's protocol.

Centrifugation:

Article Title: Functional interaction between TATA and upstream CACGTG elements regulates the temporally specific expression of Otx mRNAs during early embryogenesis of the sea urchin, Hemicentrotus pulcherrimus
Article Snippet: At the indicated time, aliquots of bombarded embryos were taken from the dishes, collected by centrifugation and stored at –80°C until being used in either luciferase assays (approximately 20 000 embryos × 3) or RT–PCR analyses (approximately 6700 embryos × 3). .. The 800 bp DNA fragment encoding the region from immediately 3′ of the transcriptional initiation site of the HpOtxE promoter to the middle of the open reading frame (ORF) of firefly luciferase was amplified from 0.3 µg of total RNA using the One Step RNA PCR Kit (AMV) (TaKaRa Biomedicals) and the primers TK1682 and TK1686.

Random Hexamer Labeling:

Article Title: Arabidopsis EMB1990 Encoding a Plastid-Targeted YlmG Protein Is Required for Chloroplast Biogenesis and Embryo Development
Article Snippet: RNA Extraction and RT-PCR Total RNA from different kinds of Arabidopsis tissues was isolated by RNAiso Plus, except the siliques and ovules were extracted using MiniBEST Plant RNA Extraction Kit (TaKaRa, Japan). .. Afterward, RNA was transcribed into cDNA using random hexamer primer with RevertAid RT Reverse Transcription Kit (Fermentas, United States), following the manufacturer’s instructions.

Quantitative RT-PCR:

Article Title: Long non-coding RNA CASC2 suppresses pulmonary artery smooth muscle cell proliferation and phenotypic switch in hypoxia-induced pulmonary hypertension
Article Snippet: .. Real time-quantitative polymerase chain reaction (qRT-PCR) Total RNA from PASMCs or rat pulmonary arterial tissues was extracted using the Trizol reagent and reversely transcribed into cDNA using Prime-Script RT-PCR kit (Takara, Dalian, China) following the manufacturer′s instructions. .. Thereafter, polymerase chain reaction was performed using a LightCycler 1.0 (Roche, Basel, Switzerland).

Article Title: Up-regulation of microRNA-340 promotes osteosarcoma cell apoptosis while suppressing proliferation, migration, and invasion by inactivating the CTNNB1-mediated Notch signaling pathway
Article Snippet: Paragraph title: Reverse transcription quantitative polymerase chain reaction (RT-qPCR) ... The primers of miR-340, Notch, CTNNB1, Bcl-2 associated protein X (Bax), Bcl-2, Bcl-2 interacting mediator of cell death (BIM), hairy and enhancer of split 1 (Hes1), Runt-related transcription factor 2 (Runx2), and osteocalcin were designed and synthesized by Takara (Dalian, Liaoning, China) ( ).

Article Title: RNA-seq profiling reveals differentially expressed genes as potential markers for vital reaction in skin contusion: a pilot study
Article Snippet: .. Quantitative real-time reverse transcriptase polymerase chain reaction (RT-qPCR) Briefly, cDNA copies of total RNA were obtained using PrimerScript RT reagent Kit (TaKaRa, Japan). .. RT-qPCR reactions were run in 48-well reaction plates with an Illumina Eco Real-Time PCR System and a SYBR green kit (TaKaRa, Japan), according to the manufacturer's recommendations.

Article Title: Overexpression of Programmed Death Ligands in Naturally Occurring Postweaning Multisystemic Wasting Syndrome
Article Snippet: Residual DNA was removed from the total RNA samples, and first-strand cDNA was synthesized from 1 μg of total RNA with a PrimeScript RT Reagent Kit with gDNA eraser (perfect real-time; Takara, code: RR047A) according to the manufacturer's protocol. .. The reaction was performed at 37°C for 15 min, followed by incubation in an 85°C water bath for 5 s. The synthesized cDNA was cooled at 4°C for several minutes and then stored at −20°C until use in real-time quantitative PCR (RT-qPCR) reactions.

Article Title: Chlamydia pneumoniae Infection Promotes Vascular Smooth Muscle Cell Migration through a Toll-Like Receptor 2-Related Signaling Pathway
Article Snippet: Paragraph title: Standard and quantitative RT-PCR. ... cDNA was prepared from RNA using 200 U of Moloney murine leukemia virus reverse transcriptase (TaKaRa, Otsu, Japan) according to a standard protocol.

Size-exclusion Chromatography:

Article Title: Lentivirus-mediated LIGHT overexpression inhibits human colorectal carcinoma cell growth in vitro and in vivo
Article Snippet: Semi-quantitative reverse transcription-PCR analysis The total RNA of HCT116/LIGHT, HCT116/GFP or the control cells was extracted using RNAiso reagent (Takara, Japan), and then converted into cDNA using a PrimeScript™ RT reagent kit (Takara), according to the manufacturer’s instructions. .. The reaction conditions of pre-denaturation were 95°C for 3 min, 95°C for 30 sec, 60°C for 30 sec and 72°C for 30 sec, with 22 cycles for GAPDH and 29 cycles for LIGHT (the cycles were based on PCR kinetics) and a total reaction volume of 20 μl.

SYBR Green Assay:

Article Title: Circular RNA AKT3 upregulates PIK3R1 to enhance cisplatin resistance in gastric cancer via miR-198 suppression
Article Snippet: For Quantitative real-time PCR (RT-PCR), 500 ng of treated RNA was directly reverse transcribed using Prime Script RT Master Mix (Takara, Japan) and either random or oligo(dT) primers. .. Reverse transcription of miRNA was performed using a New Poly(A) Tailing Kit (ThermoFisher Scientific, China). mRNA was reverse transcribed into cDNA with a PrimeScript RT Master Mix Kit (Takara, RR036A, Japan). cDNA was amplified using Universal SYBR Green Master Mix (4,913,914,001, Roche, Shanghai, China).

Article Title: RNA-seq profiling reveals differentially expressed genes as potential markers for vital reaction in skin contusion: a pilot study
Article Snippet: Quantitative real-time reverse transcriptase polymerase chain reaction (RT-qPCR) Briefly, cDNA copies of total RNA were obtained using PrimerScript RT reagent Kit (TaKaRa, Japan). .. RT-qPCR reactions were run in 48-well reaction plates with an Illumina Eco Real-Time PCR System and a SYBR green kit (TaKaRa, Japan), according to the manufacturer's recommendations.

Polymerase Chain Reaction:

Article Title: Lentivirus-mediated LIGHT overexpression inhibits human colorectal carcinoma cell growth in vitro and in vivo
Article Snippet: Semi-quantitative reverse transcription-PCR analysis The total RNA of HCT116/LIGHT, HCT116/GFP or the control cells was extracted using RNAiso reagent (Takara, Japan), and then converted into cDNA using a PrimeScript™ RT reagent kit (Takara), according to the manufacturer’s instructions. .. The specific oligonucleotide primers of the LIGHT (PCR product 128 bp) and GAPDH (PCR product 151 bp) genes were as follows: Sense, 5′-GTACGGCCCTCAGTGTTTGTG-3′ and antisense, 5′-CCCATCAGCAACAGCAAGAGA-3′; and sense, 5′-CTTAGCACCCCTGGCCAAG-3′ and antisense, 5′-GATGTTCTGGAGAGCCCCG-3′, respectively.

Article Title: Long non-coding RNA CASC2 suppresses pulmonary artery smooth muscle cell proliferation and phenotypic switch in hypoxia-induced pulmonary hypertension
Article Snippet: .. Real time-quantitative polymerase chain reaction (qRT-PCR) Total RNA from PASMCs or rat pulmonary arterial tissues was extracted using the Trizol reagent and reversely transcribed into cDNA using Prime-Script RT-PCR kit (Takara, Dalian, China) following the manufacturer′s instructions. .. Thereafter, polymerase chain reaction was performed using a LightCycler 1.0 (Roche, Basel, Switzerland).

Article Title: Up-regulation of microRNA-340 promotes osteosarcoma cell apoptosis while suppressing proliferation, migration, and invasion by inactivating the CTNNB1-mediated Notch signaling pathway
Article Snippet: The primers of miR-340, Notch, CTNNB1, Bcl-2 associated protein X (Bax), Bcl-2, Bcl-2 interacting mediator of cell death (BIM), hairy and enhancer of split 1 (Hes1), Runt-related transcription factor 2 (Runx2), and osteocalcin were designed and synthesized by Takara (Dalian, Liaoning, China) ( ). .. According to the instructions, the reaction conditions were set according to the following protocol: reverse transcription at 37°C for 15 min, three times, reverse transcriptase inactivation at 85°C for 5 s. The reaction mixture was selected for fluorescent quantitation PCR based on the instructions provided by the SYBR® Premix Ex Taq™ II reagent kit (Takara, Dalian, Liaoning, China).

Article Title: Circular RNA AKT3 upregulates PIK3R1 to enhance cisplatin resistance in gastric cancer via miR-198 suppression
Article Snippet: Paragraph title: RNA preparation, treatment with RNase R, and PCR ... For Quantitative real-time PCR (RT-PCR), 500 ng of treated RNA was directly reverse transcribed using Prime Script RT Master Mix (Takara, Japan) and either random or oligo(dT) primers.

Article Title: Arabidopsis EMB1990 Encoding a Plastid-Targeted YlmG Protein Is Required for Chloroplast Biogenesis and Embryo Development
Article Snippet: RNA Extraction and RT-PCR Total RNA from different kinds of Arabidopsis tissues was isolated by RNAiso Plus, except the siliques and ovules were extracted using MiniBEST Plant RNA Extraction Kit (TaKaRa, Japan). .. Then the cDNA was used as template for subsequent PCR analysis with specific primers (Supplementary Table ).

Article Title: RNA-seq profiling reveals differentially expressed genes as potential markers for vital reaction in skin contusion: a pilot study
Article Snippet: .. Quantitative real-time reverse transcriptase polymerase chain reaction (RT-qPCR) Briefly, cDNA copies of total RNA were obtained using PrimerScript RT reagent Kit (TaKaRa, Japan). .. RT-qPCR reactions were run in 48-well reaction plates with an Illumina Eco Real-Time PCR System and a SYBR green kit (TaKaRa, Japan), according to the manufacturer's recommendations.

Article Title: Genotype-4 hepatitis E in a human after ingesting roe deer meat in South Korea
Article Snippet: Purified RNA was used to generate the ORF2 of HEV using One-Step RT-PCR with a PLATINUM Taq Kit (Takara, Shiga, Japan). .. The second round of PCR was performed under the same conditions as the first-round PCR with 0.2 µM each of the same primers.

Article Title: Subunits of the mechano-electrical transduction channel, Tmc1/2b, require Tmie to localize in zebrafish sensory hair cells
Article Snippet: Paragraph title: cDNA generation by Reverse Transcription Polymerase Chain Reaction (RT-PCR) and semi-quantitative PCR ... To reverse transcribe cDNA we used the RNA to cDNA EcoDry Premix (Clontech, Cat # 639549).

Article Title: Functional interaction between TATA and upstream CACGTG elements regulates the temporally specific expression of Otx mRNAs during early embryogenesis of the sea urchin, Hemicentrotus pulcherrimus
Article Snippet: .. The 800 bp DNA fragment encoding the region from immediately 3′ of the transcriptional initiation site of the HpOtxE promoter to the middle of the open reading frame (ORF) of firefly luciferase was amplified from 0.3 µg of total RNA using the One Step RNA PCR Kit (AMV) (TaKaRa Biomedicals) and the primers TK1682 and TK1686. .. As a reference, the 190 bp DNA fragment encoding the 3′-untranslated region (3′-UTR) of the ubiquitin gene was amplified using the primer pair TK1591 and TK1592.

Article Title: Chlamydia pneumoniae Infection Promotes Vascular Smooth Muscle Cell Migration through a Toll-Like Receptor 2-Related Signaling Pathway
Article Snippet: cDNA was prepared from RNA using 200 U of Moloney murine leukemia virus reverse transcriptase (TaKaRa, Otsu, Japan) according to a standard protocol. .. For standard PCR, the generated cDNA was amplified by PCR using specific primers: TLR2, 5′-GCTCTCCTGTTGTGCTTCTCCAC-3′ (forward) and 5′-CAGGAGCAGATGAAATGGTTGT-3′ (reverse) ( ); GAPDH, 5′-AGTTCAACGGCACAGTCAAG-3′ (forward) and 5′-TTGTCTGTGGGACACTGCTC-3′ (reverse).

Article Title: Development of a Conventional RT-PCR Assay for Rapid Detection of Porcine Deltacoronavirus with the Same Detection Limit as a SYBR Green-Based Real-Time RT-PCR Assay
Article Snippet: RT-PCR After isolating total RNA from samples, it was reverse transcribed into cDNA by following the manufacturer's instructions in PrimeScript™ 1st Strand cDNA Synthesis Kit (Takara Biotechnology, Dalian, China). .. Using Premix Taq™ kit, the PCR assay was performed under the following conditions: 1 μ L each primer (10 μ m), 25 μ L premix, 1 μ L cDNA, and 22 μ L distilled water.

Incubation:

Article Title: Functional interaction between TATA and upstream CACGTG elements regulates the temporally specific expression of Otx mRNAs during early embryogenesis of the sea urchin, Hemicentrotus pulcherrimus
Article Snippet: Bombarded embryos were cultured in a petri dish (6 cm in diameter) and incubated at a constant temperature of 16°C. .. The 800 bp DNA fragment encoding the region from immediately 3′ of the transcriptional initiation site of the HpOtxE promoter to the middle of the open reading frame (ORF) of firefly luciferase was amplified from 0.3 µg of total RNA using the One Step RNA PCR Kit (AMV) (TaKaRa Biomedicals) and the primers TK1682 and TK1686.

Article Title: Overexpression of Programmed Death Ligands in Naturally Occurring Postweaning Multisystemic Wasting Syndrome
Article Snippet: Residual DNA was removed from the total RNA samples, and first-strand cDNA was synthesized from 1 μg of total RNA with a PrimeScript RT Reagent Kit with gDNA eraser (perfect real-time; Takara, code: RR047A) according to the manufacturer's protocol. .. The reaction was performed at 37°C for 15 min, followed by incubation in an 85°C water bath for 5 s. The synthesized cDNA was cooled at 4°C for several minutes and then stored at −20°C until use in real-time quantitative PCR (RT-qPCR) reactions.

Luciferase:

Article Title: Functional interaction between TATA and upstream CACGTG elements regulates the temporally specific expression of Otx mRNAs during early embryogenesis of the sea urchin, Hemicentrotus pulcherrimus
Article Snippet: .. The 800 bp DNA fragment encoding the region from immediately 3′ of the transcriptional initiation site of the HpOtxE promoter to the middle of the open reading frame (ORF) of firefly luciferase was amplified from 0.3 µg of total RNA using the One Step RNA PCR Kit (AMV) (TaKaRa Biomedicals) and the primers TK1682 and TK1686. .. As a reference, the 190 bp DNA fragment encoding the 3′-untranslated region (3′-UTR) of the ubiquitin gene was amplified using the primer pair TK1591 and TK1592.

Cell Culture:

Article Title: Functional interaction between TATA and upstream CACGTG elements regulates the temporally specific expression of Otx mRNAs during early embryogenesis of the sea urchin, Hemicentrotus pulcherrimus
Article Snippet: Bombarded embryos were cultured in a petri dish (6 cm in diameter) and incubated at a constant temperature of 16°C. .. The 800 bp DNA fragment encoding the region from immediately 3′ of the transcriptional initiation site of the HpOtxE promoter to the middle of the open reading frame (ORF) of firefly luciferase was amplified from 0.3 µg of total RNA using the One Step RNA PCR Kit (AMV) (TaKaRa Biomedicals) and the primers TK1682 and TK1686.

Expressing:

Article Title: Long non-coding RNA CASC2 suppresses pulmonary artery smooth muscle cell proliferation and phenotypic switch in hypoxia-induced pulmonary hypertension
Article Snippet: Real time-quantitative polymerase chain reaction (qRT-PCR) Total RNA from PASMCs or rat pulmonary arterial tissues was extracted using the Trizol reagent and reversely transcribed into cDNA using Prime-Script RT-PCR kit (Takara, Dalian, China) following the manufacturer′s instructions. .. The expression of both mRNAs and lncRNA was normalized against glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and relatively quantified using \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$ {2}^{-\Delta \Delta {C}_t} $$\end{document} 2 − ∆∆ C t method.

Article Title: Circular RNA AKT3 upregulates PIK3R1 to enhance cisplatin resistance in gastric cancer via miR-198 suppression
Article Snippet: For Quantitative real-time PCR (RT-PCR), 500 ng of treated RNA was directly reverse transcribed using Prime Script RT Master Mix (Takara, Japan) and either random or oligo(dT) primers. .. Relative gene expression levels were determined using the 2-△△CT method.

Article Title: RNA-seq profiling reveals differentially expressed genes as potential markers for vital reaction in skin contusion: a pilot study
Article Snippet: Quantitative real-time reverse transcriptase polymerase chain reaction (RT-qPCR) Briefly, cDNA copies of total RNA were obtained using PrimerScript RT reagent Kit (TaKaRa, Japan). .. GAPDH (glyceraldehyde-3-phosphate dehydrogenase) was used as an endogenous control for the RT-qPCR, and the relative expression levels were determined by the 2− △△Ct method [ ].

Reverse Transcription Polymerase Chain Reaction:

Article Title: Long non-coding RNA CASC2 suppresses pulmonary artery smooth muscle cell proliferation and phenotypic switch in hypoxia-induced pulmonary hypertension
Article Snippet: .. Real time-quantitative polymerase chain reaction (qRT-PCR) Total RNA from PASMCs or rat pulmonary arterial tissues was extracted using the Trizol reagent and reversely transcribed into cDNA using Prime-Script RT-PCR kit (Takara, Dalian, China) following the manufacturer′s instructions. .. Thereafter, polymerase chain reaction was performed using a LightCycler 1.0 (Roche, Basel, Switzerland).

Article Title: Circular RNA AKT3 upregulates PIK3R1 to enhance cisplatin resistance in gastric cancer via miR-198 suppression
Article Snippet: .. For Quantitative real-time PCR (RT-PCR), 500 ng of treated RNA was directly reverse transcribed using Prime Script RT Master Mix (Takara, Japan) and either random or oligo(dT) primers. .. Reverse transcription of miRNA was performed using a New Poly(A) Tailing Kit (ThermoFisher Scientific, China). mRNA was reverse transcribed into cDNA with a PrimeScript RT Master Mix Kit (Takara, RR036A, Japan). cDNA was amplified using Universal SYBR Green Master Mix (4,913,914,001, Roche, Shanghai, China).

Article Title: Arabidopsis EMB1990 Encoding a Plastid-Targeted YlmG Protein Is Required for Chloroplast Biogenesis and Embryo Development
Article Snippet: .. RNA Extraction and RT-PCR Total RNA from different kinds of Arabidopsis tissues was isolated by RNAiso Plus, except the siliques and ovules were extracted using MiniBEST Plant RNA Extraction Kit (TaKaRa, Japan). .. Afterward, RNA was transcribed into cDNA using random hexamer primer with RevertAid RT Reverse Transcription Kit (Fermentas, United States), following the manufacturer’s instructions.

Article Title: Genotype-4 hepatitis E in a human after ingesting roe deer meat in South Korea
Article Snippet: .. Purified RNA was used to generate the ORF2 of HEV using One-Step RT-PCR with a PLATINUM Taq Kit (Takara, Shiga, Japan). .. Briefly, RT-PCR was performed on 5 µL of purified RNA from serum in 50 µL of 2× reaction mix with 0.2 µM each primer (forward: 5' aggttggcgctctgtcgaga-3'; reverse: 5'-acagtcggctcgccattggc-3').

Article Title: Subunits of the mechano-electrical transduction channel, Tmc1/2b, require Tmie to localize in zebrafish sensory hair cells
Article Snippet: Paragraph title: cDNA generation by Reverse Transcription Polymerase Chain Reaction (RT-PCR) and semi-quantitative PCR ... To reverse transcribe cDNA we used the RNA to cDNA EcoDry Premix (Clontech, Cat # 639549).

Article Title: Functional interaction between TATA and upstream CACGTG elements regulates the temporally specific expression of Otx mRNAs during early embryogenesis of the sea urchin, Hemicentrotus pulcherrimus
Article Snippet: Paragraph title: Luciferase assay and RT–PCR analysis ... The 800 bp DNA fragment encoding the region from immediately 3′ of the transcriptional initiation site of the HpOtxE promoter to the middle of the open reading frame (ORF) of firefly luciferase was amplified from 0.3 µg of total RNA using the One Step RNA PCR Kit (AMV) (TaKaRa Biomedicals) and the primers TK1682 and TK1686.

Article Title: Development of a Conventional RT-PCR Assay for Rapid Detection of Porcine Deltacoronavirus with the Same Detection Limit as a SYBR Green-Based Real-Time RT-PCR Assay
Article Snippet: .. RT-PCR After isolating total RNA from samples, it was reverse transcribed into cDNA by following the manufacturer's instructions in PrimeScript™ 1st Strand cDNA Synthesis Kit (Takara Biotechnology, Dalian, China). .. Using Premix Taq™ kit, the PCR assay was performed under the following conditions: 1 μ L each primer (10 μ m), 25 μ L premix, 1 μ L cDNA, and 22 μ L distilled water.

Quantitation Assay:

Article Title: Up-regulation of microRNA-340 promotes osteosarcoma cell apoptosis while suppressing proliferation, migration, and invasion by inactivating the CTNNB1-mediated Notch signaling pathway
Article Snippet: The primers of miR-340, Notch, CTNNB1, Bcl-2 associated protein X (Bax), Bcl-2, Bcl-2 interacting mediator of cell death (BIM), hairy and enhancer of split 1 (Hes1), Runt-related transcription factor 2 (Runx2), and osteocalcin were designed and synthesized by Takara (Dalian, Liaoning, China) ( ). .. According to the instructions, the reaction conditions were set according to the following protocol: reverse transcription at 37°C for 15 min, three times, reverse transcriptase inactivation at 85°C for 5 s. The reaction mixture was selected for fluorescent quantitation PCR based on the instructions provided by the SYBR® Premix Ex Taq™ II reagent kit (Takara, Dalian, Liaoning, China).

Purification:

Article Title: Genotype-4 hepatitis E in a human after ingesting roe deer meat in South Korea
Article Snippet: .. Purified RNA was used to generate the ORF2 of HEV using One-Step RT-PCR with a PLATINUM Taq Kit (Takara, Shiga, Japan). .. Briefly, RT-PCR was performed on 5 µL of purified RNA from serum in 50 µL of 2× reaction mix with 0.2 µM each primer (forward: 5' aggttggcgctctgtcgaga-3'; reverse: 5'-acagtcggctcgccattggc-3').

Generated:

Article Title: Chlamydia pneumoniae Infection Promotes Vascular Smooth Muscle Cell Migration through a Toll-Like Receptor 2-Related Signaling Pathway
Article Snippet: cDNA was prepared from RNA using 200 U of Moloney murine leukemia virus reverse transcriptase (TaKaRa, Otsu, Japan) according to a standard protocol. .. For standard PCR, the generated cDNA was amplified by PCR using specific primers: TLR2, 5′-GCTCTCCTGTTGTGCTTCTCCAC-3′ (forward) and 5′-CAGGAGCAGATGAAATGGTTGT-3′ (reverse) ( ); GAPDH, 5′-AGTTCAACGGCACAGTCAAG-3′ (forward) and 5′-TTGTCTGTGGGACACTGCTC-3′ (reverse).

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