e2f2 sirna  (Santa Cruz Biotechnology)

Journal: International Journal of Molecular Sciences

Article Title: E2f2 Attenuates Apoptosis of Activated T Lymphocytes and Protects from Immune-Mediated Injury through Repression of Fas and FasL

doi: 10.3390/ijms23010311

Figure Lengend Snippet: E2F2 regulates expression of Fas and FasL at the transcriptional level: ( A ) Schematic representation of murine Fas and FasL promoter regions, indicating the localization of consensus E2F motifs detected with Consite at a 0.85 threshold level. The nucleotide positions are numbered relative to the transcriptional start site. Arrows depict the location of primers used for qPCR analysis of immunoprecipitated chromatin sequences, and letters indicate the DNA region analyzed in the qPCRs. ( B ) ChIP-qPCR analyses of Fas and FasL promoter regions in freshly purified resting (upper panel) and TCR-activated (lower panel) WT lymphocytes. Data from a representative experiment out of three independent experiments (results obtained in Exp. 2 and Exp. 3 are in ). ChIP assays were performed using anti-E2F1, anti-E2F2, and anti-SV40T (irrelevant control) antibodies, and qPCR was performed using primers specific for the Fas and FasL promoter regions. Analyses of Rbl1 promoter, a known E2F-target carrying consensus E2F binding sites, and β-actin promoter regions were carried out as positive and negative controls, respectively. Data are presented as percentage fold over of input chromatin. The values represent the mean ± SD of qPCR technical triplicates. ( C ) Promoter construct of human FAS (-678 to +51), indicating the localization of consensus E2F motifs detected with Consite at a 0.85 threshold level. The nucleotide positions of the promoter are numbered relative to the transcriptional start site. ( D ) Activation of FAS promoter-driven firefly luciferase reporter construct after silencing of E2Fs. HCT116 cells were transfected with non-target control siRNA (siCtrl) or with siRNAs specific for E2F1 (siE2F1), E2F2 (siE2F2), or their combination (siE2F1 + siE2F2). After 3 h, cells were transfected with FAS-pGL2 along with pRL-TK. Then, 48 h after siRNA transfection, FAS promoter activity was measured. Values are represented as fold-change (mean ± SD) of Relative Luciferase Levels (firefly/ Renilla = RLU) over siRNA control. n = 3, ** p < 0.0001, * p < 0.01.

Article Snippet: To silence endogenous expression of E2F1 and E2F2 , cells were transfected with 6.5 nM of siRNA for E2F1 (s4405) and E2F2 (s4409) (Thermo Fisher Scientific, Foster City, CA, USA) using Lipofectamine RNAiMAX (Thermo Fisher Scientific, Foster City, CA, USA).

Techniques: Expressing, Immunoprecipitation, ChIP-qPCR, Purification, Control, Binding Assay, Construct, Activation Assay, Luciferase, Transfection, Activity Assay

Journal: Central-European Journal of Immunology

Article Title: E2F2 stimulates CCR4 expression and activates synovial fibroblast-like cells in rheumatoid arthritis

doi: 10.5114/ceji.2021.105243

Figure Lengend Snippet: PCR primer sequences

Article Snippet: When cell confluence reached 70% in each well, anti-E2F2 siRNA or Allstars siRNA and HiPerFect transfection reagent (Qiagen, Germany) were added to serum-free culture medium.

Techniques:

Journal: Central-European Journal of Immunology

Article Title: E2F2 stimulates CCR4 expression and activates synovial fibroblast-like cells in rheumatoid arthritis

doi: 10.5114/ceji.2021.105243

Figure Lengend Snippet: E2F2 expression level in RASF with transfection of anti-E2F2 siRNA. A ) E2F2 mRNA expression level in RASF was detected using real-time PCR. B ) E2F2 protein expression was detected in RASF using Western blot analysis. C ) E2F2 protein expression was normalized to GAPDH expression. *** p < 0.001

Article Snippet: When cell confluence reached 70% in each well, anti-E2F2 siRNA or Allstars siRNA and HiPerFect transfection reagent (Qiagen, Germany) were added to serum-free culture medium.

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

Journal: Central-European Journal of Immunology

Article Title: E2F2 stimulates CCR4 expression and activates synovial fibroblast-like cells in rheumatoid arthritis

doi: 10.5114/ceji.2021.105243

Figure Lengend Snippet: PCR array analysis of the gene expression profile in RASF transfected with anti-E2F2 siRNA. A ) Detection of relative gene expression levels in RASF using a human inflammatory PCR array. B ) The PCR array result is depicted in one map

Article Snippet: When cell confluence reached 70% in each well, anti-E2F2 siRNA or Allstars siRNA and HiPerFect transfection reagent (Qiagen, Germany) were added to serum-free culture medium.

Techniques: Expressing, Transfection

Journal: Central-European Journal of Immunology

Article Title: E2F2 stimulates CCR4 expression and activates synovial fibroblast-like cells in rheumatoid arthritis

doi: 10.5114/ceji.2021.105243

Figure Lengend Snippet: mRNA expression of the screened genes by PCR array analysis in RASF. A ) C3AR1, B ) IL1RN, C ) IFNG, D ) TLR7, E ) CCR4 mRNA expression level in RASF with transfection of anti-E2F2 siRNA or Allstars siRNA. F ) CCR4 mRNA expression level in RASF with transfection of E2F2-expressing plasmids or the blank plasmids (mock). * p < 0.05, ns – no statistical significance

Article Snippet: When cell confluence reached 70% in each well, anti-E2F2 siRNA or Allstars siRNA and HiPerFect transfection reagent (Qiagen, Germany) were added to serum-free culture medium.

Techniques: Expressing, Transfection

Journal: Central-European Journal of Immunology

Article Title: E2F2 stimulates CCR4 expression and activates synovial fibroblast-like cells in rheumatoid arthritis

doi: 10.5114/ceji.2021.105243

Figure Lengend Snippet: E2F2 expression level in RASF with transfection of E2F2-expressing plasmids or blank plasmids (mock). A ) E2F2 mRNA expression level in RASF was detected using real-time PCR. B ) E2F2 protein expression was detected in RASF using Western blot analysis. C ) E2F2 protein expression was normalized to GAPDH expression. ** p < 0.01, *** p < 0.001

Article Snippet: When cell confluence reached 70% in each well, anti-E2F2 siRNA or Allstars siRNA and HiPerFect transfection reagent (Qiagen, Germany) were added to serum-free culture medium.

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

Journal: Central-European Journal of Immunology

Article Title: E2F2 stimulates CCR4 expression and activates synovial fibroblast-like cells in rheumatoid arthritis

doi: 10.5114/ceji.2021.105243

Figure Lengend Snippet: CCR4 protein expression in RASF using Western blot analysis. A ) CCR4 protein expression in RASF transfected with anti-E2F2 siRNA. B ) CCR4 expression was normalized to GAPDH expression in the siRNA-transfected RASF. C ) CCR4 expression in RASF transfected with E2F2-expressing plasmids. D ) CCR4 expression was normalized to GAPDH expression in the E2F2-expressing RASF. * p < 0.05

Article Snippet: When cell confluence reached 70% in each well, anti-E2F2 siRNA or Allstars siRNA and HiPerFect transfection reagent (Qiagen, Germany) were added to serum-free culture medium.

Techniques: Expressing, Western Blot, Transfection

Journal: Central-European Journal of Immunology

Article Title: E2F2 stimulates CCR4 expression and activates synovial fibroblast-like cells in rheumatoid arthritis

doi: 10.5114/ceji.2021.105243

Figure Lengend Snippet: The effect of E2F2 expression on apoptosis of RASF using flow cytometry assay. A ) Apoptosis of RASF with transfection of anti-E2F2 siRNA or Allstars siRNA was measured, and the result is depicted in one map. B ) Apoptosis of RASF with transfection of E2F2 expressing plasmids or blank plasmids (mock) was measured, and the result is depicted in one map. * p < 0.05

Article Snippet: When cell confluence reached 70% in each well, anti-E2F2 siRNA or Allstars siRNA and HiPerFect transfection reagent (Qiagen, Germany) were added to serum-free culture medium.

Techniques: Expressing, Flow Cytometry, Transfection

Journal: Central-European Journal of Immunology

Article Title: E2F2 stimulates CCR4 expression and activates synovial fibroblast-like cells in rheumatoid arthritis

doi: 10.5114/ceji.2021.105243

Figure Lengend Snippet: The effect of E2F2 expression on migration ability of RASF using transwell assays. A ) The cell migration of RASF with transfection of anti-E2F2 siRNA or Allstars siRNA was measured, and the result is depicted in one map. B ) The cell migration of RASF with transfection of the E2F2-expressing plasmids or the blank plasmids (mock) was measured, and the result is depicted in one map. * p < 0.05, ** p < 0.01

Article Snippet: When cell confluence reached 70% in each well, anti-E2F2 siRNA or Allstars siRNA and HiPerFect transfection reagent (Qiagen, Germany) were added to serum-free culture medium.

Techniques: Expressing, Migration, Transfection

Journal: Central-European Journal of Immunology

Article Title: E2F2 stimulates CCR4 expression and activates synovial fibroblast-like cells in rheumatoid arthritis

doi: 10.5114/ceji.2021.105243

Figure Lengend Snippet: The effect of E2F2 expression on tube-like structure formation ability in RASF using Matrigel culture assay. A ) Tube-like structure formation of RASF with transfection of anti-E2F2 siRNA or Allstars siRNA was measured, and the result is depicted in one map. B ) Tube-like structure formation of RASF with transfection of the E2F2-expressing plasmids or the blank plasmids (mock) was measured, and the result is depicted in one map. * p < 0.05, ** p < 0.01

Article Snippet: When cell confluence reached 70% in each well, anti-E2F2 siRNA or Allstars siRNA and HiPerFect transfection reagent (Qiagen, Germany) were added to serum-free culture medium.

Techniques: Expressing, Transfection

Journal: Arthritis Research & Therapy

Article Title: E2F2 directly regulates the STAT1 and PI3K/AKT/NF-κB pathways to exacerbate the inflammatory phenotype in rheumatoid arthritis synovial fibroblasts and mouse embryonic fibroblasts

doi: 10.1186/s13075-018-1713-x

Figure Lengend Snippet: Primers used for real-time polymerase chain reaction

Article Snippet: RASFs (2 × 10 5 cells in 100-mm diameter dishes or 8 × 10 4 cells in six-well plates) were transiently transfected with siRNA targeting E2F2 (SI00375410, Qiagen, Hilden, Germany) or negative control siRNA (1,027,281, Qiagen) using HiPerFect transfection reagent (Qiagen) following the manufacturer’s instructions, and all experiments were performed 24 h after transfection.

Techniques: Sequencing

Journal: Arthritis Research & Therapy

Article Title: E2F2 directly regulates the STAT1 and PI3K/AKT/NF-κB pathways to exacerbate the inflammatory phenotype in rheumatoid arthritis synovial fibroblasts and mouse embryonic fibroblasts

doi: 10.1186/s13075-018-1713-x

Figure Lengend Snippet: E2F2 affects the incidence and degree of CIA in mice. Arthritis was induced in wild-type (WT), E2f2 +/− , and E2f2 −/− mice and the incidence ( a ) and clinical pathology ( b ) of arthritis were evaluated as 0 (no swelling) to 4 (strong swelling) once per day by two independent observers under blinded conditions. All results are presented as the mean ± SEM of three independent experiments performed in triplicate. *** P < 0.001, versus the control. The severity of edema and paw redness was assessed once per day. c The degree of paw thickness was detected both in the fore paws and hind paws. d Hematoxylin and eosin-stained stained slides were scored blind by a trained observer for immune cell invasion. e The depletion of proteoglycans was determined by Safranin O-Fast Green FCF and Toluidine blue staining (100×). The regions of cartilage degeneration, which are light in staining, are marked by arrows

Article Snippet: RASFs (2 × 10 5 cells in 100-mm diameter dishes or 8 × 10 4 cells in six-well plates) were transiently transfected with siRNA targeting E2F2 (SI00375410, Qiagen, Hilden, Germany) or negative control siRNA (1,027,281, Qiagen) using HiPerFect transfection reagent (Qiagen) following the manufacturer’s instructions, and all experiments were performed 24 h after transfection.

Techniques: Control, Staining

Journal: Arthritis Research & Therapy

Article Title: E2F2 directly regulates the STAT1 and PI3K/AKT/NF-κB pathways to exacerbate the inflammatory phenotype in rheumatoid arthritis synovial fibroblasts and mouse embryonic fibroblasts

doi: 10.1186/s13075-018-1713-x

Figure Lengend Snippet: E2F2 is required for production of inflammatory factors in MEFs. MEFs were treated with different concentrations of lipopolysaccharide (LPS) for 12 h. Expression and dose-dependent effects of interleukin (IL)-1α ( a ), IL-1β ( b ), and tumor necrosis factor (TNF)-α ( c ) were detected by qRT-PCR. ** P < 0.01, *** P < 0.001, versus vehicle control. Dose-dependent effects of secreted IL-1α ( d ), IL-1β ( e ), and TNF-α ( f ) were detected by ELISA. ** P < 0.01, *** P < 0.001, versus vehicle control. IL-1α, IL-1β, and TNF-α in serum of E2f2 −/− and wild-type (WT) mice were measured by ELISA ( g ). ** P < 0.01, *** P < 0.001, versus WT

Article Snippet: RASFs (2 × 10 5 cells in 100-mm diameter dishes or 8 × 10 4 cells in six-well plates) were transiently transfected with siRNA targeting E2F2 (SI00375410, Qiagen, Hilden, Germany) or negative control siRNA (1,027,281, Qiagen) using HiPerFect transfection reagent (Qiagen) following the manufacturer’s instructions, and all experiments were performed 24 h after transfection.

Techniques: Expressing, Quantitative RT-PCR, Control, Enzyme-linked Immunosorbent Assay

Journal: Arthritis Research & Therapy

Article Title: E2F2 directly regulates the STAT1 and PI3K/AKT/NF-κB pathways to exacerbate the inflammatory phenotype in rheumatoid arthritis synovial fibroblasts and mouse embryonic fibroblasts

doi: 10.1186/s13075-018-1713-x

Figure Lengend Snippet: E2F2 participates in RA inflammation through STAT1 and PI3K/AKT/NF-κB pathways. RNA-seq was performed to screen target genes downstream of E2F2 in RASFs. a , b Heat maps indicate the most differentially expressed genes in RASFs with E2F2 knocked-down. Colored bands represent the change in gene expression: red, downregulation; blue, upregulation. c – e In-vitro verification of genes related to inflammation in RA was performed using qRT-PCR. mRNA levels of STAT1 ( c ), AKT1 ( d ), and AKT2 ( e ). f Western blot was performed to test inhibitory effects of siE2F2 on expression of E2F2, STAT1, AKT1, AKT2, p-AKT, and the p65 subunit of NF-κB. All results are presented as the mean ± SEM of three independent experiments performed in triplicate. NC knockdown scramble control, si small interfering

Article Snippet: RASFs (2 × 10 5 cells in 100-mm diameter dishes or 8 × 10 4 cells in six-well plates) were transiently transfected with siRNA targeting E2F2 (SI00375410, Qiagen, Hilden, Germany) or negative control siRNA (1,027,281, Qiagen) using HiPerFect transfection reagent (Qiagen) following the manufacturer’s instructions, and all experiments were performed 24 h after transfection.

Techniques: RNA Sequencing, Gene Expression, In Vitro, Quantitative RT-PCR, Western Blot, Expressing, Knockdown, Control

Journal: Arthritis Research & Therapy

Article Title: E2F2 directly regulates the STAT1 and PI3K/AKT/NF-κB pathways to exacerbate the inflammatory phenotype in rheumatoid arthritis synovial fibroblasts and mouse embryonic fibroblasts

doi: 10.1186/s13075-018-1713-x

Figure Lengend Snippet: STAT1 mediates E2F2 regulation of interleukin (IL)-1α, IL-1β, and tumor necrosis factor (TNF)-α expression. a – d Effect of E2F2 on STAT1. E2F2 was overexpressed by adenovirus infection or inhibited by small interfering RNA (siRNA) with or without lipopolysaccharide (LPS; 10 μg/mL). qRT-PCR ( a , b ) and Western blot ( c , d ) were performed to detect expression of STAT1. e Schematic representation of STAT1 promoters, primers for the ChIP assay, and the E2F2 binding motif in the STAT1 promoter. ChIP ( f ) and luciferase (Luc) reporter assays ( g ) were performed to show that E2F2 was recruited to the STAT1 gene promoter in RASFs in the presence of LPS. Nuclear and cytoplasmic proteins were fractionally extracted from E2F2 knocked-down RASFs ( h ) and E2f2 −/− MEFs ( i ). Effects of E2F2 on nuclear translocation of STAT1 were determined by Western blot. (Lamin A/C as a reference for nuclear extraction (N); Tubulin as a reference for cytoplasmic extraction (C).) Effect of E2F2 on nuclear translocation of STAT1 was observed using confocal fluorescence microscopy both in E2F2-silenced RASFs ( j ) and E2f2 −/− MEFs ( k ). STAT1 (green) was detected using anti-STAT1 antibody. Nuclei were stained with DAPI (blue). l In E2F2-overexpressing RASFs, IL-1α, IL-1β, and TNF-α were analyzed by qRT-PCR after silencing STAT1 in the presence of LPS stimulation (10 μg/mL). m In STAT1-overexpressing RASFs, IL-1α, IL-1β, and TNF-α were analyzed by qRT-PCR after silencing E2F2 in the presence of LPS stimulation (10 μg/mL). n In E2f2 −/− MEFs, expression of IL-1α, IL-1β, and TNF-α was detected using qRT-PCR after STAT1 overexpression in the presence of LPS stimulation (10 μg/mL). The results shown are means ± SEM of three independent experiments performed in triplicate. * P < 0.05, ** P < 0.01, *** P < 0.001, versus the control. Ad-GFP adenovirus encoding green fluorescent protein, NC knockdown scramble control, ns not significant, siE2F2 small interfering RNA knockdown of E2F2, WT wild-type

Article Snippet: RASFs (2 × 10 5 cells in 100-mm diameter dishes or 8 × 10 4 cells in six-well plates) were transiently transfected with siRNA targeting E2F2 (SI00375410, Qiagen, Hilden, Germany) or negative control siRNA (1,027,281, Qiagen) using HiPerFect transfection reagent (Qiagen) following the manufacturer’s instructions, and all experiments were performed 24 h after transfection.

Techniques: Expressing, Infection, Small Interfering RNA, Quantitative RT-PCR, Western Blot, Binding Assay, Luciferase, Translocation Assay, Extraction, Fluorescence, Microscopy, Staining, Over Expression, Control, Knockdown

Journal: Arthritis Research & Therapy

Article Title: E2F2 directly regulates the STAT1 and PI3K/AKT/NF-κB pathways to exacerbate the inflammatory phenotype in rheumatoid arthritis synovial fibroblasts and mouse embryonic fibroblasts

doi: 10.1186/s13075-018-1713-x

Figure Lengend Snippet: MyD88 mediates regulation of the PI3K/AKT/NF-κB pathway by E2F2. a , b E2F2 can regulate expression of PI3K/AKT/NF-κB. E2F2-silenced RASFs and E2f2 −/− MEFs were cultured with or without lipopolysaccharide (LPS). Western blot was performed to detect phosphorylation of AKT and NF-κB P65 both in E2F2-silenced RASFs ( a ) and E2f2 −/− MEFs ( b ). c , d Effect of E2F2 on translocation of p65. E2F2 knocked-down RASFs ( c ) and E2f2 −/− MEFs ( d ) were cultured under LPS stimulation (10 μg/mL) for 12 h; nuclear and cytoplasmic proteins were extracted separately and then Western blot was performed (Lamin A/C as a reference for nuclear extraction (N); Tubulin as a reference for cytoplasmic extraction (C).) e , f Effects of E2F2 on p65 nuclear translocation both in RASFs (Fig. ) and MEFs (Fig. ) observed using confocal fluorescence microscopy. g – j E2F2-silenced RASFs and E2f2 −/− MEFs were cultured with or without LPS. qRT-PCR ( g , h ) and Western blot ( i , j ) were performed to detect the effect of E2F2 on MyD88. k Schematic representation of the MyD88 promoter, primers for the ChIP assay, and the E2F2 binding motif in the MyD88 promoter. l , m E2F2 was recruited to the MyD88 gene promoter in RASFs in the presence of LPS. ChIP ( l ) and luciferase (Luc) reporter assay ( m ) were performed in RASFs and MEFs in the presence or absence of LPS (10 μg/mL). n MyD88 mediated the effect of E2F2 on PI3K/AKT/NF-κB pathways. Western blot showed that knockdown of MyD88 could significantly inhibit the phosphorylation of AKT and P65 in the presence or absence of E2F2 overexpression. qRT-PCR showed that inhibition of MyD88 can inhibit the expression of inflammatory factors and significantly reduce the upregulation of interleukin (IL)-1α ( o ), IL-1β ( p ), and tumor necrosis factor (TNF)-α ( q ) by E2F2. The effect of inhibitors of PI3K/AKT/NF-κB pathways (LY294002 and PDTC) on E2F2; qRT-PCR was used to detect the effect of inhibitors on expression of IL-1α ( r , u ), IL-1β ( s , v ), and TNF-α ( t , w ) in response to LPS. The results shown are means ± SEM of three independent experiments performed in triplicate. ** P < 0.01, *** P < 0.001, versus the control. Ad-GFP adenovirus encoding green fluorescent protein, NC knockdown scramble control, si small interfering, WT wild-type

Article Snippet: RASFs (2 × 10 5 cells in 100-mm diameter dishes or 8 × 10 4 cells in six-well plates) were transiently transfected with siRNA targeting E2F2 (SI00375410, Qiagen, Hilden, Germany) or negative control siRNA (1,027,281, Qiagen) using HiPerFect transfection reagent (Qiagen) following the manufacturer’s instructions, and all experiments were performed 24 h after transfection.

Techniques: Expressing, Cell Culture, Western Blot, Phospho-proteomics, Translocation Assay, Extraction, Fluorescence, Microscopy, Quantitative RT-PCR, Binding Assay, Luciferase, Reporter Assay, Knockdown, Over Expression, Inhibition, Control

Journal: Arthritis Research & Therapy

Article Title: E2F2 directly regulates the STAT1 and PI3K/AKT/NF-κB pathways to exacerbate the inflammatory phenotype in rheumatoid arthritis synovial fibroblasts and mouse embryonic fibroblasts

doi: 10.1186/s13075-018-1713-x

Figure Lengend Snippet: STAT1/MyD88 complexes mediate E2F2 regulation of inflammatory cytokines. a – d STAT1/MyD88 complex is found in RASFs and MEFs. E2F2-silenced RASFs and E2f2 −/− MEFs were cultured with or without lipopolysaccharide (LPS; 10 μg/mL). Co-IP ( a , b ) was performed to test the binding. Confocal immunofluorescence ( c , d ) was performed to confirm the result (magnification 10 × 40, MyD88 (green) and STAT1 (red)). e – g Effect of STAT1/MyD88 complexes on the expression of cytokines. qRT-PCR was performed to detect expression of interleukin (IL)-1α ( e ), IL-1β ( f ), and tumor necrosis factor (TNF)-α ( g ) in STAT1/MyD88 knockdown RASFs with or without E2F2 overexpression in the presence of LPS (10 μg/mL). The results shown are means ± SEM of three independent experiments performed in triplicate. ** P < 0.01, *** P < 0.001, versus the control. h Model depicting the role of E2F2 in RA pathogenesis. Ad-GFP adenovirus encoding green fluorescent protein, NC knockdown scramble control, si small interfering, WT wild-type

Article Snippet: RASFs (2 × 10 5 cells in 100-mm diameter dishes or 8 × 10 4 cells in six-well plates) were transiently transfected with siRNA targeting E2F2 (SI00375410, Qiagen, Hilden, Germany) or negative control siRNA (1,027,281, Qiagen) using HiPerFect transfection reagent (Qiagen) following the manufacturer’s instructions, and all experiments were performed 24 h after transfection.

Techniques: Cell Culture, Co-Immunoprecipitation Assay, Binding Assay, Immunofluorescence, Expressing, Quantitative RT-PCR, Knockdown, Over Expression, Control

Journal: Oncotarget

Article Title: Downregulated miR-31 level associates with poor prognosis of gastric cancer and its restoration suppresses tumor cell malignant phenotypes by inhibiting E2F2

doi: 10.18632/oncotarget.9288

Figure Lengend Snippet: ( A ) qRT-PCR. Level of E2F2 mRNA after miR-31 negative control or miR-31 mimic transfection into gastric cancer cells. ( B , C ) Western blot. Level of endogenous E2F2 protein after miRNA negative control or miR-31 mimic transfection into gastric cancer cells. The relative protein level was normalized to GAPDH and data were presented as mean ± sd. ( D ) Sequence alignment. miR-31 sequences and its predicted binding site in 3′-UTR of E2F2. Plasmids contain wild-type or mutant sequences. ( E ) Luciferase reporter assay. The vector containing wide-type E2F2 3′UTR or mutant E2F2 3′-UTR was co-transfected into SGC-7901 and MGC-803 cells together with miRNA negative control or miR-31 mimic transfection. Luciferase activity ratio was presented as firefly luciferase value/renilla luciferase value and then normalized to that of the empty plasmid. Each column represented mean ± sd.

Article Snippet: miR-31 mimic, miRNA mimic control, siRNA against E2F2 and siRNA control were synthesized by Ribobio (Guangzhou, China) and transfected into gastric cancer cell lines using Lipofectamine RNAiMAX (Invitrogen) according to the manufacturer's instructions.

Techniques: Quantitative RT-PCR, Negative Control, Transfection, Western Blot, Sequencing, Binding Assay, Mutagenesis, Luciferase, Reporter Assay, Plasmid Preparation, Activity Assay

Journal: Oncotarget

Article Title: Downregulated miR-31 level associates with poor prognosis of gastric cancer and its restoration suppresses tumor cell malignant phenotypes by inhibiting E2F2

doi: 10.18632/oncotarget.9288

Figure Lengend Snippet: ( A , B ) qRT-PCR analysis of E2F2 expression in 40 pairs of gastric cancer and corresponding normal tissues. Expression of E2F2 protein was normalized to that of β-actin. The level of E2F2mRNA was significantly higher than that of the adjacent tissues. ( C ) Western blot. Level of E2F2 protein was normalized to GAPDH. ( D ) Immunohistochemistry. E2F2 protein expression in gastric cancer tissues was analyzed by immunohistochemistry. a, normal gastric tissue; b, moderately differentiated gastric adenocarcinoma; c, poor differentiated gastric adenocarcinoma; d, superficial muscular infiltration; e, serosal layer infiltration; f, whole layer infiltration (all ×100). ( E ) Expression of E2F2 protein in different differentiation status of gastric cancer. (poorly differentiated = 22, moderately differentiated = 18). ( F ) Expression of E2F2 in different T stage (depth of cancer invasion) of gastric cancer, including T1–2 (mucous and muscular layer) 6 cases, T3 (serosal layer) 27 cases and T4 (whole layer) 7 cases. ( G ) Expression of E2F2 in different N stage (lymph node metastases) of gastric cancer (N0 = 4, N1 = 10, N2 = 9, N4 = 17). ( H ) Kaplan-Meier curve of overall survival of gastric cancer patients with high ( n = 29) vs. low ( n = 11) E2F2 levels ( p = 0.047). ( I ) Comparison of miR-31 with E2F2 level in gastric cancer ( r = 0.122, p = 0.027).

Article Snippet: miR-31 mimic, miRNA mimic control, siRNA against E2F2 and siRNA control were synthesized by Ribobio (Guangzhou, China) and transfected into gastric cancer cell lines using Lipofectamine RNAiMAX (Invitrogen) according to the manufacturer's instructions.

Techniques: Quantitative RT-PCR, Expressing, Western Blot, Immunohistochemistry, Comparison

Journal: Oncotarget

Article Title: Downregulated miR-31 level associates with poor prognosis of gastric cancer and its restoration suppresses tumor cell malignant phenotypes by inhibiting E2F2

doi: 10.18632/oncotarget.9288

Figure Lengend Snippet: Tumor cells were transiently transfected with negative control siRNA orE2F2 siRNA and then subjected to different assays. ( A ) RT-PCR. Level of E2F2 mRNA was assessed by RT-PCR after transfection with negative control or E2F2 siRNA into gastric cancer cells. ( B ) Western blot. Level of E2F2 protein was assessed after transfection with negative control or E2F2 siRNA into gastric cancer cells. The relative protein levels were normalized to GAPDH and data were presented as mean ± sd. ( C ) Cell viability CCK8 assay. Cell viability was assessed after 24, 48, 72 and 96 h transfection with negative control or E2F2 siRNA into gastric cancer cells. Data were presented as mean ± sd of three independent experiments. * p < 0.05 and ** p < 0.01. ( D ) Apoptosis assay. Level of apoptosis was assessed after 48 h transfection with negative control or E2F2 siRNA into gastric cancer cells. Data were presented as mean ± sd of three independent experiments with duplicated samples.* p < 0.05 and ** p < 0.01. ( E , F ) Flow cytometric cell cycle assay. Cell cycle distribution was analyzed additional culture of 12 and 24 h after 48 h transfection with negative control or E2F2 siRNA into gastric cancer cells. Experiments were repeated at least three times with similar results and the data were expressed as mean ± sd. * p < 0.05 and ** p < 0.01. ( G ) Tumor cell migration assay. Fluorescent images (×200) (left) and quantification (right) of migration level of SGC-7901 and MGC-803 cells after 24 h transfection with negative control or E2F2 siRNA. ( H ) Tumor cell invasion assay. Fluorescent images (left) and quantification (right) of invasion level of SGC-7901 and MGC-803 cells after 24 h transfection with negative control or E2F2 siRNA.

Article Snippet: miR-31 mimic, miRNA mimic control, siRNA against E2F2 and siRNA control were synthesized by Ribobio (Guangzhou, China) and transfected into gastric cancer cell lines using Lipofectamine RNAiMAX (Invitrogen) according to the manufacturer's instructions.

Techniques: Transfection, Negative Control, Reverse Transcription Polymerase Chain Reaction, Western Blot, CCK-8 Assay, Apoptosis Assay, Cell Cycle Assay, Cell Migration Assay, Migration, Invasion Assay

Journal: Oncotarget

Article Title: Downregulated miR-31 level associates with poor prognosis of gastric cancer and its restoration suppresses tumor cell malignant phenotypes by inhibiting E2F2

doi: 10.18632/oncotarget.9288

Figure Lengend Snippet: Correlations between miR-31 and E2F2 expressions and clinical characteristics in patients with gastric cancer

Article Snippet: miR-31 mimic, miRNA mimic control, siRNA against E2F2 and siRNA control were synthesized by Ribobio (Guangzhou, China) and transfected into gastric cancer cell lines using Lipofectamine RNAiMAX (Invitrogen) according to the manufacturer's instructions.

Techniques: Expressing

Journal: Oncotarget

Article Title: miR-155 regulates the proliferation and invasion of clear cell renal cell carcinoma cells by targeting E2F2

doi: 10.18632/oncotarget.7951

Figure Lengend Snippet: ( A ) miR-155 expression levels were significantly upregulated in ccRCC cancer tissues compared with normal tissues. ( B – D ) Correlations between miR-155 expression level and the clinical stage, T stage, Fuhrman grades of ccRCC respectively. ( E ) miR-155 expressions in various RCC cell lines compared with that in HKC. ( F ) Downregulation of E2F2 mRNA levels in ccRCC cancer tissues; here, normal tissues served as controls. ( G ) Correlations between E2F2 expression level and the clinical stage of ccRCC. ( H ) Negative correlation of E2F2 mRNA levels and miR-155 levels ( n = 54, r 2 = 0.4121, P < 0.0001). ( I ) Representative images of E2F2 IHC in ccRCC cancer tissues and their paired normal tissues. ( J ) Western blot of E2F2 showed alterations in protein levels consistent with variations in mRNA levels in clinical samples. Data represent the mean ± SD. (* P < 0.05; ** P < 0.01; *** P < 0.001).

Article Snippet: Short interfering RNAs (siRNAs) against E2F2 (siE2F2) and the negative control (siNC) with non-functioning sequences were also synthesized by Genepharma.

Techniques: Expressing, Western Blot

Journal: Oncotarget

Article Title: miR-155 regulates the proliferation and invasion of clear cell renal cell carcinoma cells by targeting E2F2

doi: 10.18632/oncotarget.7951

Figure Lengend Snippet: ( A ) Alteration of E2F2 expression levels in 786-O cells 48 h after siRNA treatment compared with siNC. ( B ) Alteration of E2F2 expression levels in ACHN cells 48 h after transfection with lentiviral E2F2 plasmids compared with the empty vector. ( C ) MTS assay revealed that transfection of the siRNA of E2F2 significantly accelerated proliferation velocity in 786-O cells while E2F2 overexpression attenuated proliferation in ACHN cells. ( D ) Effect of E2F2 overexpression or suppression on the colony formation of ACHN and 786-O cells, respectively. The number of foci of > 50 cells was counted after 14 d. ( E ) Representative photographs of transwell assays (magnification, ×100) of 786-O and ACHN cells identifying E2F2 as a tumor suppressor. ( F ) E2F2 knockdown in 786-O cells significantly increased the number of viable cells; in ACHN cells, E2F2 overexpression largely decreased cell viability. Data represent the mean ± SD. Each experiment was performed in triplicate. (* P < 0.05; ** P < 0.01; *** P < 0.001).

Article Snippet: Short interfering RNAs (siRNAs) against E2F2 (siE2F2) and the negative control (siNC) with non-functioning sequences were also synthesized by Genepharma.

Techniques: Expressing, Transfection, Plasmid Preparation, MTS Assay, Over Expression, Knockdown

Journal: Oncotarget

Article Title: miR-155 regulates the proliferation and invasion of clear cell renal cell carcinoma cells by targeting E2F2

doi: 10.18632/oncotarget.7951

Figure Lengend Snippet: ( A ) Sequence alignment of the E2F2 3′UTR with wild-type (WT) versus mutant (MUT) potential miR-155 targeting sites. ( B ) mRNA levels of E2F2 were examined by qRT-PCR in 786-O and ACHN cells with different interferences. ( C ) Transfection of the miR-155 mimic or miR-155 inhibitor in 786-O and ACHN greatly changed E2F2 protein levels. ( D ) Immunofluorescence staining results showed the inverse effect of miR-155 on E2F2 in different cell lines. ( E ) Luciferase reporter assay showed decreased reporter activity after transfection of the wild-type E2F2 3′UTR reporter construct in 293T cells overexpressing miR-155. The E2F2 3′UTR MUT and control constructs showed no effect on reporter activity. Here, the Renilla luciferase construct was used as an internal control. The normalized luciferase activity of the control construct in each experiment was set to 1. Data represent the mean ± SD. (* P < 0.05; ** P < 0.01; *** P < 0.001).

Article Snippet: Short interfering RNAs (siRNAs) against E2F2 (siE2F2) and the negative control (siNC) with non-functioning sequences were also synthesized by Genepharma.

Techniques: Sequencing, Mutagenesis, Quantitative RT-PCR, Transfection, Immunofluorescence, Staining, Luciferase, Reporter Assay, Activity Assay, Construct, Control

Journal: Oncotarget

Article Title: miR-155 regulates the proliferation and invasion of clear cell renal cell carcinoma cells by targeting E2F2

doi: 10.18632/oncotarget.7951

Figure Lengend Snippet: ( A ) miR-155 and E2F2 mRNA level changes in 786-O cells with the miR-155 mimic (versus the control) and the E2F2 plasmid (versus the control) 48 h after transfection. ( B ) The mRNA levels of miR-155 and E2F2 in ACHN were analyzed by qRT-PCR after the use of miR-155 inhibitor and siE2F2. ( C ) E2F2 protein level alterations in 786-O cells with the miR-155 mimic (versus the control) and the E2F2 plasmid (versus the control) 48 h after transfection. After co-transfection of ACHN cells with siRNA duplexes (siE2F2 or the negative control) and miR-155 inhibitors (miR-155 or the negative control), the levels of E2F2 protein and EMT-related proteins were measured by Western blot. ( D ) E2F2 overexpression counteracted the positive proliferative effects of miR-155 in 786-O cells. ( E ) E2F2 inhibition hampered the negative proliferative function of miR-155 inhibitor in ACHN cells. ( F ) Cell migration and invasion in 786-O cells were analyzed by transwell assays. Cells were co-transfected with lentiviral particles (E2F2 or the empty vector) and miR-155 (mimic or the NC mimic). ( G ) Knockdown of E2F2 may reverse the effect of the miR-155 inhibitor on migration and invasion in ACHN cells. Data represent the mean ± SD. (* P < 0.05; ** P < 0.01; *** P < 0.001).

Article Snippet: Short interfering RNAs (siRNAs) against E2F2 (siE2F2) and the negative control (siNC) with non-functioning sequences were also synthesized by Genepharma.

Techniques: Control, Plasmid Preparation, Transfection, Quantitative RT-PCR, Cotransfection, Negative Control, Western Blot, Over Expression, Inhibition, Migration, Knockdown

Journal: Oncotarget

Article Title: miR-155 regulates the proliferation and invasion of clear cell renal cell carcinoma cells by targeting E2F2

doi: 10.18632/oncotarget.7951

Figure Lengend Snippet: ( A ) Mouse tumors were obtained and dissected 4 weeks after subcutaneous injection of the transfected 786-O/miR-155 plasmid or 786-O/EV transfected cells. ( B , C ) Comparison of tumor weights and volumes between the 786-O/miR-155 and 786-O/EV groups. ( D ) Protein levels of E2F2 were immunohistochemically evaluated in the experimental and control groups. ( E ) EMT-related markers, including E-cadherin, ZEB1, and vimentin, were detected by Western blot between the groups. Data represent the mean ± SD. (* P < 0.05; ** P < 0.01; *** P < 0.001).

Article Snippet: Short interfering RNAs (siRNAs) against E2F2 (siE2F2) and the negative control (siNC) with non-functioning sequences were also synthesized by Genepharma.

Techniques: Injection, Transfection, Plasmid Preparation, Comparison, Control, Western Blot