Journal: International journal of oncology

Article Title: HBV triggers APOBEC2 expression through miR‑122 regulation and affects the proliferation of liver cancer cells.

doi: 10.3892/ijo.2019.4870

Figure Lengend Snippet: Figure 1. APOBEC2 expression in liver cancer cells following HBV infection. Huh7 and HepG2 cells at 70% confluence were transfected with pGEMHBV or pEGFP‑C1 for 24 h (mRNA analysis) or 48 h (protein analysis). Expression of APOBEC2 (A) mRNA and (B) protein expression in Huh7 cells. Expression of APOBEC2 (C) mRNA and (D) protein expression in HepG2 cells. pEGFP‑C1 was used as a negative control for pGEMHBV. β‑actin served as an internal control. Data are presented as the mean ± standard error of the mean (n=3). *P<0.05, **P<0.01, ***P<0.001 vs. pEGFP‑C1. APOBEC2, apolipoprotein B mRNA‑editing enzyme catalytic subunit 2; EGFP, enhanced green fluorescent protein; HBV, hepatitis B virus.

Article Snippet: Polyclonal APOBEC2 (1:500; cat. no. 20121-1-AP; ProteinTech Group, Inc.) and cleaved-caspase-3 antibodies (1:500; cat. no. WL01857; Wanleibio Co., Ltd.), and monoclonal antibodies against β-actin (cat. no. TA-09; Beijing Zhongshan Golden Bridge Biotechnology Co., Ltd.; OriGene Technologies, Inc.) were used for immunoblotting overnight at 4 ̊C.

Techniques: Expressing, Infection, Transfection, Negative Control, Control, Virus

Journal: International journal of oncology

Article Title: HBV triggers APOBEC2 expression through miR‑122 regulation and affects the proliferation of liver cancer cells.

doi: 10.3892/ijo.2019.4870

Figure Lengend Snippet: Figure 2. Verification of the inhibition efficiency of shRNAs on APOBEC2 expression. Four shRNAs specific for APOBEC2 were designed and transfected into Huh7 cells, and total RNA and protein were extracted at 24 and 48 h post‑transfection, respectively. Then, reverse transcription‑quantitative PCR and western blot analyses were conducted to detect the inhibition efficiency. Compared with shNC, shAPOBEC2‑4 effectively inhibited APOBEC2 (A) mRNA and (B) protein expression; APOBEC2 mRNA and protein expression decreased by ~70 and ~34%, respectively, following APOBEC2 knockdown. Therefore, shAPOBEC2‑4 was selected for further experimentation in the present study. shNC served as a control. Data are presented as the mean ± standard error of the mean (n=3). *P<0.05, **P<0.01. APOBEC2, apolipoprotein B mRNA‑editing enzyme catalytic subunit 2; NC, negative control; shRNAs, short hairpin RNA.

Article Snippet: Polyclonal APOBEC2 (1:500; cat. no. 20121-1-AP; ProteinTech Group, Inc.) and cleaved-caspase-3 antibodies (1:500; cat. no. WL01857; Wanleibio Co., Ltd.), and monoclonal antibodies against β-actin (cat. no. TA-09; Beijing Zhongshan Golden Bridge Biotechnology Co., Ltd.; OriGene Technologies, Inc.) were used for immunoblotting overnight at 4 ̊C.

Techniques: Inhibition, Expressing, Transfection, Western Blot, Knockdown, Control, Negative Control, shRNA

Journal: International journal of oncology

Article Title: HBV triggers APOBEC2 expression through miR‑122 regulation and affects the proliferation of liver cancer cells.

doi: 10.3892/ijo.2019.4870

Figure Lengend Snippet: Figure 3. Modulation of APOBEC2 expression affects liver cancer cell viability and apoptosis. Huh7 cells at 70% confluence were transfected with pAPOBEC2 or shAPOBEC2 for 48 h to increase or decrease cellular APOBEC2 expression, respectively. The effects of APOBEC2 on cells were determined via MTT assay and western blotting. (A) APOBEC2 overexpression enhanced Huh7 cell viability compared with pEGFP‑C1. (B) APOBEC2 suppression reduced Huh7 cell viability compared with shNC. (C) APOBEC2 overexpression increased cleaved‑caspase‑3 expression compared with pEGFP‑C1. (D) APOBEC2 suppres sion reduced cleaved‑caspase‑3 expression compared with shNC. pEGFP‑C1 and shNC were used as NCs for pAPOBEC2 and shAPOBEC2, respectively. β‑actin served as an internal control. Data are presented as the mean ± standard error of the mean (n=3). *P<0.05, **P<0.01. APOBEC2, apolipoprotein B mRNA‑editing enzyme catalytic subunit 2; EGFP, enhanced green fluorescent protein; NC, negative control; shRNAs, short hairpin RNA.

Article Snippet: Polyclonal APOBEC2 (1:500; cat. no. 20121-1-AP; ProteinTech Group, Inc.) and cleaved-caspase-3 antibodies (1:500; cat. no. WL01857; Wanleibio Co., Ltd.), and monoclonal antibodies against β-actin (cat. no. TA-09; Beijing Zhongshan Golden Bridge Biotechnology Co., Ltd.; OriGene Technologies, Inc.) were used for immunoblotting overnight at 4 ̊C.

Techniques: Expressing, Transfection, MTT Assay, Western Blot, Over Expression, Control, Negative Control, shRNA

Journal: International journal of oncology

Article Title: HBV triggers APOBEC2 expression through miR‑122 regulation and affects the proliferation of liver cancer cells.

doi: 10.3892/ijo.2019.4870

Figure Lengend Snippet: Figure 4. Expression of APOBEC2 and miR‑122 in different liver cell lines. Total RNA and protein were extracted from Huh7 and HepG2 cells, and respectively subjected to reverse transcription‑quantitative PCR and western blot analyses to evaluate miR‑122 and APOBEC2 expression in these cells. (A and B) APOBEC2 mRNA and protein expression. (C) miR‑122 expression. β‑actin served as an internal control. Data are presented as the mean ± stan dard error of the mean (n=3). *P<0.05, **P<0.01, ***P<0.001 vs. HepG2. APOBEC2, apolipoprotein B mRNA‑editing enzyme catalytic subunit 2; miR‑122, microRNA.

Article Snippet: Polyclonal APOBEC2 (1:500; cat. no. 20121-1-AP; ProteinTech Group, Inc.) and cleaved-caspase-3 antibodies (1:500; cat. no. WL01857; Wanleibio Co., Ltd.), and monoclonal antibodies against β-actin (cat. no. TA-09; Beijing Zhongshan Golden Bridge Biotechnology Co., Ltd.; OriGene Technologies, Inc.) were used for immunoblotting overnight at 4 ̊C.

Techniques: Expressing, Western Blot, Control

Journal: International journal of oncology

Article Title: HBV triggers APOBEC2 expression through miR‑122 regulation and affects the proliferation of liver cancer cells.

doi: 10.3892/ijo.2019.4870

Figure Lengend Snippet: Figure 5. Validation of miR‑122 targeting APOBEC2 mRNA. The expression levels of miR‑122 and APOBEC2 in Huh7 cells transfected with miR‑NC, miR‑122, AMO‑NC or AMO‑122 were determined. Analysis of (A and B) miR‑122 and APOBEC2 mRNA, and (C and D) APOBEC2 protein expression 24 and 48 h post‑transfection by reverse transcription‑quantitative PCR and western blotting, respectively. β‑actin served as an internal control. (E) Schematic diagram of potential binding sites for miR‑122 in the 3'UTR of APOBEC2 mRNA. The locations of putative miR‑122 binding sites in the 3'UTR of APOBEC2 mRNA are marked by arrows at 1,334 nt. (F) WT and MUT nucleotides in the putative target sequence for miR‑122 in the APOBEC2 mRNA. The MUT region indicated by the red line was generated by over‑lapping PCR to minimize complementarity between miR‑122 and the 3'UTR of APOBEC2 mRNA. (G) Relative luciferase activity of Huh7 cells transfected with miR‑122 mimics + APOBEC2‑WT or APOBEC2‑MUT. Huh7 cells were co‑transfected with miR‑122 mimics or miR‑NC and a luciferase reporter plasmid harboring WT or MUT miR binding sites. The effects of miR‑122 on luciferase activity were determined by luciferase reporter assays. The activity of Renilla luciferase was normalized to that of firefly luciferase. Data are presented as the mean ± standard error of the mean (n=3). *P<0.05, **P<0.01, ***P<0.001. APOBEC2, apolipoprotein B mRNA‑editing enzyme catalytic subunit 2; AMO, anti‑miR oligonucleotide; mfe, minimum free energy; miR, microRNA; MUT, mutated; NC, negative control; nt, nucleotide, the number counts from the first nucleotide at the 5' end of APOBEC2 mRNA; UTR, untranslated region; WT, wild type.

Article Snippet: Polyclonal APOBEC2 (1:500; cat. no. 20121-1-AP; ProteinTech Group, Inc.) and cleaved-caspase-3 antibodies (1:500; cat. no. WL01857; Wanleibio Co., Ltd.), and monoclonal antibodies against β-actin (cat. no. TA-09; Beijing Zhongshan Golden Bridge Biotechnology Co., Ltd.; OriGene Technologies, Inc.) were used for immunoblotting overnight at 4 ̊C.

Techniques: Biomarker Discovery, Expressing, Transfection, Western Blot, Control, Binding Assay, Sequencing, Generated, Luciferase, Activity Assay, Plasmid Preparation, Negative Control

Journal: International journal of oncology

Article Title: HBV triggers APOBEC2 expression through miR‑122 regulation and affects the proliferation of liver cancer cells.

doi: 10.3892/ijo.2019.4870

Figure Lengend Snippet: Figure 6. HBV and miR‑122 regulate APOBEC2 expression. Huh7 cells were respectively transfected with miR‑122, AMO‑122 or the corresponding NCs + pGEMHBV. Total RNA was extracted after transfection for 24 h, and the expression of (A) miR‑122 and (B) APOBEC2 was determined via reverse transcription‑quantitative PCR analysis. Data are presented as the mean ± standard error of the mean (n=3). *P<0.05, **P<0.01, ***P<0.001. APOBEC2, apoli poprotein B mRNA‑editing enzyme catalytic subunit 2; AMO, anti‑miR oligonucleotide; EGFP, enhanced green fluorescent protein; HBV, hepatitis B virus; miR, microRNA; NC, negative control.

Article Snippet: Polyclonal APOBEC2 (1:500; cat. no. 20121-1-AP; ProteinTech Group, Inc.) and cleaved-caspase-3 antibodies (1:500; cat. no. WL01857; Wanleibio Co., Ltd.), and monoclonal antibodies against β-actin (cat. no. TA-09; Beijing Zhongshan Golden Bridge Biotechnology Co., Ltd.; OriGene Technologies, Inc.) were used for immunoblotting overnight at 4 ̊C.

Techniques: Expressing, Transfection, Virus, Negative Control

Journal: International journal of oncology

Article Title: HBV triggers APOBEC2 expression through miR‑122 regulation and affects the proliferation of liver cancer cells.

doi: 10.3892/ijo.2019.4870

Figure Lengend Snippet: Figure 7. Network of the HBV‑miR‑122‑APOBEC2 signaling cascade and its involvement in liver cancer. The genes in the side boxes are miR‑122 targets that have been reported to be directly involved in cell prolifera tion, the suppression of cell apoptosis and/or the metastasis of liver cancer. APOBEC2, apolipoprotein B mRNA‑editing enzyme catalytic subunit 2; HBV, hepatitis B virus; miR, microRNA; SRF, serum response factor; igf1R, insulin‑like growth factor 1 receptor; NDRG3, N‑myc downstream‑regulated gene 3; G‑6PD, glucose‑6‑phosphate‑dehydrogenase; ADAM, a disintegrin and metalloprotease family.

Article Snippet: Polyclonal APOBEC2 (1:500; cat. no. 20121-1-AP; ProteinTech Group, Inc.) and cleaved-caspase-3 antibodies (1:500; cat. no. WL01857; Wanleibio Co., Ltd.), and monoclonal antibodies against β-actin (cat. no. TA-09; Beijing Zhongshan Golden Bridge Biotechnology Co., Ltd.; OriGene Technologies, Inc.) were used for immunoblotting overnight at 4 ̊C.

Techniques: Virus

Journal: International journal of oncology

Article Title: HBV triggers APOBEC2 expression through miR‑122 regulation and affects the proliferation of liver cancer cells.

doi: 10.3892/ijo.2019.4870

Figure Lengend Snippet: Figure 8. HBV and APOBEC2 modulate the expression of IL‑6, IKKε and TNF‑α. Huh7 cells at 70% confluence were transfected with pGEMHBV or pAPOBEC2 for 24 h. mRNA expression of (A) IL‑6, (B) IKKε and (C) TNF‑α in Huh7 cells treated with pGEMHBV or pAPOBEC2, as determined by reverse transcription‑quantitative polymerase chain reaction. pEGFP‑C1 served as a negative control for pGEMHBV and pAPOBEC2. Data are presented as the mean ± standard error of the mean (n=3). *P<0.05, **P<0.01 and ***P<0.001. APOBEC2, apolipoprotein B mRNA‑editing enzyme catalytic subunit 2; EGFP, enhanced green fluorescent protein; HBV, hepatitis B virus; IKK, IκB kinase; IL‑6, interleukin‑6; TNF‑α, tumor necrosis factor‑α.

Article Snippet: Polyclonal APOBEC2 (1:500; cat. no. 20121-1-AP; ProteinTech Group, Inc.) and cleaved-caspase-3 antibodies (1:500; cat. no. WL01857; Wanleibio Co., Ltd.), and monoclonal antibodies against β-actin (cat. no. TA-09; Beijing Zhongshan Golden Bridge Biotechnology Co., Ltd.; OriGene Technologies, Inc.) were used for immunoblotting overnight at 4 ̊C.

Techniques: Expressing, Transfection, Polymerase Chain Reaction, Negative Control, Virus

Journal: Scientific Reports

Article Title: Ovarian steroids modulate mRNA expression of ECM associated genes and collagen deposition induced by TGF β1 in equine endometrium in vitro

doi: 10.1038/s41598-024-84250-1

Figure Lengend Snippet: The effect of progesterone (P4; 10 − 7 M) or 17β-estradiol (E2; 10 − 9 M) on mRNA expression of collagen type 1 alpha chain 1 ( COL1A1) ( A ), collagen type 3 alpha chain 1 ( COL3A1) ( B ), matrix metalloproteases (MMP): MMP-1 ( C ), MMP-2 ( D ), MMP-3 ( E ), MMP-9 ( F ), and MMP-13 ( G ), and tissue inhibitors of matrix metalloproteases (TIMPs): TIMP-1 ( H ) and TIMP-2 ( I ) in equine endometrial fibroblasts. Data were analyzed by Student’s t-test and expressed as mean ± SEM. Asterisks designate statistical differences between treatments (* p < 0.05; ** p < 0.01; *** p < 0.001; **** P < 0.0001).

Article Snippet: The primary antibody against COL1 (1:1000 diluted; RRID: AB_2891017, 20121, Novotec, Lyon, France) was incubated overnight at 4ºC, and the secondary antibody horseradish peroxidase (HRP)-conjugated anti-rabbit (1:20,000; RRID: AB_2617138; P0448, DakoCytomation, Carpinteria, CA, USA) was incubated during 1.5 h at room temperature.

Techniques: Expressing

Journal: Scientific Reports

Article Title: Ovarian steroids modulate mRNA expression of ECM associated genes and collagen deposition induced by TGF β1 in equine endometrium in vitro

doi: 10.1038/s41598-024-84250-1

Figure Lengend Snippet: Relative collagen type I alpha chain 2 ( COL1A2 ) mRNA expression of equine endometrial explants obtained from mares in the follicular phase (FP; n = 5) and treated for 24 ( A ) or 48 h ( B ) with medium alone (control), transforming growth factor β1 (TGF-β1; 10 ng/mL), TGF-β1 (10ng/mL) + 17β-estradiol (E2; 10 − 9 M) or E2 alone (10 − 9 M). Results were analyzed by one-way analysis of variance (ANOVA), followed by a Tukey’s multiple comparisons test and considered significant at p < 0.05 and shown as mean ± SEM. Asterisks represent significant differences relative to the respective control and asterisks above lines designate differences between treatments (* p < 0.05; ** p < 0.01; **** P < 0.0001).

Article Snippet: The primary antibody against COL1 (1:1000 diluted; RRID: AB_2891017, 20121, Novotec, Lyon, France) was incubated overnight at 4ºC, and the secondary antibody horseradish peroxidase (HRP)-conjugated anti-rabbit (1:20,000; RRID: AB_2617138; P0448, DakoCytomation, Carpinteria, CA, USA) was incubated during 1.5 h at room temperature.

Techniques: Expressing, Control

Journal: Scientific Reports

Article Title: Ovarian steroids modulate mRNA expression of ECM associated genes and collagen deposition induced by TGF β1 in equine endometrium in vitro

doi: 10.1038/s41598-024-84250-1

Figure Lengend Snippet: Relative collagen type I (COL1) protein abundance in equine endometrial explants obtained from mares in the follicular phase (FP; n = 5) and treated for 24 ( A ) or 48 h ( B ) with medium alone (control), transforming growth factor β1 (TGF-β1; 10 ng/mL), TGF-β1 (10ng/mL) + 17β-estradiol (E2; 10 − 9 M) or E2 (10 − 9 M), respectively. Results were analyzed by one-way analysis of variance (ANOVA), followed by a Tukey’s multiple comparisons test and considered significant at p < 0.05 and shown as mean ± SEM. Asterisks represent significant differences relative to the respective control and asterisks above lines designate differences between treatments (* p < 0.05; ** p < 0.01; *** P < 0.001).

Article Snippet: The primary antibody against COL1 (1:1000 diluted; RRID: AB_2891017, 20121, Novotec, Lyon, France) was incubated overnight at 4ºC, and the secondary antibody horseradish peroxidase (HRP)-conjugated anti-rabbit (1:20,000; RRID: AB_2617138; P0448, DakoCytomation, Carpinteria, CA, USA) was incubated during 1.5 h at room temperature.

Techniques: Control

Journal: Scientific Reports

Article Title: Ovarian steroids modulate mRNA expression of ECM associated genes and collagen deposition induced by TGF β1 in equine endometrium in vitro

doi: 10.1038/s41598-024-84250-1

Figure Lengend Snippet: Relative collagen type I alpha chain 2 ( COL1A2 ) mRNA expression in equine endometrial explants obtained from mares in the mid-luteal phase (MLP; n = 5), and treated for 24 ( A ) or 48 h ( B ) with medium alone (control), transforming growth factor β1 (TGF-β1; 10 ng/mL), TGF-β1 (10ng/mL) + progesterone (P4; 10 − 7 M) or P4 alone (10 − 7 M). Results were analyzed by one-way analysis of variance (ANOVA), followed by a Tukey’s multiple comparisons test and considered significant at p < 0.05 and shown as mean ± SEM. Asterisks represent significant differences relative to the respective control and asterisks above lines designate differences between treatments (* p < 0.05; ** p < 0.01; *** P < 0.001).

Article Snippet: The primary antibody against COL1 (1:1000 diluted; RRID: AB_2891017, 20121, Novotec, Lyon, France) was incubated overnight at 4ºC, and the secondary antibody horseradish peroxidase (HRP)-conjugated anti-rabbit (1:20,000; RRID: AB_2617138; P0448, DakoCytomation, Carpinteria, CA, USA) was incubated during 1.5 h at room temperature.

Techniques: Expressing, Control

Journal: Scientific Reports

Article Title: Ovarian steroids modulate mRNA expression of ECM associated genes and collagen deposition induced by TGF β1 in equine endometrium in vitro

doi: 10.1038/s41598-024-84250-1

Figure Lengend Snippet: Relative collagen type I (COL1) protein abundance in equine endometrial explants obtained from mares in the mid-luteal phase (MLP; n = 5) and treated for 24 ( A ) or 48 h ( B ) with medium alone (control), transforming growth factor β1 (TGF-β1; 10 ng/mL), TGF-β1 (10ng/mL) + progesterone (P4; 10 − 7 M) or P4 (10 − 7 M), respectively. Results were analyzed by one-way analysis of variance (ANOVA), followed by a Tukey’s multiple comparisons test and considered significant at p < 0.05 and shown as mean ± SEM. Asterisks represent significant differences relative to the respective control and asterisks above lines designate differences between treatments (* p < 0.05; *** P < 0.001).

Article Snippet: The primary antibody against COL1 (1:1000 diluted; RRID: AB_2891017, 20121, Novotec, Lyon, France) was incubated overnight at 4ºC, and the secondary antibody horseradish peroxidase (HRP)-conjugated anti-rabbit (1:20,000; RRID: AB_2617138; P0448, DakoCytomation, Carpinteria, CA, USA) was incubated during 1.5 h at room temperature.

Techniques: Control