Journal: Nucleic acids research

Article Title: A-MYB substitutes for B-MYB in activating cell cycle genes and in stimulating proliferation.

doi: 10.1093/nar/gkae370

Figure Lengend Snippet: Figure 2. A-MYB regulates Cyclin B2 by indirectly binding to its CHR promoter element through MuvB. ( A ) siRNA knockdown was performed in HCT116 ( n = 5) and U2OS ( n = 4) cells. Tw enty -f our hours af ter knoc kdo wn, cells w ere transfected with luciferase reporter constructs carrying the wild-type (wt) or CHR-mutated (CHRmut) Cyclin B2 promoter. Forty-eight hours after knockdown, luciferase activity was quantified from cell lysates. Differences between B-MYB KD and A-MYB + B-MYB dKD are statistically not significant. ( B ) To rescue HCT116 cells from siRNA knockdown of A-MYB and B-MYB , cells were transfected with the mouse A-Myb-overexpressing plasmid mA-Myb-GFP or the empty vector pEGFP as well as luciferase reporter constructs carrying the wild-type (wt) or CHR-mutated (CHRmut) Cyclin B2 promoter in parallel to the siRNA transfections. Forty-eight hours after knockdown, luciferase activity was quantified from cell lysates ( n = 3). ( C ) For luciferase assa y s after mouse A-Myb o v ere xpression, HCT116, RPE-1, Hep3B or HeLa cells were transfected with the mAmyb-GFP overexpression vector as well as luciferase reporter constructs carrying the wild-type (wt) or CHR-mutated (CHRmut) Cyclin B2 promoter. Forty-eight hours after knockdown, luciferase activity was quantified from cell lysates ( n = 12). ( D ) Binding of A-MYB to LIN37 was tested in CoIP assays with HCT116 native protein extracts. As controls, E2F4 representing the DREAM complex and B-MYB as MMB component were precipitated (representative experiment of n = 4). ( E ) Binding of A-MYB to the MuvB core components LIN9, LIN37 and LIN54 was tested in CoIP assays with RPE-1 native protein extracts. As controls, LIN9 and LIN37 representing the MuvB core complex and B-MYB as an MMB component were precipitated (representative experiment of n = 3). ( F ) Binding of the MuvB core to A-MYB was tested in CoIP assa y s with RPE-1 native protein extracts. As negative control, p130 representing the DREAM complex was precipitated (representative experiment of n = 3). ( G ) T98G cells were synchronized in the cell cycle by density arrest and released for 15 h or 24 h into the cell cycle to obtain cell populations enriched in G 0 , S and G 2 / M phase, respectively. From each time point, native protein extracts were isolated and subjected to a LIN37 CoIP. Binding of DREAM and MMB components B-MYB, E2F4, and LIN37 as well as A-MYB was analyzed by Western blot (representative from n = 3). ( H ) Binding of A-MYB to wild-type (wt) and CHR mutant (CHRmut) Cyclin B2 promoter probes in DNA-affinity purification was analyzed by Western blot (representative of n = 3). Mean ± SD are given, and significances were calculated by two-way ANO V A (* P < 0.05; ** P < 0.01; *** P < 0.001).

Article Snippet: Cell culture and cell count analysis Human fibroblast BJ cells (DSMZ, Braunschweig, Germany), human colorectal carcinoma HCT116 cells (provided by Bert Vogelstein), human embryonic kidney HEK293 cells (DSMZ), human papillomavirus-related cervical adenocarcinoma HeLa cells (DSMZ), human pediatric hepatocellular carcinoma Hep3B cells (DSMZ), human retinal pigment epithelial hTERT immortalized RPE-1 cells (DSMZ), human glioblastoma T98G cells (DSMZ), and human bone osteosarcoma U2OS cells (DSMZ) were cultivated in Dulbecco’s modified Eagle’s medium (DMEM; Lonza).

Techniques: Binding Assay, Knockdown, Transfection, Luciferase, Construct, Activity Assay, Plasmid Preparation, Over Expression, Negative Control, Isolation, Western Blot, Mutagenesis, Affinity Purification

Journal: International Journal of Molecular Sciences

Article Title: Dysregulation of miRISC Regulatory Network Promotes Hepatocellular Carcinoma by Targeting PI3K/Akt Signaling Pathway

doi: 10.3390/ijms231911300

Figure Lengend Snippet: The relative miR-221 and AEG-1 mRNA levels in HCC cell lines. The miR-221 ( A ) and AEG-1 ( B ) relative mRNA levels analyzed in HCC cell line panels, the relative miR-221 expression in miR-221 mimic- ( C ), miR-221 inhibitor- ( D ), and AEG-1 siRNA- ( E ) transfected HCC cells. The relative mRNA levels of AEG-1 in miR-221 mimic/inhibitor- ( F ) and AEG-1 siRNA-transfected groups in HCC cells ( G ). The RNU6 and GAPDH were used as internal controls. Error bars presented as mean ± s.d and p -values represented as *** p < 0.001, **** p < 0.0001. ns represented as non-significance.

Article Snippet: Human HCC cell lines (HepG2, Huh7, and Hep3B) were obtained from the National Centre for Cell Science (NCCS), Pune, India, and cultured in DMEM with 10% FBS (fetal bovine serum) and 1% PS (penicillin streptomycin) (Invitrogen, Carlsbad, CA, USA).

Techniques: Expressing, Transfection

Journal: International Journal of Molecular Sciences

Article Title: Dysregulation of miRISC Regulatory Network Promotes Hepatocellular Carcinoma by Targeting PI3K/Akt Signaling Pathway

doi: 10.3390/ijms231911300

Figure Lengend Snippet: AEG-1 and miR-221 regulate angiogenesis and cell cycle regulatory mRNA expressions in the HCC cell lines. The regulatory mRNAs expressions which regulate angiogenesis (LSF and MMP9) and cell cycle (p57, p53, and RB) were analyzed in miR-221 mimic-, miR-221 inhibitor-, AEG-1 siRNA-, and their corresponding control-transfected HepG2, Huh7, and Hep3B cells by using qRT- PCR. The GAPDH was used as an internal control. Error bars presented as mean ± s.d and p -values represented as * p < 0.05, ** p < 0.01, and *** p < 0.001. ns represented as non-significance.

Article Snippet: Human HCC cell lines (HepG2, Huh7, and Hep3B) were obtained from the National Centre for Cell Science (NCCS), Pune, India, and cultured in DMEM with 10% FBS (fetal bovine serum) and 1% PS (penicillin streptomycin) (Invitrogen, Carlsbad, CA, USA).

Techniques: Control, Transfection, Quantitative RT-PCR

Journal: International Journal of Molecular Sciences

Article Title: Dysregulation of miRISC Regulatory Network Promotes Hepatocellular Carcinoma by Targeting PI3K/Akt Signaling Pathway

doi: 10.3390/ijms231911300

Figure Lengend Snippet: AEG-1/miR-221 regulates the apoptosis and autophagy regulatory mRNAs in the HCC cell lines. The apoptosis and autophagy regulatory mRNAs (OPN, Bcl-2, PTEN, LC3A, PI3K, and Akt) expressions were analyzed in miR-221 mimic-, miR-221 inhibitor, and AEG-1 siRNA-transfected HCC cells by using qRT-PCR, and GAPDH used as an internal control. Error bars presented as mean ± s.d. and p -value represented as * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 compared with corresponding controls.

Article Snippet: Human HCC cell lines (HepG2, Huh7, and Hep3B) were obtained from the National Centre for Cell Science (NCCS), Pune, India, and cultured in DMEM with 10% FBS (fetal bovine serum) and 1% PS (penicillin streptomycin) (Invitrogen, Carlsbad, CA, USA).

Techniques: Transfection, Quantitative RT-PCR, Control

Journal: International Journal of Molecular Sciences

Article Title: Dysregulation of miRISC Regulatory Network Promotes Hepatocellular Carcinoma by Targeting PI3K/Akt Signaling Pathway

doi: 10.3390/ijms231911300

Figure Lengend Snippet: Silencing of AEG-1 and miR-221 inhibits HCC cell migration and proliferation in vitro. The effect of the miR-221/AEG-1 measured on HCC cell migration and cell proliferation in the miR-221 mimic-, miR-221 inhibitor-, AEG-1 siRNA-, and their control-transfected HepG2, Huh7, and Hep3B cells by invasion ( A ), migration ( B ), and MTT assay ( C ) in vitro. Images analyzed using Image J (NIH) (scale bar, 100 µm). Error bars presented as mean ± s.d and p -value represented as ** p < 0.01, *** p < 0.001 compared to the corresponding controls.

Article Snippet: Human HCC cell lines (HepG2, Huh7, and Hep3B) were obtained from the National Centre for Cell Science (NCCS), Pune, India, and cultured in DMEM with 10% FBS (fetal bovine serum) and 1% PS (penicillin streptomycin) (Invitrogen, Carlsbad, CA, USA).

Techniques: Migration, In Vitro, Control, Transfection, MTT Assay

Journal: International Journal of Molecular Sciences

Article Title: Dysregulation of miRISC Regulatory Network Promotes Hepatocellular Carcinoma by Targeting PI3K/Akt Signaling Pathway

doi: 10.3390/ijms231911300

Figure Lengend Snippet: The effects of AEG-1/miR-221 on HCC cell cycle regulation and apoptosis. The effects of AEG-1/miR-221 on apoptosis ( A ) and cell cycle ( B ) analyzed in the miR-221 mimic-, and miR-221 inhibitor-, AEG-1 siRNA-, and their corresponding control-transfected HCC cells in vitro by flow cytometry analysis. Error bars presented as the mean ± s.d. and p -values represented as ** p < 0.01, *** p < 0.001 compared to the corresponding controls.

Article Snippet: Human HCC cell lines (HepG2, Huh7, and Hep3B) were obtained from the National Centre for Cell Science (NCCS), Pune, India, and cultured in DMEM with 10% FBS (fetal bovine serum) and 1% PS (penicillin streptomycin) (Invitrogen, Carlsbad, CA, USA).

Techniques: Control, Transfection, In Vitro, Flow Cytometry

Journal: International Journal of Molecular Sciences

Article Title: Dysregulation of miRISC Regulatory Network Promotes Hepatocellular Carcinoma by Targeting PI3K/Akt Signaling Pathway

doi: 10.3390/ijms231911300

Figure Lengend Snippet: The effect of AEG-1/miR-221 was analyzed on cell cycle and angiogenesis regulatory proteins in HCC cell lines. The relative AEG-1 protein expression in THLE-2, HepG2, Huh7, and Hep3B cells ( A ) and miR-221 mimic-, miR-221 inhibitor-, and AEG-1 siRNA-transfected HCC cells ( B ). The regulatory protein expressions which regulate angiogenesis (LSF and MMP 9) and cell cycle (p57, p53, and RB) were analyzed in miR-221 mimic-, miR-221 inhibitor-, AEG-1 siRNA-, and their controls-transfected HepG2 ( C ), Huh 7 ( D ), and Hep3B ( E ) cells by western blot. p -Values presented as * p < 0.05, ** p < 0.01,*** p < 0.001, **** p < 0.0001 and error bars presented as the mean ± s.d. ns—(non-significant) compared to the controls.

Article Snippet: Human HCC cell lines (HepG2, Huh7, and Hep3B) were obtained from the National Centre for Cell Science (NCCS), Pune, India, and cultured in DMEM with 10% FBS (fetal bovine serum) and 1% PS (penicillin streptomycin) (Invitrogen, Carlsbad, CA, USA).

Techniques: Expressing, Transfection, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: Dysregulation of miRISC Regulatory Network Promotes Hepatocellular Carcinoma by Targeting PI3K/Akt Signaling Pathway

doi: 10.3390/ijms231911300

Figure Lengend Snippet: Ectopic expression of AEG-1/miR-221 dysregulates apoptosis and autophagy regulatory protein levels in HCC cell lines. The relative expressions of apoptosis (OPN and Bcl-2), autophagy (LC3A/B), PTEN, and PI3K/Akt proteins were analyzed by western blotting in the mock control, miR-221 mimic, miR-221 inhibitor, AEG-1 siRNA, and corresponding controls transfected HepG2 ( A ), Huh7 ( B ), and Hep3B ( C ) cells using β-actin as an internal control. Error bars are represented as mean ± s.d. and * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 compared to the control group. ns: non-significance.

Article Snippet: Human HCC cell lines (HepG2, Huh7, and Hep3B) were obtained from the National Centre for Cell Science (NCCS), Pune, India, and cultured in DMEM with 10% FBS (fetal bovine serum) and 1% PS (penicillin streptomycin) (Invitrogen, Carlsbad, CA, USA).

Techniques: Expressing, Western Blot, Control, Transfection