Journal: Molecular cancer research : MCR

Article Title: AGO2 mediates MYC mRNA stability in hepatocellular carcinoma

doi: 10.1158/1541-7786.MCR-19-0805

Figure Lengend Snippet: AGO2 regulates MYC mRNA transcript. (A) Relative mRNA expression of MYC mRNA of Huh1 and MHCC97H cells transduced with shCtrl and shAGO2 lentivirus. (**p<0.005, Data are mean ± SEM). (B) Immunohistochemistry analyses of MYC and AGO2 in tumor xenograft tissues. (Images are at 200X magnification and scale bar is 20 μM). (C) RBP-RNA immunoprecipitation analysis. Anti-AGO2 or mouse anti-IgG were used to pull down RNAs cross-linked to AGO2 followed by qRT-PCR of MYC. Fold enrichment of transcript levels first adjusted to 2% input and normalized to IgG (**p<0.005, Data are mean ± SD of triplicates). (D) Relative luciferase (normalized to Renilla) activity of Huh1 and MHCC97H cells transduced with shAGO2 compared to shCtrl (**p<0.005, Data are mean ± SD of triplicates). (E) MYC mRNA transcript levels in shCtrl or shAGO2 after Actinomycin D (ActD) or 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB) treatment over specified time (Data are mean ± SEM).

Article Snippet: HHT4 cells were cultured on fibronectin (BD Biosciences) coated plates. pLKO.1 shAGO2 and pLKO.1 shCtrl were purchased from OpenBiosystems (TRC lentivirus). pGFP-C-shlentil (shCtrl, shAGO2 1, and shAGO2 2) were purchased from Origene and packaged using HEK293T cells.

Techniques: Expressing, Transduction, Immunohistochemistry, Immunoprecipitation, Quantitative RT-PCR, Luciferase, Activity Assay

Journal: Molecular cancer research : MCR

Article Title: AGO2 mediates MYC mRNA stability in hepatocellular carcinoma

doi: 10.1158/1541-7786.MCR-19-0805

Figure Lengend Snippet: AGO2 stabilizes MYC transcript in Huh1 cells with impaired miRNA processing. (A) Representative immunoblot of Huh1.sgCtrl (Ctrl) and sg.DICER1 (DICER1−/−) cells. (B-C) Huh1 DICER1−/− cells express higher levels of MYC mRNA and lower Let-7 miRNA expression (**p<0.005, Data are mean ± SEM). (D) RNA immunoprecipitation of anti-AGO2 and anti-IgG in wildtype (Ctrl), DICER1−/− HCC cells (**p<0.005, Data are mean ± SEM). (E) MYC mRNA levels in Ctrl and DICER1−/− cells following Actinomycin D (ActD) treatment. (F) Ctrl and DICER1−/− cells were treated with shCtrl or shAGO2 lentivirus and MYC mRNA expression was measured (**p<0.005, Data are mean ± SEM).

Article Snippet: HHT4 cells were cultured on fibronectin (BD Biosciences) coated plates. pLKO.1 shAGO2 and pLKO.1 shCtrl were purchased from OpenBiosystems (TRC lentivirus). pGFP-C-shlentil (shCtrl, shAGO2 1, and shAGO2 2) were purchased from Origene and packaged using HEK293T cells.

Techniques: Western Blot, Expressing, Immunoprecipitation

Journal: Oncology Letters

Article Title: Inhibition of autophagy enhances apoptosis induced by bortezomib in AML cells

doi: 10.3892/ol.2020.12370

Figure Lengend Snippet: Downregulation of Beclin-1 enhances apoptosis and reduces cell viability induced by bortezomib in NB4 cells. Cells were infected with lentiviruses expressing shRNAs (non-targeting control or Beclin-1). Puromycin-resistant cells were pooled after each infection. (A) Cells transfected with the control shRNA and shBeclin-1 were treated with or without bortezomib (20 nM) for 24 h, and the expression levels of cleaved caspase-3, cleaved PARP, Bcl-2, p62 and Beclin-1, and LC3-I to LC3-II conversion were determined by western blotting. (B) Ratio of Bcl-2 and β-actin expression levels. Data are presented as the mean ± standard deviation of three independent repeats. *P<0.05 and **P<0.01. (C) Ratio of LC3-II and β-actin expression levels. Data are presented as the mean ± standard deviation of three independent repeats. **P<0.01. (D) Cells transfected with the control shRNA and shBeclin-1 were treated with bortezomib (20 nM) for 0 and 24 h, and cell viability was assessed using a water-soluble tetrazolium salts-8 assay. Data are presented as the mean ± standard deviation of three independent repeats. ***P<0.001. shRNA/sh, short hairpin RNA; CTRL, control; PARP, poly(ADP-ribose) polymerase.

Article Snippet: Lentiviral particles containing short hairpin RNA (shRNA/sh) Beclin-1 (cat. no. 131209BZ; 5′-CCGACTTGTTCCTTACGGAAA-3′) and shRNA negative control (shCTRL; cat. no. 131127CZ; 5′-TTCTCCGAACGTGTCACGTTTC-3′) expression vectors were obtained from Shanghai GenePharma Co., Ltd., and were then cloned into pGLV3/H1/GFP-Puro vector (Shanghai GenePharma Co., Ltd.).

Techniques: Infection, Expressing, Transfection, shRNA, Western Blot, Standard Deviation