Journal: Molecular medicine reports

Article Title: β-arrestin2 regulates TRAIL-induced HepG2 cell apoptosis via the Src-extracellular signal-regulated signaling pathway.

doi: 10.3892/mmr.2016.5216

Figure Lengend Snippet: Figure 3. β‑arrestin2 mediated activation of the TRAIL‑induced Src‑ERK signaling pathway. (A) HepG2 cells were treated with TRAIL for 0.25, 0.5, 1, 2 or 4 h and the cell lysates were subjected to immunoblotting with the indicated antibodies. The band intensities were quantified by densitometry using LI‑COR Odyssey Analysis software 1.2. *P<0.05, **P<0.01 vs. the TRAIL‑ group. HepG2 cells were transfected with (B) pBS‑U6‑β‑arrestin2 or control plasmid for 72 h or (C) pcDNA3.0 and GFP‑Arrb2 plasmids for 48 h. (B and C) Cells were stimulated with TRAIL for 1 or 2 h and the levels of p‑, total Src and ERK were detected by western blotting. Equal protein loading was confirmed by GAPDH. (D) Cells were pretreated with 0.1% DMSO or 5 µM PP2 or 5 µM PP3 for 2 h, then treated with 200 ng/ml TRAIL. Cell lysates were prepared and subjected to western blotting using the indicated antibodies. (E) Cell viability was detected using a CCK‑8 and experiments were independently repeated three times. *P<0.05 vs. DMSO. (F) Cells were stained with DAPI and the nuclear morphology was observed by immunofluorescent microscopy (magnification, x200). (G) Cells were pretreated with 0.1% DMSO or 20 µM U0126 for 2 h, then exposed to 200 ng/ml TRAIL for 24 h. Cell viability was detected by CCK‑8 and experiments were independently repeated for three times. *P<0.05 vs. DMSO. (H) Cells were stained with DAPI and the nuclear morphology was observed by immunofluorescent microscopy (magnification, x200). TRAIL, tumor necrosis factor‑related apoptosis‑inducing ligand; p, phosphorylated; RNAi, RNA interference; Arrb, β‑arrestin; GFP, green fluorescent protein; ERK, extracel lular signal‑regulated kinase; DMSO, dimethyl sulfoxide; CCK‑8, Cell Counting Kit‑8; DAPI, 4',6‑diamidino‑2‑phenylindole.

Article Snippet: Monoclonal rabbit antibodies against poly ADP ribose polymerase (PARP; 9532s, 1:1,000), pro-caspase-3 (9665s; 1:1,000), cleaved caspase-3 (9664s; 1:500), Src (2109s; 1:1,000), phosphorylated (p)-Src (Tyr416; 6943s; 1:1,000), ERK (9102s; 1:1,000), p‐ERK (Thr202/Tyr204; 4376s; 1:1,000), DR5 (8074s; 1:1,000), β-arrestin2 (3857s; 1:1000) and β-actin (4970s; 1:1,000) were obtained from Cell Signaling Technology, Inc. (Danvers, MA, USA).

Techniques: Activation Assay, Western Blot, Software, Transfection, Control, Plasmid Preparation, Staining, Microscopy, CCK-8 Assay