pflag-cmv4 plasmid  (Merck KGaA)

Journal: Oncotarget

Article Title: Eupafolin enhances TRAIL-mediated apoptosis through cathepsin S-induced down-regulation of Mcl-1 expression and AMPK-mediated Bim up-regulation in renal carcinoma Caki cells

doi: 10.18632/oncotarget.11604

Figure Lengend Snippet: Caki cells were treated with the indicated concentrations of eupafolin for 24 h. The protein levels of Mcl-1, Bcl-xL, Bcl-2, Bim, Bax, DR5, FADD, c-FLIP, cIAP1, cIAP2, XIAP, and actin were determined by Western blotting. The level of actin was used as a loading control.

Article Snippet: The Caki cells were transfected in a stable manner with the pFLAG-CMV4/Mcl-1 plasmid or control plasmid pcDNA 3.1 vector using Lipofectamine2000 as prescribed by the manufacturer (Invitrogen, Carlsbad, CA).

Techniques: Western Blot

Journal: Oncotarget

Article Title: Eupafolin enhances TRAIL-mediated apoptosis through cathepsin S-induced down-regulation of Mcl-1 expression and AMPK-mediated Bim up-regulation in renal carcinoma Caki cells

doi: 10.18632/oncotarget.11604

Figure Lengend Snippet: A. Caki cells were treated with 30 μM eupafolin for the indicated time periods. The protein and mRNA levels of Mcl-1 were determined by Western blotting (left panel), RT-PCR (middle panel) and real-time PCR (right panel), respectively. The level of actin was used as a loading control. B. Caki cells were pre-treated with 20 μg/mL cycloheximide (CHX) for 30 min, and then treated with 30 μM eupafolin for the indicated time periods. The protein levels of Mcl-1 were determined by Western blotting. The level of actin was used as a loading control. The band intensity of the Mcl-1 protein was measured using ImageJ (public domain JAVA image-processing program; http://rsb.info.nih.gov/ij , lower panel). C. Caki cells were pre-treated with lactacystin 2.5 μM for 30 min, and then treated with 30 μM eupafolin for 24 h. The protein levels of Mcl-1 and actin were determined by Western blotting. The level of actin was used as a loading control. D. Caki cells were pre-treated with chloroquine (CQ, 10 μM) and bafilomycin A (10 nM) for 30 min, and then added 30 μM eupafolin for 24 h. The protein levels of Mcl-1 and actin were determined by Western blotting. The level of actin was used as a loading control. E. Caki cells were pretreated with the 2 μM ZFL, 20 μg/ml E64D, and 2 μM pepstatin A for 30 min, and then added with 30 μM eupafolin for 24 h. The protein levels of Mcl-1 and actin were determined by Western blotting. The level of actin was used as a loading control. F. Caki cells were pretreated with the indicated concentrations of ZFL for 30 min, and then added with 30 μM eupafolin for 24 h. G and H. Caki cells were transiently transfected with cathepsin S (Cat S, G), cathepsin B (Cat B, H) siRNA or control siRNA. After transfection, cells were treated with 30 μM eupafolin for 24 h. The protein levels of Mcl-1, cathepsin B, cathepsin S, and actin were determined by Western blotting. The level of actin was used as a loading control. I. Vector cells (Caki/vector) and Mcl-1-overexpressed cells (Caki/Mcl-1) were treated with 50 ng/mL TRAIL in the presence or absence of 30 μM eupafolin for 24 h. Apoptosis was analyzed as a sub-G1 population by FACS. The protein levels of PARP and actin were determined by Western blotting. The level of actin was used as a loading control. The values in panel (B) represent the mean ± SD from three independent samples. * p < 0.05 compared to CHX.

Article Snippet: The Caki cells were transfected in a stable manner with the pFLAG-CMV4/Mcl-1 plasmid or control plasmid pcDNA 3.1 vector using Lipofectamine2000 as prescribed by the manufacturer (Invitrogen, Carlsbad, CA).

Techniques: Western Blot, Reverse Transcription Polymerase Chain Reaction, Real-time Polymerase Chain Reaction, Transfection, Plasmid Preparation

Journal: Oncotarget

Article Title: Eupafolin enhances TRAIL-mediated apoptosis through cathepsin S-induced down-regulation of Mcl-1 expression and AMPK-mediated Bim up-regulation in renal carcinoma Caki cells

doi: 10.18632/oncotarget.11604

Figure Lengend Snippet: A. U251MG (glioma cells) and DU145 cells (prostate cancer cells) were treated with 50 ng/ml TRAIL in the presence or absence of 30 μM eupafolin for 24 h. The level of apoptosis was assessed by measuring the sub-G1 fraction using flow cytometry (upper panel). The protein levels of PARP, Mcl-1, Bim, and actin were determined by western blotting. The level of actin was used as the loading control (lower panel). B. Caki, mesangial cells (MC) and mouse renal tubular epithelial (TCMK-1) cells were treated with50 ng/ml TRAIL in the presence or absence of 30 μM eupaforin for 24 h. The cell morphology was examined using interference light microscopy. The level of apoptosis was assessed by measuring the sub-G1 fraction using flow cytometry. C. MC and TCMK1 cells were treated with the indicated concentrations of eupafolin for 24 h. The protein levels of Mcl-1, Bim and actin were determined by western blotting. The level of actin was used as the loading control.

Article Snippet: The Caki cells were transfected in a stable manner with the pFLAG-CMV4/Mcl-1 plasmid or control plasmid pcDNA 3.1 vector using Lipofectamine2000 as prescribed by the manufacturer (Invitrogen, Carlsbad, CA).

Techniques: Flow Cytometry, Western Blot, Light Microscopy