MM cells (5 x103) were treated for 48 h with CP at the indicated concentrations in 96-well plates. Subsequently, the inhibitory effect of CP on MM cell line growth was assessed using a WST-1 assay (Premix WST-1 Cell Proliferation Assay, Takara Bio, Otsu, Japan) and an Infinite M1000 PRO microplate reader (Tecan Japan, Kawasaki, Japan) as described previously [34 (link)]. The half-maximal inhibitory concentration (IC50) was defined as the drug concentration resulting in 50% cell survival relative to that of DMSO-treated cells. To analyze the proliferation of MM cells with or without BMSCs, we used a BrdU assay (BrdU cell proliferation assay reagent, Cell Signaling Technology, Danvers, MA) [5 (link)]. Initially, this assay was established using BrdU incorporated into cellular DNA during cell proliferation, and it is similar to the 3H-incorporation assay but does not use radioactive reagents. Therefore, the BrdU assay is suitable for this proliferation assay.
Apoptosis was evaluated by PARP, caspase-3, caspase-8, and caspase-9 western blotting and quantified using an Annexin V/7-AAD staining kit (BD Biosciences, San Jose, CA), as per the manufacturer’s instructions, followed by analysis on a BD FACS Canto II using FACSDiva (BD Biosciences, Tokyo, Japan) [35 ].
Apoptosis was evaluated by PARP, caspase-3, caspase-8, and caspase-9 western blotting and quantified using an Annexin V/7-AAD staining kit (BD Biosciences, San Jose, CA), as per the manufacturer’s instructions, followed by analysis on a BD FACS Canto II using FACSDiva (BD Biosciences, Tokyo, Japan) [35 ].
Free full text: Click here
