Article Title: Mitochondrial Kv1.3 Channels as Target for Treatment of Multiple Myeloma
Figure Lengend Snippet: Mitochondrial Kv1.3 is expressed in the multiple myeloma cell lines L-363 and RPMI-8226. PAPTP and PCARBTP efficiently kill human multiple myeloma cells. ( a ) Whole cell lysates (left, 50 µg protein/lane) were lysed in RIPA-Buffer, mitochondria from L-363, RPMI-8226 and Jurkat cells (right, 30 µg protein/lane) were enriched as described; both were supplemented with 5× reducing SDS sample buffer, boiled and separated on 8.5% SDS-polyacrylamide gels and blotted on nitrocellulose membranes. Membranes were divided and, after blocking, membranes were incubated 1 h with anti-Kv1.3 or anti-Tim23 primary antibodies, respectively. After extensively washing, blots were incubated with secondary antibodies for 1 h, washed and developed with the Roti–Lumin system. Multiple myeloma cell lines RPMI-8226 and L-363 were treated with increasing concentrations PAPTP ( b ) or PCARBTP ( c ), solvent control (0.1% DMSO), 2 µM staurosporine as a positive control and 2 µM margatoxin as a membrane-impermeable Kv1.3 blocker. After 24 h, cells were stained with Annexin V-APC/7AAD and examined for cell death by flow cytometry. Results are reported as percentages with respect to untreated samples ± SD, ( n = 3 independent experiments, each experiment in triplicate). Graph insert EC50: EC50 values of the indicated compounds in RPMI-8226 and L-363 were calculated by using GraphPad Prism version 9.3 for Windows (GraphPad Software, San Diego, CA, USA).
Article Snippet: Membranes were blocked with 4% BSA/PBS for 1 h and incubated with anti-Kv1.3-antibody (Alomone Labs, Jerusalem, Israel, #APC-101) and anti-Tim23-antibody (BD, Franklin Lakes, NJ, USA, #611223).
Techniques: Blocking Assay, Incubation, Positive Control, Staining, Flow Cytometry, Software