Figure 1

BTK knockdown in INA6 cells reduces in vitro clonogenicity but has no effect on short-term growth and promotes growth in coculture with osteoclasts. (a) After 3 days of culture, in vitro growth of INA6 cells infected with BTK-KD or scrambled control (CONT) shRNA was assessed with MTT assays, which reflect the overall number of viable cells. Cells were plated at three densities (5 × 10³, 10 × 10³ and 25 × 10³ cells/well). (b) Left panel, clonogenicity of BTK-KD and CONT cells assessed with 14-day clonogenic assays. Right panel, Representative photographs of typical colonies of BTK-KD and CONT cells at × 5 and × 10 original microscopic magnifications. The clonogenic assay was repeated three times. (c) Upper panel: flow cytometry cell cycle analysis of BTK-KD and CONT cells taken from standard cell culture. Lower panel: flow cytometry analysis of BrdU incorporation by BTK-KD and CONT cells taken from standard cell culture. (d) Growth of luciferase-expressing BTK-KD or CONT INA6 cells on osteoclasts, measured by bioluminescence. Cells were plated at three MM densities (1 × 10⁴, 2 × 10⁴ and 3 × 10⁴ cells/well) on top of mature osteoclasts and MM growth measured after 3 days.