Colony formation assay in methylcellulose-based media of CB33 and UH3 cell lines containing siRNA directed against A20 or firefly luciferase. LCLs were embedded in 0.75% methylcellulose at 104 cells per dish. Each of the siRNAs was lentiviral vector–transduced. The assay was performed 3 times in independent infections, and each assay was performed in triplicate. The insertless vector CSII-H1-pgk-Puro served as vector control (v.c). Three kinds of siRNAs were constructed, each for a different sequence of A20 (siRNA A20 nos. 1, 2, and 3). Three kinds of luciferase siRNA (siRNA luciferase nos. 1, 2, and 3) were also generated as controls. (A) Representative colony assay of CB33. (B) The bar graph depicts the number of colonies 2 weeks after culture. The data represent the mean ± SD. CB33 carrying the A20 knockdown was plated in methylcellulose in the presence of 1 μM and 4 μM of dimethyl sulfoxide (control) or BMS-345541. (C) Representative colony assay. (D) The data represent the mean relative NF-κB luciferase activity normalized against Renilla luciferase activity ± SD after 3-day exposure of BMS-345541 in liquid culture. The experiments were performed in triplicate. NC indicates negative control; and VC, vector-only control. (E) The bar graph represents the number of colonies observed in the methylcellulose assay. Colonies were counted after 2 weeks of culturing. The data represent mean ± SD. Each assay was performed in triplicate.
