Figure 2.

SRX3305 modulates critical B-cell survival pathways in CLL. (A) CLL cell lines (OSU-CLL, MEC-1, and MEC-2) were treated with DMSO vehicle (Veh), SRX3305 (0.5, 1, 2 µM), ibrutinib (Ibr, 1 µM), idelalisib (Idel, 1 µM), or JQ1 (0.5 µM). After 4 h, whole-cell lysates were collected and analyzed for phosphorylation and expression of the given proteins, indicative of modulation of BTK (p-BTK/BTK), PI3K (p-PRAS40/PRAS40), and BET/BRD4 (MYC). GAPDH serves as a loading control (n = 3 independent experiments per cell line). Representative immunoblots are shown. (B) OSU-CLL and MEC-1 cells were treated with DMSO vehicle (Veh), SRX3305 (0.5, 1, 2 µM), 1 µM ibrutinib (Ibr; irreversible BTK inhibitor), or 1 µM BMS-986142 (BMS; reversible BTK inhibitor). Cells were either incubated with treatment continuously for 4 h (Continuous Tx), or treatment was washed out after 1 h and replaced with fresh media for the remaining 3 h of incubation (Wash-out Tx). The stimulation of BCR signaling was performed via the addition of 10 μg/mL anti-IgM for the last 15 min (+α-IgM). Whole-cell lysates were then analyzed for expression or phosphorylation of the indicated proteins (n = 3 independent experiments per cell line). β-Tubulin serves as a loading control. Representative immunoblots are shown for each cell line. Numbers below the bands represent densitometric quantification relative to loading control, total protein (for phosphorylated proteins), and normalized to Veh (for (A)) or +α-IgM Veh (for (B)).