Figure 3.
Inhibitory effects of UBX-382 on BCR-mediated downstream signaling. (A) Evaluation of inhibitory effects on cell proliferation by ibrutinib (yellow circle), ARQ-531 (green diamond), and UBX-382 (red square) in TMD-8 cells. The cells were seeded and treated with increasing doses of ibrutinib, ARQ-531, and UBX-382 for 3 days. Cell proliferation was measured by the Cell Titer-Glo assay in duplicates. Data are expressed as the mean ± SEM. (B) Secreted CCL3 and CCL4 levels in the TMD-8 cells. Secretion of CCL3 (left) and CCL4 (right) by the TMD-8 cells following anti–immunoglobulin M (IgM) stimulation for 8 hours and 10 nM UBX-382, ibrutinib, acalabrutinib, ARQ-531, Binder, or MT-802 for 24 hours. Each graph indicates the mean concentration of CCL3 and CCL4 produced by the TMD-8 cells cultured in a medium with DMSO, UBX-382, ibrutinib, acalabrutinib, ARQ 531, Binder, or MT-802 in the presence of 10 μg/mL anti-IgM. The experiments were performed in duplicates. Data represent the mean ± SEM. (C,D) Comparison of antiproliferative effect following treatment with ibrutinib (yellow circle), ARQ-531, and UBX-382 in WSU-DLCL2, OCI-Ly3, or U2932 cells. The cells were seeded and treated with increasing concentrations of ibrutinib, ARQ-531, and UBX-382 for 5 days. The assay was performed in duplicates. Data are expressed as the mean ± SEM. (E) Suppression of BCR-mediated signaling axis by UBX-382. U2932 cells were treated with 100 nM ibrutinib, acalabrutinib, ARQ-531, Binder, and UBX-382 for 6 and 24 hours and stimulated with 10 μg/mL anti-IgM for 15 minutes. Phosphorylation and total protein levels of BTK, SYK, MEK, and ERK were visualized via immunoblotting using indicated specific antibodies. GAPDH was used as a loading control. The quantitative results of the band intensity are labeled under the corresponding protein band (bottom). n/a, not applicable.

Inhibitory effects of UBX-382 on BCR-mediated downstream signaling. (A) Evaluation of inhibitory effects on cell proliferation by ibrutinib (yellow circle), ARQ-531 (green diamond), and UBX-382 (red square) in TMD-8 cells. The cells were seeded and treated with increasing doses of ibrutinib, ARQ-531, and UBX-382 for 3 days. Cell proliferation was measured by the Cell Titer-Glo assay in duplicates. Data are expressed as the mean ± SEM. (B) Secreted CCL3 and CCL4 levels in the TMD-8 cells. Secretion of CCL3 (left) and CCL4 (right) by the TMD-8 cells following anti–immunoglobulin M (IgM) stimulation for 8 hours and 10 nM UBX-382, ibrutinib, acalabrutinib, ARQ-531, Binder, or MT-802 for 24 hours. Each graph indicates the mean concentration of CCL3 and CCL4 produced by the TMD-8 cells cultured in a medium with DMSO, UBX-382, ibrutinib, acalabrutinib, ARQ 531, Binder, or MT-802 in the presence of 10 μg/mL anti-IgM. The experiments were performed in duplicates. Data represent the mean ± SEM. (C,D) Comparison of antiproliferative effect following treatment with ibrutinib (yellow circle), ARQ-531, and UBX-382 in WSU-DLCL2, OCI-Ly3, or U2932 cells. The cells were seeded and treated with increasing concentrations of ibrutinib, ARQ-531, and UBX-382 for 5 days. The assay was performed in duplicates. Data are expressed as the mean ± SEM. (E) Suppression of BCR-mediated signaling axis by UBX-382. U2932 cells were treated with 100 nM ibrutinib, acalabrutinib, ARQ-531, Binder, and UBX-382 for 6 and 24 hours and stimulated with 10 μg/mL anti-IgM for 15 minutes. Phosphorylation and total protein levels of BTK, SYK, MEK, and ERK were visualized via immunoblotting using indicated specific antibodies. GAPDH was used as a loading control. The quantitative results of the band intensity are labeled under the corresponding protein band (bottom). n/a, not applicable.

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