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. 2021 Jul 22;22(3):680. doi: 10.3892/ol.2021.12941

Figure 5.

Figure 5.

RCS inhibits BTZ-induced cytoplasmic ubiquitin condensation in CAL27 cells. (A) CAL27 cells were treated with BTZ (5 nM) and/or RCS (5 µM) for 24 and 48 h. Immunostaining using anti-acetylated tubulin and anti-tubulin mAbs was performed. Immunoblotting with anti-β-actin mAb was performed as a loading control. (B) CAL27 cells were treated with BTZ (5 and 10 nM) and/or RCS (10 µM) for 24 h. Fluorescence immunostaining was performed with anti-ubiquitin (Ub) mAb. Arrowheads indicate the speckled pattern of Ub-proteins. Arrows indicate the aggregate of Ub-proteins. DAPI staining indicates the position of the nucleus (blue). Scale bar, 10 µm. (C) Immunoblotting with anti-Ub mAb in CAL27 cells after treatment with BTZ (5 nM) and/or RCS (5 µM) for 48 h (top). Immunoblotting with anti-β-actin mAb was performed as an internal control. Same membrane was subsequently stained with CBB to compare protein loading amounts (bottom). RCS, ricolinostat; BTZ, bortezomib; mAb, monoclonal antibody; Ub, ubiquitin; CBB, Coomassie Brilliant Blue.