Real-time detection of cytoplasmic foci induced by proteotoxic stresses. (A) TanGIBLE can detect cytoplasmic foci composed of defective proteins. HeLa cells expressing C-terminally Flag-tagged TanGIBLE were treated with 10 μg/ml puromycin for 2 h, followed by 1% Triton X-100 extraction and 4% paraformaldehyde fixation. Fixed cells were stained with anti-Flag polyclonal (shown in red to detect TanGIBLE) and anti-puromycin (shown in green to detect puromycin-labeled defective proteins) antibodies. 10S TanGIBLE was used as a negative control. Scale bar: 20 μm. (B) Live cell detection of endogenous defective proteins induced by puromycin using EGFP-fused TanGIBLE. HeLa cells expressing EGFP-fused authentic TanGIBLE showed diffuse fluorescent signals throughout the cytoplasm. However, small cytoplasmic foci appeared within 30 min of puromycin treatment (5 μg/ml) and increased in size and number thereafter (upper panels). EGFP-fused 10S TanGIBLE was used as a negative control (middle panels). Authentic TanGIBLE did not cause cytoplasmic foci formation in the absence of puromycin (lower panels). Video images were available in Video 1 (EGFP-TanGIBLE) and Video 2 (EGFP-10S TanGIBLE). Scale bar: 20 μm. (C) Live cell detection of endogenous defective proteins induced by bortezomib treatment using EGFP-fused TanGIBLE. In HeLa cells expressing EGFP-TanGIBLE, small cytoplasmic foci appeared after 4 h of bortezomib treatment (10 μM) and increased in size thereafter. EGFP-fused 10S TanGIBLE was used as the negative control. Scale bar: 20 μm.