Research Paper Volume 11, Issue 3 pp 986—1007

Figure 1. Aggregation, cytotoxicity, and ROS analyses on TBP/Q₇₉-GFP-expressing 293 cells. (A) Cytotoxicity of SG-Tang (0.1−100 μg/ml) in uninduced cells using the MTT assay (n = 3). To normalize, the relative untreated cell viability was set as 100%. (B) Experimental flow chart. TBP/Q₇₉-GFP 293 cells were plated on dishes, grown for 24 h, and treated with SAHA (0.1 μM) or SG-Tang (0.001-1000 µg/ml) for 8 h. Then doxycycline (Dox, 10 µg/ml) and oxaliplatin (5 µM) were added to the medium for 6 days, followed by aggregation, IC₅₀ cytotoxicity (by HCA), and ROS (by flow cytometry) measurements. (C) Representative fluorescence microscopy images of TBP/Q₇₉-GFP cells untreated or treated with SAHA (0.1 μM) and SG-Tang (100 µg/ml) for 6 days, with nuclei counterstained in blue (top row) or aggregates marked in red (bottom row). (D) Aggregation analysis (n = 3) of TBP/Q₇₉-GFP-expressing cells untreated or treated with SAHA (0.1 μM) or SG-Tang (0.001−1000 μg/ml). To normalize, the relative aggregation level in untreated cells was set as 100%. The red line represents 81% aggregation for 0.1 μM SAHA treatment. P values: comparisons between SAHA/SG-Tang treated and untreated (**: P < 0.01 and ***: P < 0.001), or between SG-Tang treated and SAHA treated (^#: P < 0.05, ^##: P < 0.01 and ^###: P < 0.001). (E) IC₅₀ cytotoxicity of SG-Tang (1−1000 μg/ml) in induced TBP/Q₇₉-GFP 293 cells by the percentage of survived cells (n = 3). To normalize, the relative survived cell number in untreated cells was set as 100%. (F) The induced GFP and ROS levels were measured by flow cytometry (n = 3). P values: comparisons between induced and uninduced cells, or between SG-Tang (100 µg/ml) treated and untreated cells.