Fig 2: Target identification and target validation of TRIP. (A) Representative images of TS-FITGE in HeLa cells. Images of the Cy3 channel (green, PBS-treated) and Cy5 channel (red, 10 µM of TRIP-treated) are overlaid. The area in the white box is magnified. (B) Cellular thermal shift assay (CETSA) for specific binding of TRIP to HSP60 in HeLa cells. (C) Sensorgrams of surface plasmon resonance (SPR) assay showing the binding kinetics of TRIP (2.5–40 µM) to immobilized HSP60. The dissociation constant (KD) value was calculated as the ratio of rate constants (Kd/Ka). The inset shows a steady-state response against various concentrations of TRIP (N = 3). (D) Representative images of melting curves (left) and first derivatives (right) of differential scanning fluorimetry (DSF) analysis demonstrating the binding of TRIP to HSP60. Before heating, 4 µM of purified HSP60 was incubated with 100× Syprox-Orange in the absence or presence of 100 or 200 µM of TRIP for 30 min (N = 4). (E) Chaperonin functional assay investigating the functional inhibition of TRIP to HSP60. Heated luciferase proteins were incubated with HSP60/HSP10 heterodimer in the absence or presence of TRIP. The luminescence signal was normalized by the vehicle-treated sample (N = 5). (F) Depletion of HSP60 using siHSP60-2 in HeLa cells showing sensitization of HeLa cells to TRIP, not cisplatin (N = 6). Data are presented as the mean ± SD.

Fig 3: TRIP induced mitochondrial stress-mediated p-JNK/AP-1/CHOP apoptosis. (A,B) Flow cytometry data investigating (A) an increase of mitochondrial reactive oxygen species (ROS) and (B) mitochondrial depolarization upon either TRIP treatment or depletion of HSP60 using siRNA in HeLa cells (N = 3). HeLa cells were treated with 10 µM of TRIP for 8 h or transfected with siRNA for 48 h. (C) Immunoblot data showing CHOP activation via different signaling pathways in HeLa cells. HeLa cells were treated with either 200 nM of thapsigargin or 10 µM of TRIP at various time points. (D) TRIP-mediated AP-1 activation in HeLa cells. 10 µM of each compound was treated for 24 h (upper), and 10, 20, and 40 µM of TRIP were treated for 24 h (lower). Luciferase signal was normalized by the vehicle-treated sample (N = 6). Statistical analysis was performed compared to the vehicle group. (E) Flow cytometry data showing the alteration in the number of apoptotic cells upon treatment with either TRIP or etoposide in HeLa cells. HeLa cells were treated with either 10 µM of TRIP for 24 h or 50 µM of etoposide for 24 h. Data are presented as the mean ± SD (ns, not significant, p > 0.05; *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001).