Fig 2: Western blot analysis of expression of Bcl-2 and Bax proteins. GAPDH was used as an internal control. Following the knockdown of TROP2, the expression of Bax was enhanced, whereas Bcl-2 protein expression levels were decreased compared with the NC. Bcl-2, B-cell lymphoma 2; Bax, Bcl-2-associated X protein; NC, negative control.

Fig 3: Effects of RT on the expression of the apoptotic-related proteins: Mcl-1, Bcl-2, and Bax in the NSCLC cell line (H460) and patient-derived primary cancer cell lines (ELC12, ELC16, ELC17, and ELC20). (a) H460, ELC12, and ELC16 cell lines were seeded and treated with 0–1 μM of RT for 24 h. Then, immunofluorescence analysis was performed using an antibody against Mcl-1. The Alexa Fluor 488 conjugated secondary antibody and Hoechst 33342 were added and the images were visualized under a fluorescence microscope. (b) ELC12, ELC16, ELC17, and ELC20 cell lines were seeded and treated with 0–1 μM of RT for 24 h. Western blot analysis was performed to detect the Mcl-1, Bcl-2, and Bax protein levels. The blots were reprobed with β-actin to confirm an equal loading of each of the protein samples and densitometry was used to calculate the relative protein levels. Densitometric values of protein expression levels were presented as the fold changes relative to β-actin. (c) H460 cells were seeded and treated with 0–1 μM of RT, TM-(–)-45, TM-(–)-18, TM-(–)-4a, TM-(–)-52, and TM-(–)-55 for 24 h. After that, Western blot analysis was performed to detect the Mcl-1, Bcl-2, and Bax protein levels. Data represent the mean ± SEM (n = 3) (* 0.01 ≤ p < 0.05, ** p < 0.01, compared with the untreated control).

Fig 4: A protein array was used to examine any change in factors involved in apoptotic signaling. Proteins were collected 2 h following the final injury. N = 3 biological replicates were used. Several proteins showed a trend with injury and treatment, however, the only proteins that were significantly changed were Bax (*P < 0.05) and 14-3-3, by antibody array analysis (a). With Western immunoblots (b and c), we observed a significant decrease in Bax expression following injury in the presence of treatment compared to vehicle alone (*P < 0.05; B). We did not observe any change in heat shock protein 90 (HSP90; c).

Fig 5: Effect of AS-IV on palmitic acid (PA)-induced ER stress and podocyte apoptosis. Podocytes were pretreated with or without AS-IV at the indicated concentrations (0, 10, 20, 40 and 80 μM, respectively) for 12 h followed by 250 μM palmitate exposure for 24 h. (A-B) Representative confocal microscopic images of (a) ER-tracker and (b) nephrin in podocytes with different cultural treatment. Scale bars, 10 μm; Original magnification, ×400. c Representative immunoblots and densitometry quantification of BIP, CHOP, cleaved caspase 3, 9, Bax and Bcl-2 expression in podocyte with different cultural treatment. d Representative flow cytometry images and quantitative analysis of apoptotic podocytes with different cultural treatment. Data are presented as means ± SEM. n = 3 for each group (a–d). *p < 0.05, **p < 0.01, compared with BSA-treated podocyte; ## p < 0.01, compared with PA-treated podocyte