Figure 1.

Mitochondrial stress-mediated FOSB activation in non-small cell lung cancer (NSCLC) cells. (A) Total lysates from control and NSCLC cells without (Co) or with TP4 treatment (T, 6.71 μM) were analyzed by Western blot using antibodies against GAPDH and FOSB. Quantitative analysis of FOSB (normalized to GAPDH) is shown below the blot. (B) Cell viability was determined by the ATP assay for NSCLC cells transfected with FOSB or FOSΔB plasmid. Eight replicate wells were analyzed for each dose. (C) Total lysates from A549 cells transfected with control (Neg-si) or FOSB small interfering RNAs (siRNAs) were analyzed by Western blot using antibodies against GAPDH and FOSB. (D) Quantitative analyses of FOSB levels (C), normalized to GAPDH. (E) Viability of A549 cells transfected with FOSB siRNAs were determined by ATP assay. Eight replicate wells were analyzed for each condition. (F) Intracellular localization of biotinylated-TP4 in A549 cells. Cells were stained with biotin, prohibitin (upper), giantin (middle), and calreticulin (lower) antibodies. Hoechst33342 was used to stain nuclei. Boxed regions are magnified in the panels to the right of the merged images. Yellow arrows indicate colocalization of biotinylated-TP4 with mitochondria. Bar: 40 μm. (G) Mitochondrial fractions from A549 cells without (Co) or with TP4 treatment (T, 5.03 μM) were analyzed by Western blot using antibodies against organelle markers and TP4. (H) Mitochondria in A549 cells were stained by MitoTracker Red CMXRos dye. Fluorescence intensity of the mitochondria in each cell was quantified after 5.03 and 6.71 μM TP4 treatment for 3 h. Bar: 20 μm. (I) Mitochondrial Ca²⁺ levels were measured kinetically (every 30 s for 30 min) using Rhod-2 AM dye after treatment with the indicated doses of TP4. (J) Ca²⁺ levels were measured by the addition of Fluo-4 dye after treatment with the indicated doses of TP4 for 15–60 min. Eight wells were analyzed for each treatment in an independent repeat. (K) Total lysates from A594 cells without (Co) or with 6.71 μM TP4 treatment (T) for 45 min to 6 h were analyzed by Western blot using antibody against GAPDH, FOSB, and ERK. The relative amounts of FOSB + FOSΔB, ERK1/2, and p-ERK1/2 in each lane are expressed as RDU and normalized to GAPDH signal. Experiments were independently repeated with comparable results. (L) Total lysates from control, BAPTA/AM-treated, TP4-treated cells, and combination-treated cells were analyzed by Western blot using antibodies against GAPDH and FOSB. Quantitative analyses of the blots shown in left; levels of FOSB + FOSΔB were normalized to GAPDH. (N) Cell viability was measured in cells treated with BATPA/AM and TP4. Eight wells were analyzed for each independent replicate. Quantitative results are presented as the mean ± SD. (n = 3, two tailed t-test in (A,D,E,H,J,L,M), one-way ANOVA followed by Bonferroni’s test in (B): * p < 0.05; ** p < 0.01; *** p < 0.001). Co in (A,G,K): control group.