Role of activated JNK signaling upon CDDP stress.

<p>(A) Levels of phosphorylated-JNK were analyzed by immunoblot in untreated HOS cells, in CDDP exposed (at IC₅₀ dose) HOS cells for 24h and in OS-R cells. Densitometric scanning was performed with ImageJ software and normalized to total protein. β-Actin served as a loading control. (B) JNK signaling was inhibited in HOS cells with SP600125 (SP, 25μM). SP was added 2h prior to CDDP treatment (IC₅₀ for 24h). Cleaved PARP-1 levels were then analyzed by immunoblot in untreated HOS, only SP-treated cells, only CDDP-treated cells, and in HOS cells treated with SP plus CDDP. β-Actin served as a loading control. Densitometric scanning was performed with ImageJ software. (C) A fold change in caspase-3 activity was analyzed colorimetrically in HOS cells. JNK signaling was inhibited with SP (25μM; added 2h prior to CDDP) followed by CDDP (IC₅₀) treatment for 24h. Activity in control HOS cells was considered as "1". (D) Representative phase contrast images for HOS-untreated (i), CDDP (IC₅₀)-treated (24h) (ii), only SP-treated (iii), and cells with prior JNK inhibition followed by CDDP (IC₅₀) treatment (24h) (iv). The scale bar represents 30μm. (E) Autophagy was inhibited in HOS cells with prior treatment of CQDP (10μM, 24h) followed by CDDP (IC₅₀) exposure. Protein expression of autophagic markers- LC3B-II, ATG-5 and ATG-12 were analyzed by immunoblot. Densitometric scanning was performed with ImageJ and represented. β-Actin served as a loading control. (F) To confirm an increase in autophagic flux upon JNK inhibition HOS cells were treated with SP (25μM; 2h) followed by CQDP (10μM) for 24h. Immunoblot analysis was performed for anti-LC3B-II. Difference in protein level of LC3B-II is represented with a bar diagram. β-Actin served as a loading control.