Effect of autophagy inhibition on MAPK signaling.

<p>(A) Levels of phosphorylated-JNK were analyzed by immunoblot after CDDP exposure (IC₅₀) for 1h (A), 6h (A) and 24h (B) in HOS cells. A prior treatment of CQDP (10μM, 24h) was given wherever mentioned before CDDP exposure. Densitometric scanning was performed with ImageJ. β-Actin served as a loading control. (C and D) Levels of phosphorylated-ERK1/2 were analyzed by immunoblot after CDDP exposure (IC₅₀) for 1h (C), 6h (C) and 24h (D) in HOS cells. A prior treatment of CQDP (10μM, 24h) was given wherever mentioned before CDDP exposure. Densitometric scanning was performed with ImageJ software. β-Actin served as a loading control. (E) Cell viability was measured 24h after treatment by trypan blue assay. Cells were either untreated or treated with the following:- SP (25μM); CQDP (10μM); SP (25μM) plus CQDP (10μM); CDDP (IC₅₀); CQDP (10μM) plus CDDP (IC₅₀); SP (25μM) plus CDDP (IC₅₀); or SP (25μM) plus CQDP (10μM) plus CDDP (IC₅₀). (F) A fold change in caspase-3 activity was analyzed colorimetrically 24h after treatment in HOS cells. Cells were either untreated or treated with the following:- SP (25μM); CQDP (10μM); SP (25μM) plus CQDP (10μM); CDDP (IC₅₀); SP (25μM) plus CDDP (IC₅₀); or SP (25μM) plus CQDP (10μM) plus CDDP (IC₅₀). Activity in control HOS cells was considered as "1". (G) Representative phase contrast images of HOS cells treated with CDDP (IC₅₀) for 24h or along with combinations of inhibitors, SP600125 (25μM) or CQDP (10μM) or both (i-v). The scale bar represents 30μm.