AITC induces shift in G/F-actin, translocation/localization in mitochondria and dephosphorylation of cofilin. (A) U937 cells were treated with 20 μM AITC for different time intervals as indicated, the levels of F-actin and G-actin were measured using the G-actin/F-actin In Vivo Assay Kit as described in Methods. Densitometric analysis of the blots were performed by using Quantity One software (Bio-Rad laboratories, Munchen, Germany) and G/F-actin ratio was calculated. (B) Levels of F-actin (green) and G-actin (red) in cells treated with AITC were imaged by confocal microscopy. Scale bar represents 5 μm. (C) U937 cells were treated without or with various concentrations of AITC for 24 h, or treated with 20 μM AITC for different time intervals as indicated. Cell lysates, mitochondrial and cytosolic fractions were prepared and subjected to Western blot analysis using antibodies against phospho-cofilin (p-cofilin), cofilin and actin. Blots were subsequently stripped and reprobed with antibody against GADPH and COX IV (mitochondrial fraction) to ensure equivalent loading. (D) U937 cells were pretreated with the caspase inhibitor Z-VAD-FMK (20 μM) for 2 h, followed by treatment with 20 μM AITC for 24 h. Cell lysates, mitochondrial and cytosolic fractions were prepared and subjected to Western blot analysis. (E-F) Cells were untreated (control) or treated with 20 μM AITC for 12 h, cofilin was labeled by an anti-cofilin antibody followed by Alexa 488-conjugated goat anti-mouse antibody (green); fluorescent deoxyribonuclease I conjugates (red) for staining G-actin; Fluorescent phallotoxins for staining F-actin; mitochondria were stained by Mitotracker Red or Green as described in Methods. Fluorescence images were collected at similar focal planes by confocal microscopy. Scale bar represents 5 μm. (G) Cell lysates, cytosolic and mitochondrial fractions of control and AITC-treated cells were prepared and subjected to immunoprecipitation using an anti-cofilin antibody, followed by immunoblotting analysis.