Figure 3.

Nimbolide induced cellular reorganization through actin depolymerization

(A) Phase contrast microscopic image of MDA-MB-231 cells treated with nimbolide for 30 min, 1 h, and 3 h showing change in cellular morphology. (B) Live cell fluorescence microscopic images of MDA-MB-231 cells treated with nimbolide showing reduction in actin cytoskeletal network with increase in time. MDA-MB-231 cells were incubated with SiR-actin (fluorescent, cell membrane permeable molecule binds specifically to F-actin) for 1 h and treated with nimbolide for further 90 min. The cellular events were captured every 10 s using Leica microscope (with 63× objective) (F-actin, red). (C) Actin depolymerization assay was performed using MDA-MB-231 cell lysates treated with nimbolide for 30 min, 1 h, and 3 h. Cell lysates were added to the pyrene-labeled actin filaments and change in fluorescence intensity was recorded using BMG microplate reader. (D) Actin depolymerization was analyzed quantitatively by analyzing the reduction in pyrene fluorescence using ClARIOstar software version V5.61. Data represent mean ± SD of three independent experiments. ∗p < 0.05 versus control. (E) Nimbolide treatment reduced the expression of F-actin. F-actin was isolated from MDA-MB-231 cells treated with nimbolide at indicated time points, run on SDS-PAGE, and probed with actin antibody. The expression of F-actin was analyzed using densitometry; the data represent mean ± SD of three independent experiments. ∗p < 0.05 versus control. (F) Immunofluorescent analysis of α-actinin and actin in MDA-MB-231 cells after treatment with nimbolide (n = 3).