Figure 10

Experimental validation of differential permeation of Withaferin-A (Wi-A) and Withanone (Wi-N) through cell membrane. (A) Human cancer cells (U2OS) treated with Wi-A/Wi-N were immunostained with anti-WiNA antibody showed their presence in cells and predominantly in the nucleus. Cells treated for 24 h showed Wi-A, but not Wi-N. Cells treated for 48 h showed both Wi-A and Wi-N. (B) Normal cells treated with Wi-A for 48 h showed high intensity of Wi-A staining in the nucleus; Wi-N treated cells showed faint cytoplasmic staining. (C) High resolution images showing the presence of Wi-A predominantly in the nucleus of U2OS and TIG-3 cells. Wi-N showed weak nuclear staining U2OS cells, and pan-cytoplasmic staining in normal cells. (D) Wi-A/Wi-N treated cells were examined for DNA damage marker protein γH2AX showed its higher upregulation and activation in Wi-A, as compared to Wi-N treated U2OS cells. (E) Upregulation of γH2AX, p53 and downregulation of CARF in Wi-A, but not Wi-N, treated cells. (F,G) Wi-A, but not Wi-N, treated normal (TIG-3 and MRC5) cells showed increase in γH2AX, pATR and pCHK1.