Figure 4

Akt signaling mediates inhibitory actions of curcumin during chondrogenesis. Chick limb bud mesenchymal cells were cultured with or without 20 µM curcumin. (A) Changes in the levels of Akt or pAKt in control and curcumin-treated cultures were determined by Western blotting on the indicated days. (B-E) Chondrogenic progenitors were electroporated with vectors expressing either a myristoylated constitutively active form of Akt (myr-Akt), a dominant inhibitory mutant Akt (mutant-Akt) or an empty vector (mock) using a square wave generator (BTX-830; Gentronics, San Diego, CA) with 20 msec, 200 square pulses. (B) Cells were stained with PNA or Alcian blue on day 3 or 5 of culture, respectively. (C) Percentages of apoptotic cells were quantified by flow cytometric analysis on day 3 of culture. (D) Changes in the levels of pFAK³⁹⁷ FAK, and integrin β1 were determined by Western blotting. (E) Cells were immunostained for F-actin using Alexa488-phalloidin on 4 days of culture. Notice many rounding cells inside dotted circle in control- or curcumin/myr-Akt-treated culture.