Figure 3.

Akt pathway activation leads to survival, pathway reactivation, and histone remodeling of magnolol‐treated cells. (A) WM1366 and WM164 cells were exposed to DMSO, 30 µmol L⁻¹ magnolol, 10 µmol L⁻¹ SC79 or combination of 30 µmol L⁻¹ magnolol/10 µmol L⁻¹ SC79 for 72 h. Medium was aspirated following the treatment and cells fixed with 4% paraformaldehyde (PFA). Cells were stained with 0.5% crystal violet (n = 3, biological replicates). (B) In a separate experiment, WM1366 and WM164 cells were exposed to DMSO, 30 µmol L⁻¹ magnolol, 10 µmol L⁻¹ SC79 or a combination of 30 µmol L⁻¹ magnolol/10 µmol L⁻¹ SC79 for 48 h. Proteins were subjected to immunoblotting with p‐mTOR, p‐Akt, p‐ERK, t‐ERK. Actin was used as a loading control. (C) WM164 and WM1366 cells were treated with DMSO, 30 µmol L⁻¹ magnolol, 1 µmol L⁻¹ MK2206 or 30 µmol L⁻¹ magnolol/1 µmol L⁻¹ MK2206 for 72 h. Cells were stained with 0.5% crystal violet and fixed with 4% PFA. (D) In a separate experiment, WM164 and WM1366 cells were treated with the above‐mentioned drugs for 48 h. Proteins were isolated and immunoblotted with p‐mTOR, t‐mTOR, p‐Akt, t‐Akt, p‐ERK, t‐ERK. (E) WM1366 and WM164 cells were exposed to DMSO, 30 µmol L⁻¹ magnolol, 10 µmol L⁻¹ SC79 or combination of 30 µmol L⁻¹ magnolol/10 µmol L⁻¹ SC79 for 48 h in a 24‐well plate. Following treatment cells were fixed with 4% PFA and blocked with 0.3% TritonX100, 5% goat serum in 1% BSA and blotted for H3K4me3 and H3K9me3. Expression of these histone marks was determined by immunofluorescence. Representative merged images of DAPI (blue) and antibody (red) are shown (10X magnification). (F) In a separate experiment, WM164 cells were exposed to DMSO, 25 nmol L⁻¹ dabrafenib/5 nmol L⁻¹ trametinib, 25 nmol L⁻¹ dabrafenib/5 nmol L⁻¹ trametinib/25 µmol L⁻¹ magnolol or 25 nmol L⁻¹ dabrafenib/5 nmol L⁻¹ trametinib/25 µmol L⁻¹ magnolol/10 µmol L⁻¹ SC79. WM1366 cells were exposed to DMSO, 10 µmol L⁻¹ SC79, 7.5 nmol L⁻¹ docetaxel/25 µmol L⁻¹ magnolol or 7.5 nmol L⁻¹ docetaxel/25 µmol L⁻¹ magnolol/10 µmol L⁻¹ SC79 for 48 h. Similarly, these cells were stained for H3K4me3 and H3K9me3 by immunofluorescence