Non specific scramble sirna control

Ishikawa cells were seeded overnight in the growth media in 6‐well plates and transfected the next day with 10 nmol/L non‐specific scramble siRNA control (#6568; Cell Signaling Technology) or FOXO1‐specific siRNA (#6256; Cell Signaling Technology) by use of Lipofectamine 2000 reagent (Invitrogen) according to the manufacturer's recommendations. At 48 h after transfection, cells were incubated in RPMI‐1640 medium supplemented with 10% FBS in the absence or presence of 2 mM metformin for 24 h. Cell number was counted before cells harvested with RIPA buffer for Western blot analysis of total protein. Silencing of FOXO1 was verified by Western blot analysis with FOXO1 antibody (1:1000; Cell Signaling Technology).

Metformin, compound C, and human recombinant insulin were from Sigma‐Aldrich (St. Louis, MO, USA). Anti‐pan‐FOXO1 (N18), anti‐ki‐67, and Anti‐p‐AMPK (Thr172) antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA, USA) for IHC. Anti‐p‐AMPK, anti‐pan‐AMPK (Thr172), anti‐p‐FOXO1 (Ser256), anti‐p‐AKT (Ser473), and anti‐pan‐AKT antibodies were from Cell Signaling Technology (Beverly, MA, USA). Anti‐pan‐FOXO1 (H128) antibody for Western blotting and immunofluorescence staining was from Santa Cruz Biotechnology. The BCA Protein Assay Kit for quantification of total protein and the Enhanced Chemiluminescence Detection Kit were from Pierce (Rockford, IL, USA). The Histostain‐Plus Kit was from Zhongshan Golden Bridge Biotechnology (Beijing, China). The annexin V–FITC/propidium iodide Apoptosis Detection Kit was from BD Biosciences (San Jose, CA, USA). The Lipofectamine 2000 reagent were from Invitrogen (Burlington, ON, Canada). The non‐specific scramble siRNA control and FOXO1‐specific siRNA were purchased from Cell Signaling Technology. All other chemicals were purchased from Sigma‐Aldrich.