Pycr1

The expression of PYCR1 protein was examined using IHC staining with primary antibody (PYCR1, 1:2000, Proteintech). The slide was then photographed under a microscope and analyzed by two observers who were blinded to the clinical and pathological data. PYCR1 expression was scored semi-quantitatively by multiplying the staining intensity (0, no staining; 1, weak staining; 2, moderate staining; 3, strong staining) and the staining rate (graded on the percentage of positive tumor cells; 0, none; 1, 1–25%; 2, 26–50%; 3, 51–75%; 4, >75%). Protein expression level was then classified into two categories: low expression (grades 0–3) and high expression (grades 4–12).

For the immunoblot analysis, A375 and M14 cells were collected and lysed with RIPA buffer after 24 hours of transfection. Forty micrograms of lysates was separated on a 10% SDS-PAGE gel, and then transferred to a polyvinylidene fluoride membrane. The membrane was blocked with 5% of fat-free milk for 1 hour, and probed with primary (1:1,000) and secondary (1:5,000) antibodies. The subsequent immunoblot analysis was performed using ECL. The primary antibodies used for the immunoblot analysis were PYCR1 (Proteintech), AKT (Cell Signaling Technology), p-AKT (Cell Signaling Technology), Caspase3-p17 (Proteintech), Bax (Proteintech), Bcl2 (Proteintech), p70 (Abcam) and α-Tubulin (Abcam). The densitometry was measured using ImageJ software, and the protein levels were normalized to α-Tubulin.

Cells were harvested and lysed in RIPA containing proteinase and phosphatase inhibitors. The concentration of proteins in cell lysates were quantified by BCA kit (Applygen Technologies, China) following the instruction. Equal amounts of cell lysates (20 µg protein/lane) were electrophoresed on SDS-polyacrylamide gels and transferred to nitrocellulose membrane. Membranes were blocked and then probed with the appropriate primary antibody [PYCR1, 1:5,000 (Proteintech); PARP, 1:1,000 (Proteintech); Caspase-3, 1:1,000 (CST); cleaved-Caspase 3, 1:1000 (CST); p-PI3K, 1:2,000 (Proteintech); p-Akt, 1:4,000 (Proteintech); Akt, 1:1,000 (Proteintech); β-actin, 1:8,000 (Applygen)] in blocking buffer overnight at 4 °C. The bound antibodies were detected with IRDye 800CW-conjugated goat anti-rabbit IgG or anti-mouse IgG (at 1:10,000 dilution; LI-COR Bioscience, Lincoln, NE, USA) and visualized with Odyssey 290 infrared imaging system (LI-COR Bioscience).