Ccdc106

Total protein was extracted using lysis buffer (Pierce, Rockford, IL, USA) and quantified with the Bradford method [19 (link)]. Fifty μg total protein per sample were separated via 10% SDS-PAGE, and transferred onto polyvinylidene fluoride membranes (PVDF; Millipore, Billerica, MA, USA). Membranes were incubated overnight at 4°C with the following primary antibodies: CCDC106 (1:100, ab105354, Abcam, Cambridge, UK), GAPDH (1:5000, Sigma, St. Louis, MO, USA), Myc-tag, Cyclin A2, Cyclin B1, Cyclin D1, Cyclin D2, Cyclin D3, Cyclin E1, Cyclin E2, Cyclin H, p-P38, P38, p-ERK, ERK, p-AKT, and AKT (1:1000; Cell Signaling Technology, Danvers, MA, USA). Membranes were washed and incubated with peroxidase-conjugated anti-mouse or anti-rabbit IgG (Santa Cruz Biotechnology) at 37°C for 2 h. Bound proteins were visualized using electrochemiluminescence (Pierce, Rockford, IL, USA) and detected with a bio-imaging system (DNR Bio-Imaging Systems, Jerusalem, Israel).

Lysis buffer (Pierce, Rockford, IL, USA) was used to extract total protein. Fifty μg of each protein sample were separated on SDS-PAGE, and were transferred onto polyvinylidene fluoride membranes (PVDF, Millipore, Billerica, MA, USA). Primary antibodies used targeted: CCDC106 (1:1000, ab105354, Abcam, Cambridge, UK), GAPDH (1:1000, Sigma, St. Louis, MO, USA), ATF4 (1:1000, Proteintech, Wuhan, China) and Cyclin A2, Cyclin B1, Cyclin D1, Cyclin D2, Cyclin D3, Cyclin E1, Cyclin E2, Cyclin H, p21, p27, E-cadherin, N-cadherin, Vimentin, Slug, Snail, Claudin-1, Claudin-4, MMP2, MMP7, MMP9 and ZEB1 (1:1000; all from Cell Signaling Technology, Danvers, MA, USA). Antibodies were incubated with the PVDF membranes overnight at 4°C. After washing the membranes with PBS, appropriate secondary antibodies, peroxidase-conjugated anti-mouse or anti-rabbit IgG (1:5000, Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) were incubated at 37°C for 2 h. Electrochemiluminescence (Pierce, Rockford, IL, USA) and a bio-imaging system (DNR BioImaging Systems, Jerusalem, Israel) were used to detect and analyze the bound antibodies.

The formalin-fixed and paraffin-embedded tissues were cut into sections at a thickness of 4 μm. Antibodies were incubated overnight at 4°C at the following dilutions: 1:200 for CCDC106 (Abcam, Cambridge, MA, USA); 1:100 for p53 (Cell Signaling Technology, Danvers, MA, USA); 1:200 for p21 (Proteintech, Wuhan, China) and 1:500 for ATF4 (Proteintech, Wuhan, China). A biotin-labeled secondary antibody (Ultrasensitive; MaiXin, Fuzhou, China) was incubated at room temperature for 30 min, and color was developed using 3,3’-diaminobenzidine (DAB) Chromogen solution (Dako, Tokyo, Japan).
The expression of CCDC106, p53, p21 and ATF4 was scored according to the number of positively stained cells and their staining intensity. Expression was divided into four grades according to the color intensity: 0 (no color), 1 (light yellow particles), 2 (medium strength yellow particles) and 3 (dark yellow or tawny particles). According to the staining area of cells, there were four grades: 1 (1%-25%), 2 (26%-50%), 3 (51%-75%) and 4 (76%-100%). A final score of 0–12 was obtained by multiplying the intensity and percentage scores.