N cad

Antibodies for EMT, cell cycle, and DNA damage repair pathway were purchased as follows: ZEB-1 (CST:3396), N-CAD (CST:13116), E-CAD (CST:3195), Slug (CST:9585), Snail (CST:3879), Vimentin (CST:5741), β-catenin (CST:8480), CCND1 (CST:92G2), p21 (CST:2947 s), CDK4 (Abcam: Ab108357), CDK6 (Abcam:Ab124821), total ATM (CST:2873), phospho-ATM (Ser1981) (Abcam:5883), phospho-Chk2 (Thr68) (CST:2197), phospho-H2AX (Ser139) (CST:9718), RAD51 (Abcam:ab133534), and β-actin (CST:3700). All the western blot primary antibodies were 1:1000 diluted except RAD51 (1:10000 diluted). The antibody of HMGA2 (Proteintech, 20795-1-AP) was used in Immunohistochemistry (IHC).

Antibodies that recognize Cyclin D1 (Cell Signaling Technology, #2922), CDK4 (Cell Signaling Technology, #12790), β-actin (Sigma, A1978), NCAD (Abcam, ab98952), ECAD (Cell Signaling Technology, #3195), FOXO3 (Abcam, ab12162) and CTNNB1 (Ser33/37/Thr41) (Cell Signaling Technology, #4270) were used in the study. Horseradish peroxidase (HRP) labelled secondary antibody (Beyotime Biotech) was used in western blots. PS341 was purchased from Santa Cruz, sc-217785.

pFLAG-YAP1 was a gift from Yutaka Hata (addgene plasmid # 66853) and pEGFP-C3-hYAP1 was a gift from Marius Sudol (addgene plasmid # 17843), addgene (Watertown, MA, USA). The following antibodies were used in this study: mouse anti-YAP (dilution- 1:1000 in 5% Milk+TBST, Santa Cruz Biotechnology, SCBT, cat# sc101199, Dallas, TX, USA), rabbit anti-phospho-YAP-Y407 (custom generated by Life Technologies, Carlsbad, CA, USA), mouse anti-NEK1 (1:1000 in TBST, SCBT, cat# sc-398813, Dallas, TX, USA), rabbit anti-phospho-NEK1 pT141 (lab-generated by Life Technologies, Carlsbad, CA, USA), HRP-conjugated anti-β-tubulin (1:1000 in TBST, SCBT, cat# sc-23949, Dallas, TX, USA,), anti-FLAG (1:1500 in 5% Milk+TBST, Cell Signaling Technology, CST, cat# 14793S, D6W5B, Danvers, MA, USA), anti-GAPDH (1:1300 in 5% BSA+TBST, CST, cat# 2118S (14C10), anti-GFP (Thermo Fisher, cat# MA5-15256 (GF28R), Waltham, MA, USA), anti-phospho-ATR (T1989) (1:1000 in 5% Milk+TBST, CST, cat# 58014S), anti-BAX (1:1000 in TBST, SCBT, cat# sc-23959 (6A7),), E-Cad (1:1000 in TBST, CST, cat# 3195S,), N-Cad (CST, cat# 13116S), and rabbit anti-actin (1:1000 in TBST, Abcam, cat# ab1801, Cambridge, MA, USA). Secondary HRP-conjugated antibodies, anti-rabbit (CST, cat# 7074S) and anti-mouse (CST, cat# 7076S) were used to probe immunoblots.

Cells were fixed in 4% paraformaldehyde in DPBS for 20 minutes and permeabilized with 1% Triton X-100 for 20 minutes at room temperature. After 60 minutes blocking with 2% normal goat serum, cells were incubated with primary antibody overnight at 4°C, washed, and stained with secondary antibodies (1:300, goat anti-rabbit IgG-Cy3; or 1:300, goat anti-mouse IgG-Cy3) for 60 minutes at room temperature and then washed three times with phosphate-buffered saline (PBS). The primary antibodies for respective cells include OCT4 (1:200, Abcam), NANOG (1:200, Abcam), PAX6 (1:200, Abcam), SOX2 (1:200, Abcam), NESTIN (1:200, Abcam), SOX1 (1: 200, Abcam), ZO-1 (1:100, Abcam), N-CAD (1: 100, Abcam), MAFB (1:300, Sigma), SOX9 (1:300, EMD Millipore), PRDM1 (1:200, Cell Signaling Technology), and NR2F1 (1:300, R&D Systems). DAPI (4', 6-diamidino-2-phenylindole) (1:500) was used as counter-staining for nuclei. The images were captured and analyzed with the Olympus IX73 and Image J.

Antibodies that recognize pAKT (Ser473) (Cell Signaling Technology, #9271), AKT (Cell Signaling Technology, #9272), pERK (Thr202/Tyr204) (Cell Signaling Technology, #4370), ERK (Cell Signaling Technology, #4695), p85α (Santa Cruz, sc-376112), p110α (Cell Signaling Technology, #4249), β-actin (Sigma, A1978), NCAD (Abcam, AB98952), ECAD (Cell Signaling Technology, #3195), VIM (RV202) (Abcam, AB8978), ZEB1 (D80D3) (Cell Signaling Technology, #3396), pGSK3β (Ser9) (Cell Signaling Technology, #9323), GSK3β (Y174) (Abcam, AB32391), and non-phospho CTNNB1 (Ser33/37/Thr41) (Cell Signaling Technology, #4270) were used in the study. Horseradish peroxidase (HRP) labelled secondary antibody (Beyotime Biotech) was used in western blots. Protein A-Sepharose 4B beads (Invitrogen, 10-1041) was used for immunoprecipitation studies.

Cells were harvested and proteins were extracted from cells as previously described 46 (link). The protein concentration was determined using a protein assay kit (Bio-Rad, Hercules, CA, USA). Equal amounts of proteins (30 µg) were resolved by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred onto a polyvinylidene fluoride (PVDF) membrane (Millipore, Bedford, MA, USA). After blocking with 5% non-fat milk, the membrane was incubated overnight at 4 ºC with the primary antibody. The primary antibodies used in our study were as follows: α-SMA, 1:1000; FAP, 1:1000; FSP1, 1;300; vimentin, 1:1000; E-cad, abcam, #ab216833, 1:500; N-cad, abcam, #ab216833, 1:500; FN1, proteintech, 15613-I-AP, 1:500; Slug, Santa Cruz, sc-166476, 1:200; Snail, Santa Cruz, sc-393172, 1:200, and ZEB1, CST, #70512, 1:1000. Then HRP-conjugated secondary antibodies (1:3000) purchased from ZhongShanJinQiao (BeiJing, China) were incubated and the signal was detected with enhanced chemiluminescence.