Ki 67

Protein was extracted from hUC-MSC samples or liver tissue using radioimmunoprecipitation assay (RIPA) lysis buffer. The primary antibodies were insulin receptor substrate 1 (IRS-1), phosphoinositide 3-kinase (PI3K, 1:1200, Immunoway Biotechnology), protein kinase B (AKT, 1:1200, Immunoway Biotechnology), phosphoinositide-dependent protein kinase 1(PDK1, 1:1200, Immunoway Biotechnology), glucose transporter type 4 (GLUT4, 1:1200, Immunoway Biotechnology), B-cell lymphoma-2-associated X (BAX, 1:1200, Immunoway Biotechnology), proliferating cell nuclear antigen (PCNA, 1:1200, Immunoway Biotechnology), B-cell lymphoma-2 (BCL-2), cell cycle-associated proteins (Ki-67, cyclin A, cyclin E, 1:1200, Immunoway Biotechnology), A 160 kDa substrate of the Akt Ser/Thr kinase (AS160, 1:1200, Immunoway Biotechnology), cysteine protease protein (Caspase3, 1:1200, Immunoway Biotechnology) glyceraldehyde phosphate dehydrogenase (GAPDH, 1:3000), and β-actin (1:3000). The secondary antibodies conjugated to horseradish peroxidase (1:3000, Immunoway Biotechnology). The normalization of other proteins was performed using β-actin and GAPDH western blot assays [19 (link)].

Tumor xenografts were fixed in paraformaldehyde (Servicebio, G1101, Wuhan, China) for 12 h and embedded in paraffin. After that, the KeyGEN One-Step IHC Assay (KeyGen, KGOS60-KGO300, Nanjing, China) was used to stain 5-μm-thick paraffin sections, which were then incubated with the following antibodies: Brdu (Servicebio, GB12051, Wuhan, China), Ki-67(ImmunoWay, YT2467, TX, USA), PTPN1(Abcam, ab75856, Cambridge, UK), MAP3K11(Abcam, ab51068, Cambridge, UK), JNK1/2/3 (Bimake, A5005, TX, USA), and c-Jun (Bimake, A5730, TX, USA). The paraffin sections were then photographed.

Western blot assays were performed to detect the protein expression of Reg3A, Ki67, PCNA, and β-actin (ImmunoWay, USA). Cells were lysed with Lysis Buffer (CST, USA) on ice for 30 min, which was centrifuged at 14,000 rpm for 10 min at 4°C. The protein samples were normalized using a BCA assay kit (Shanghai, China). Equal amounts of protein lysates were electrophoresed on SDS-PAGE gels and then transferred onto nitrocellulose membranes. After blocked 1 h at room temperature, membranes were incubated with primary antibody overnight at 4°C. Membranes were then washed in PBST (PBS with 0.1% Tween) and incubated with horseradish peroxidase-labeled secondary antibody for 1 h at room temperature. After being washed in PBST three times, membranes were visualized by enhanced chemiluminescence reagents (KeyGen, China).