br Transwell cell migration assay
2.6. Transwell cell migration assay
Forty-eight hours after transient transfection with Con-siRNA, Bach1-siRNA, Ad-GFP or Ad-Bach1, A2780 cells were harvested using trypsin and counted. Forty thousand cells were added to the top chambers of the Transwell plates (8 μm pore size; Corning, NY, USA) in 200 μL of serum-free medium. In the lower compartments of the chambers, 500 μL of RPMI 1640 medium supplemented with 20% FBS was added as an inducer to promote cell migration. After 48 h of in-cubation at 37 °C in a 5% CO2 incubator, the cells that migrated through the filters were fixed, stained with a 0.1% crystal violet solution, pho-tographed, and counted. The experiment was repeated three times, and statistical significance was analyzed using Student's t-test .
2.7. Western blot analysis
Cell lysates (30 μg of protein) from each group were separated on a sodium dodecyl sulfate (SDS)-polyacrylamide gel and transferred onto a PVDF membrane (Invitrogen, Carlsbad, CA). Membranes were blocked
in 5% milk solution for 2 h at room temperature and were then in-cubated with primary antibody overnight at 4 °C with agitation. Detection was performed using an HRP-labeled secondary antibody and enhanced chemiluminescence (ECL, Pierce, Rockford, IL, USA) reagents according to the manufacturer's instructions. The 1360614-48-7 used in this study are listed in Supplementary Table 2.
2.8. Xenograft experiments
All studies involving experiments with animals were approved by the Ethics Committee of Experimental Research at Fudan University Shanghai Medical College, adopting the “Guide for the Care and Use of Laboratory Animals” published by the United States National Institutes of Health.
Six-week-old nude mice (six mice per group, grouped as WT, KO, Lenti-Con and Lenti-Bach1) were injected subcutaneously in the bi-lateral flank area with 3 × 106 cells in 100 μL of phosphate-buﬀered saline (PBS) mixed with 100 μL of Matrigel. Tumor growth was mon-itored every 3 days, and the tumor volume was calculated according to formula V(mm3) = a × b2/2, where a is the largest diameter and b is the perpendicular diameter. Three weeks after injection, the mice were euthanized with an intraperitoneal injection of 2% sodium pento-barbital (50 mg/kg), and the tumor weight was measured. A portion of the tumors was fixed in 10% buﬀered formalin solution for subsequent histological examination, and the remaining tissue was snap frozen in liquid nitrogen and stored at −70 °C for molecular studies.
For the in vivo tumor metastasis assay, eight nude mice per group were injected intraperitoneally (i.p.) with 5 × 106 cells in 200 μL of PBS [24,25]. Five weeks after i.p. injection, the visceral organs (liver, in-testine, mesentery, kidney, ovary and diaphragm) were observed. The visceral organs were removed, and a portion of the liver tumors were fixed in 10% buﬀered formalin solution for subsequent histological examination. The remaining tissue was snap frozen in liquid nitrogen and stored at −70 °C for molecular studies.
2.9. Immunohistochemical (IHC) staining
Thirty paraﬃn-embedded specimens from tissues representing dif-ferent stages of ovarian cancer and 5 specimens from adjacent non-cancer tissues were obtained from the Obstetrics & Gynecology Hospital of Fudan University (Shanghai Red House Ob & Gyn Hospital). The study was approved by the Ethics Committee (permit no. 2017–25).
Slides of tissue from patients or xenografts were incubated with anti-Bach1 rabbit polyclonal (1:200 dilution; DF8317, Aﬃnity), anti-Slug (1:100 dilution; DF6202, Aﬃnity), anti-Ki67 (1:100 dilution; GB13030-2, Goodbiotechnology, Wuhan,China) or anti-HMGA2 (1:250 dilution; SAB2701959, Sigma-Aldrich, Buchs, Switzerland) antibodies at 4 °C overnight, with normal rabbit serum used as the negative con-trol, followed by incubation with a horseradish peroxidase-conjugated anti-rabbit secondary antibody (Santa Cruz, CA, USA) at 37 °C for 1 h. The signals were detected using a diaminobenzidine (DAB) substrate kit (Vector Laboratories, Burlingame, CA, USA). Counterstaining was per-formed with hematoxylin. An immunohistochemical score (IHS) was used to evaluate Bach1, Slug or HMGA2 expression in human specimens as previously described . The expression of Bach1 and Slug in mouse xenografts was quantified using ImagePro Plus (IPP) according to three parameters: integrated optical density (IOD), area and mean optical density = IOD sum/area.
2.10. In vitro coimmunoprecipitation (co-IP) assay
The Bach1-FLAG and HMGA2-HA plasmids were cotransfected into HEK293T cells using Lipofectamine 2000 (Invitrogen) and cultured in 10 cm dishes for 48 h to obtain 90% confluence. The cell lysates were transferred to microcentrifuge tubes and centrifuged at 12,000×g for 15 min at 4 °C. The supernatants were transferred to another tube and