Cell culture and reagents
The immortalized lung epithelial cells NL20 and MRC5 and human NSCLC A549, H1299 and H460 cells were purchased from American Type Culture Collection (ATCC, Manassas, VA). All cells were grown in a humidified incubator at 37℃ with 5% CO2 and subjected to mycoplasma analysis routinely. The cell culture medium of RPMI-1640 and Fetal Bovine Serum (FBS) were obtained from Invitrogen (Grand Island, NY). The natural products bergenin, MG 132, and cycloheximide (CHX) were obtained from Selleck Chemicals (Houston, TX). Transfection reagent lipofectamine 2000 was purchased from Thermo Fisher Scientific (Waltham, MA). Antibodies against survivin, cleaved-caspase 3, cleaved-PARP, cleaved-caspase 3, cytochrome C, Bax, VDAC1, p-Survivin Thr34, p-Akt Ser473, p-Wee1 Ser642, p-CDK1 Tyr15 and p-CDK1 Thr161, ubiquitin, Flag-tag, HA-tag, α-Tubulin and β-actin were purchased from Cell Signaling Technology, Inc. (Beverly, MA). The anti-ki 67 and FbxL 7 antibodies were products of Abcam (Cambridge, United Kingdom).
The MTS assay, used to analyze cell viability, was performed as described previously14. Briefly, human NSCLC cells were seeded into the 96-well plates (2×103 cells/well) and maintained overnight. Cells were incubated with various concentrations of bergenin for 24, 48, and 72 h. Then cell viability was analyzed according to the standard protocol after adding the MTS reagent (#G3580, Madison, WI) to the cell culture medium.
Anchorage-independent cell growth.
The Anchorage-independent cell growth assay was performed as described previously15. Briefly, Eagle’s basal medium consists of 0.6% agar, 10% FBS and various concentration of bergenin, which were loaded in a 6-well plate as an agar base. Human NSCLC cells were resuspended at a concentration of 8000 cells/ml and inoculated in 6-well plates containing 0.6% Basal Medium Eagle (0.3% agar, 10% FBS, and different doses of bergenin. Colonies were counted after maintaining for 2 weeks.
Clinical tissue sample collections.
A total of 39 cases of NSCLC tissues and matched adjacent non-tumor tissues were collected from 39 patients in the department of pathology, The Third Xiangya Hospital of Central South University, Changsha, Hunan, China. All the patients signed written informed consent and did not receive treatment before surgery.
The cells were treated with bergenin and lysed in RIRA buffer (#89901, Thermo Fisher Scientific) at 4℃ for around 30min followed by centrifugation at 12,000 × rpm for 10min to collect supernatant as the whole-cell extract (WCE). Then the BCA protein assay kit (#23228, Thermo Fisher Scientific) was used to detect the WCE concentration. A total of 20ug WCE was subjected to SDS-PAGE gels and transferred to the PVDF membrane. Subsequently, the membranes are blocked with 5% non-fat milk for 1 h and incubated with primary and second antibodies. The ECL reagent (#34579, Thermo Fisher Scientific) was used to visualize the target protein expression.
Total proteins from NSCLC cells cultured in 10 cm culture dishes were used for the Co-IP assay. Briefly, primary antibodies against HA or Flag were added to each cell lysate and incubated overnight at 4℃ with rotation. Protein A/G agarose beads were added and incubated with mild rotation at 4°C for 2 h to bind to the primary antibody. The agarose beads were washed with PBS and cell lysis buffer to wash off unbound antibodies. The agarose beads were resuspended in 20 µl 1x SDS-PAGE loading buffer and boiled and centrifuged to collect the supernatant for western blot analysis.
Generation of survivin knockdown stable cell lines.
To generate survivin knockdown stable cells, 293T cells were co-transfected with pLKO.1-shsurvivin lentivirus plasmids (TRCN0000073718, TRCN0000073721, Millipore Sigma), PSPAX2 and PMD2-G. The supernatant containing viral was collected at 72 h after transfection. NSCLS cells were grown at 70%-80% confluence and infected with the lentivirus and polybrene (5 µg/ml). The infected cells were cultured with fresh medium containing puromycin (1µg/ml) and maintained for 1 week for colony selection.
In vivo tumor growth.
The in vivo tumorigenesis was approved by the Institutional Animal Care and Use Committee (IACUC) of the Third Xiangya Hospital of Central South University (Changsha, China). A549 and H1299 cells (2×106) were injected into the right flank of 6-week-old athymic nude mice (n = 5). Tumor growth was monitored, and volume was measured every 2 days. The tumor-bearing mice were randomly divided into two groups (n = 5) when tumor volume reached around 100 mm3. The treatment group was initiated bergenin (30 mg/kg) via intraperitoneal injection every two days, and the control group was administrated the vehicle control. The mice were euthanized with CO2 (3 L/min) for 5 min at the endpoint. Tumor volume was calculated following the formula: length × width × width/2.
Immunohistochemical staining (IHC).
The IHC staining was performed as described previously16. Tumor tissues obtained from clinical samples and xenograft tumors were subjected to IHC analysis. Briefly, the tissue sections were deparaffinized and rehydrated. The antigen retrieval was performed subsequently by submerging into sodium citrate buffer (10 mM, pH 6.0) and boiled for 10 min. Then the tissue slides were treated with 3% H2O2 in methanol for 10 min, washed with PBS and blocked with 10% goat serum albumin, and incubated with primary antibodies overnight at 4℃. After hybridization with secondary antibodies at room temperature, the target protein was visualized using the DAB substrate and counterstained by hematoxylin.