Twist1

Western blotting analysis was conducted as described previously in our study (16 (link)). Total cellular proteins were harvested in IP lysis buffer (Beyotime, Shanghai, China) with a cocktail of proteinase and phosphatase inhibitors (Bimake.cn) and were measured by a BCA protein assay kit (Thermo Fisher Scientific, USA). Protein lysates were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to nitrocellulose (NC) membrane (Millipore, USA). Then the membranes were blocked with 5% skimmed milk in Tris-Buffered saline (TBS) for 1 hour at RT and incubated with primary antibodies overnight at 4°C followed by incubation with DyLight fluorescent dye labelled secondary antibodies for 1 hour at RT. Finally, immunoblot signals were detected using an Odyssey Imaging System (LI-COR Biosciences, USA). Primary antibodies used in this analysis were listed as follows: TRIM50 (1:100 dilution, Abcam, ab174880), E-cadherin (1:5000, Proteintech, 20874-1-AP), N-cadherin (1:2000, Proteintech, 22018-1-AP), Vimentin (1:2000, Proteintech, 10366-1-AP), Snail1 (1:1000, Proteintech, 13099-1-AP), Snail2 (1:2000, Proteintech, 12129-1-AP), Twist1 (1:1000, Proteintech, 25465-1-AP), Twist2 (1:1000, Proteintech, 11752-1-AP), ZEB1 (1:1000, Proteintech, 21544-1-AP), ZEB2 (1:2000, Proteintech, 14026-1-AP), and β-actin (1:5000, MultiSciences, ab008).

Western blot analysis was performed to determine the effects of FOXD3 silencing on MAPK signaling pathway and EMT transition in thyroid cancer cells and tumor tissues [12 (link)]. ATC cells were washed with cold PBS and lysed with 1%NP40 and the supernatant was used as total protein lysate. On the other hand, human thyroid tumor samples were homogenized in liquid nitrogen and lysed for 30 min in ice-cold protein extraction buffer. Equal amounts of total protein (50 μg) were separated on a 10% SDS-PAGE and transferred onto nitrocellulose membrane. Then, after blocking, the membranes were incubated with primary monoclonal antibodies overnight at 4°C. The following primary antibodies were used: FOXD3 (Millipore), β-actin (Sigma), TWIST1 (Proteintech) and p-ERK, ERK, E-cadherin and cleaved caspase-3 (Cell signaling). Next, the membrane was incubated with fluorescent labeled secondary antibody for 1 h. Finally, the protein bands were visualized and quantified using the Odyssey Infrared Imaging System (LI-COR, USA).

Western Blot was performed to evaluate the protein levels as describe before28 (link). Briefly, cells were homogenized with NP-40 lysis buffer and then centrifuged the lysate at 12,000 rpm for 20 min to obtain supernatant. After protein concentration was determined by BCA protein assay kit (Thermo), equal amounts of total protein were resolved by 12% SDS-PAGE followed by transferring to PVDF membranes. Blots were blocked for 1 h in 5% BSA and then incubated overnight with the primary antibodies: TBL1XR1 (HPA019182, Sigma), Vimentin (10366-1-AP, Proteintech), Snai1 (13099-1-AP, Proteintech), Slug (12129-1-AP, Proteintech), TWIST1 (18125-1-AP, Proteintech), N-cadherin (13769-1-AP, Proteintech), E-cadherin (20874-1-AP, Proteintech), Cyclin D1 (60186-1-Ig, Proteintech), c-Myc (10828-1-AP, Proteintech) and β-actin (sc-1616, Santa Cruz). After washing three times with TBST, the membranes were incubated for another 1 h with secondary antibodies conjugated with horseradish peroxidase. The immunoreactivity was visualized using the enhanced chemiluminescence (ECL) reagent and autoradiographic film.

Ginsenoside CK (Lot#: BP0651) was obtained from Chengdu Biopurify Phytochemicals Ltd. (Sichuan, China). Cell culture media (DMEM (Lot#: SH30243.01) and RPMI 1640 (Lot#: SH30809.01)) were purchased from HyClone (Logan, UT, USA). MTT (Lot#: ST316), DMSO (Lot#: ST038) and RIPA lysis buffer (Lot#: P0013B) were purchased from Beyotime Biotechnology (Shanghai, China). CoCl₂ (Lot#: C804815) was obtained from Macklin Biochemical Co., Ltd. (Shanghai, China). Trypsin (Lot#: T8150), penicillin/ streptomycin (Lot#: P1400), crystal violet (Lot#: G1063), PMSF (Lot#: P0100), phosphatase inhibitor cocktail (Lot#: P1260) and BCA protein assay reagent kits (Lot#: PC0020) were purchased from Solarbio Science & Technology Co., Ltd. (Beijing, China). FBS (Lot#: 04-001-1ACS) was purchased from Biological Industries (Kibbutz Beit Haemek, Israel). Rabbit antibodies against E-cadherin, Vimentin, N-cadherin, Snail, HIF-1α, Twist1, IκB-α, p65, Lamin B1, ICAM-1, CA9 and c-Myc and mouse antibodies against HIF-1α and VEGF were obtained from Proteintech Group, Inc (Chicago, IL, USA). Anti-phospho-IκB-α and anti-phospho-p65 rabbit antibodies were purchased from Cell Signaling Technology (Boston, MA, USA), and anti-MMP-2 and anti-MMP-9 rabbit antibodies were purchased from Abways Technology (Shanghai, China). Details about antibodies are shown in Table S1.

Total proteins were extracted from cells with RIPA buffer (Sigma Aldrich), separated by SDS‐PAGE gels and transferred to polyvinylidene difluoride (PVDF) membrane (Millipore). The membranes were blocked with 5% nonfat milk and incubated with primary antibodies (β‐CATENIN, AKT, pAKT, mTOR, pmTOR, MEK, pMEK (Cell Signaling Technology), MMP2, SNAI1 (Abcam, Cambridge, UK), IL32, TWIST1, GAPDH (Proteintech) overnight at 4°C, followed by incubation with secondary antibodies for 1 hour at room temperature. The immunoreactive protein bands were detected by ECL detection system. GAPDH was used as a loading control.

The cells were prepared and treated with VP as RT-qPCR above. After VP light activation for 6 h or 12 h, total protein was extracted from cells for Western blot analysis as described previously 15 (link). Antibodies against YAP1 (1:2000 dilution), E-cadherin (1:1000 dilution), Snail (1:1000 dilution), β-catenin (1:2000 dilution), EGFR (1:4000 dilution), programmed death ligand-1 (PD-L1) (1:1000 dilution), Twist1 (1:1000 dilution) and β-actin (1:4000 dilution) were obtained from Proteintech. An antibody against Oct4 (1:1000 dilution) was obtained from BioSS. Antibodies against HPV-16 E6 (1:1000 dilution) and E7 (1:200 dilution) were obtained from Abcam (Cambridge, UK) and Santa Cruz (CA, USA), respectively. A horseradish peroxidase-linked secondary antibody (1:4000 dilution) was obtained from Proteintech.