Tunicamycin br In the current study we investigate the e ect
In the current study, we investigate the eﬀect of activin A on E-cadherin expression in ovarian cancer cells and explore the underlying molecular mechanisms. Our results show that activin A treatment downregulates E-cadherin expression in two human ovarian cancer cell lines, SKOV3 and OVISE. Inhibition of ALKs or siRNA-mediated knockdown of ALK4 blocks the inhibitory eﬀect of activin A on E-cadherin expression. Knockdown of SMAD2, SMAD3 or SMAD4 at-tenuates the activin A-induced downregulation of E-cadherin. Moreover, activin A upregulates the expression of E-cadherin tran-scriptional repressors (SNAIL and SLUG), the knockdown of which abolishes activin A-induced E-cadherin downregulation. Importantly, forced-expression of E-cadherin decreases both basal and activin A-stimulated cell migration. This study demonstrates that activin A in-duces human ovarian cancer cell migration by inducing SNAIL- and SLUG-mediated E-cadherin downregulation.
2. Materials and methods
The SKOV3 cell line was obtained from American Type Culture Collection (ATCC, Manassas, VA). OVISE cells were generously pro-vided by Dr. David Huntsman (University of British Columbia, Vancouver, BC). Cells were cultured in a 1:1 (v/v) mixture of M199/ MCDB105 medium (Sigma-Aldrich, Oakville, Ontario, Canada) sup-plemented with 10% fetal bovine serum (FBS; Hyclone Laboratories Inc., Logan, UT) and 1% penicillin/streptomycin (Gibco). Cultures were maintained at 37 °C in a humidified Tunicamycin of 5% CO2 in air and were serum starved for 24 h prior to treatment.
2.2. Antibodies and reagents
2.3. Reverse transcription quantitative real-time PCR (RT-qPCR)
Total RNA was extracted using TRIzol (Invitrogen, Thermo Fisher Scientific, CA) according to the manufacturer's instructions. Reverse transcription was performed with 3 μg RNA, random primers and Moloney murine leukemia virus (M-MLV) reverse transcriptase (Promega, Madison, WI). RT-qPCR was performed using an Applied Biosystems 7300 Real-Time PCR System equipped with 96-well optical reaction plates. TaqMan gene expression assays for ACVR1B (ALK4; Hs00244715_m1) and GAPDH (Hs02758991_g1) were purchased from Applied Biosystems (Thermo Fisher Scientific). Each 20 μL TaqMan RT-qPCR reaction contained 1 × TaqMan Gene Expression MasterMix (Applied Biosystems), 80 ng cDNA, and 1 × specific TaqMan gene ex-pression assay containing primers and probe. Alternatively, each 20 μL SYBR Green RT-qPCR reaction contained 1 × SYBR Green PCR Master Mix (Applied Biosystems), 80 ng cDNA and 250 nM of each specific primer. Thermal cycling conditions were 50°C for 2 min, 95 °C for 10 min, and 40 cycles of 95 °C for 15 s and 60 °C for 1 min. Relative quantification of mRNA levels was performed by the comparative Cq method with GAPDH as the reference gene and using equation 2– Cq. The primers used in this study were: E-cadherin (CDH1), 5′-ACA GCC CCG CCT TAT GAT T-3′ (sense) and 5′-TCG GAA CCG CTT CCT TCA-3′ (antisense); N-cadherin (CDH2), 5′-GGA CAG TTC CTG AGG GAT CA-3′ (sense) and 5′-GGA TTG CCT TCC ATG TCT GT-3′ (antisense); SNAIL (SNAI1), 5′-CCCCAATCGGAAGCCTAACT-3′ (sense) and 5′-GCTGGAA GGTAA ACT CTG GAT TAG A-3′ (antisense); SLUG (SNAI2), 5′-TTC GGACCC ACA CAT TAC CT-3′ (sense) and 5′-GCAGTGAGGGCAAGA
Cells were lysed in lysis buﬀer (Cell Signaling Technology, MA) supplemented with protease inhibitor cocktail (Sigma). Equal amounts of protein were separated by 10% SDS-PAGE and transferred to PVDF membranes (Bio-Rad). After blocking with 5% non-fat dry milk in Tris-buﬀered saline (TBS) for 1 h at room temperature, membranes were incubated at 4 °C overnight with the indicated primary antibodies di-luted 1:1000 in TBS with 5% milk. Membranes were then washed and probed with a horseradish peroxidase (HRP)-conjugated secondary antibody for 1 h at room temperature. Immunoreactive proteins were detected using enhanced chemiluminescent substrate (Pierce, Thermo Fisher Scientific) and X-ray film. Densitometric quantification was performed using ImageJ software, and GAPDH was for normalization.