Treat cells with GABAB receptor agonist

For cell proliferation assay, a total of 1×10⁴ MCF7-V or MCF-GB_1e cells were seeded into 24-well plates and grown in complete DMEM for two days at 37°C with 5% CO₂. Cells were then incubated or not with GABA_B receptor agonist baclofen (100 and 200 μM) or antagonist {"type":"entrez-protein","attrs":{"text":"CGP54626","term_id":"875260408","term_text":"CGP54626"}}CGP54626 (5 and 10 μM) at 37°C for four consecutive days. The media were replaced every two days, and cell proliferation was examined every 24 h by using CCK-8 solution.

To examine the level of pAkt (S473), 4×10⁵ of MCF7-V or MCF-GB_1e cells were seeded into 3.5-cm plates, maintained in complete DMEM for 2 days at 37°C with 5% CO₂ followed by serum starvation for 24 h. Cells were then incubated or not with baclofen (100 and 200 μM) or {"type":"entrez-protein","attrs":{"text":"CGP54626","term_id":"875260408","term_text":"CGP54626"}}CGP54626 (5 and 10 μM) for 24 h at 37°C, and the TCLs were analyzed by immuoblot assay with antibodies against pAkt (S473), Akt, GB₁ and β-actin.

For clonogenic assay, 200 of MCF7-V or MCF-GB_1e cells were seeded into 24-well plates and treated or not with baclofen (100 and 200 μM) or {"type":"entrez-protein","attrs":{"text":"CGP54626","term_id":"875260408","term_text":"CGP54626"}}CGP54626 (5 and 10 μM) for 10–14 days. The media were replaced every 3 days. When colonies were visible to the naked eyes, the plates were washed, fixed, and stained with 1% crystal violet. Images were captured and the number of colonies was counted.