Transient transfection of mimics or inhibitors miR-200b and miR-34c, GATA3 siRNA (GATA3 si), control siRNA (con si) (San Cruz Biotech), GATA3 plasmid (GATA3 P), and control plasmid (con P) was performed using a Neon Transfection System (Invitrogen Corporation, Carlsbad, CA) according to the manufacturer’s protocol. Briefly, cells were electroporated and incubated with indicated miRNAs, siRNAs, and plasmids. Experiments were initiated forty-eight hours after the transfection.
For establishing stable transfectants, the plasmids of p38WT, p38D179, and control plasmids were transfected into MCF7 cells using a Neon Transfection machine (Life Technologies). Positive colonies were selected in standard cell culture media containing G418 (400 μg/ml). Short hairpin RNA (shRNA) of p38γ MAPK (p38γsh) or scrambled control shRNA (Santa Cruz Biotechnology) was transfected into MCF7, MCF7-ErbB2, and BT474 cells using a Neon Transfection machine (Life Technologies). Positive colonies were selected in standard cell culture media containing 4 μg/ml puromycin. Cell lysates were collected and analyzed by immunoblotting for the verification of the overexpression or silencing of p38γ MAPK.
For establishing stable transfectants, the plasmids of p38WT, p38D179, and control plasmids were transfected into MCF7 cells using a Neon Transfection machine (Life Technologies). Positive colonies were selected in standard cell culture media containing G418 (400 μg/ml). Short hairpin RNA (shRNA) of p38γ MAPK (p38γsh) or scrambled control shRNA (Santa Cruz Biotechnology) was transfected into MCF7, MCF7-ErbB2, and BT474 cells using a Neon Transfection machine (Life Technologies). Positive colonies were selected in standard cell culture media containing 4 μg/ml puromycin. Cell lysates were collected and analyzed by immunoblotting for the verification of the overexpression or silencing of p38γ MAPK.