Fig 3.

Pax3 regulates Brn-2 transcription. (A) Luciferase assay of B16, SKmel28, and 501mel cells transfected with a Brn-2-luciferase reporter together with a Pax3 expression vector or control vector as indicated. Data from a representative experiment are presented as fold changes compared to the control (set to 1). Columns represent means from four experiments; bars indicate SD; *, P < 0.05. (B) Western blot using anti-Brn-2 or anti-Pax3 antibodies of 501mel transfected with an siRNA of Pax3 or scrambled (control) as indicated. Erk2 was used as a loading control. (C) Relative expression of Pax3 and Brn-2 mRNA as determined using RT-qPCR from two independent clones of B16F10 depleted for Pax3 using shRNA compared to the WT B16F10. Values were normalized to GAPDH and are shown as fold changes. (D) Western blot of Pax3 protein in parental and shPax3 B16-expressing clones 1 and 2. ERK was used as a loading control. (E) Chromatin immunoprecipitation (ChIP) of B16 cells using an anti-Pax3 or anti-Lef1 antibody as a control. Amplification of the Brn-2 promoter was detected by semiquantitative PCR using primers specific for the Brn-2 promoter. (F and G) ChIP of Pax3 and RNA PolII followed by qPCR at the Brn-2 promoter or an unrelated region 1.8 kb upstream using IgG as a control in the indicated cell lines.