Fig 6.

Pax3 is implicated in melanoma cell migration and invasiveness. (A) Relative Brn-2, Pax3, and Mitf mRNA levels of 501mel and SKmel28 cells were determined by RT-qPCR. Results represent the means from three independent experiments with error bars indicating standard deviations. (B) Western blot of 501mel, B16, and Skeml28 cells after 24 h of treatment with LY294002 using anti-Brn-2 and anti-Pax3 antibodies. Anti-p-S6 was used as a control of LY294002 efficiency, and total S6 (tS6) and Erk2 were used for the loading control. p-Erk was used for PI3K inhibition specificity. (C) Pax3 knockdown decreases the wound-healing ability of the B16 cell line in a scratch/wound assay. (D) Pax3 knockdown decreases invasiveness potential of B16 cells. After 24 h, the number B16ShPax3 clone 1 invading cells was counted and compared to the control (parental cell line). (E) Brn-2 expression is sustained upon LY294002 exposure when Pax3 is overexpressed. Brn-2 and Pax-3 expression analysis was assessed by Western blotting of Skmel28 parental or Skmel28 cells expressing mCherry-Pax3 protein. Anti-p-S6 was used as a control of LY294002 efficiency, and total S6 (tS6) and Erk2 were used for the loading control. Quantification was achieved using ImageJ software. (F) LY294002 does not decrease invasion of Skmel28 melanoma cells when Pax3 is overexpressed. After 24 h, the number of Skmel28 parental cells or those expressing mCherry-Pax3 treated with LY294002 was counted and compared to the control (DMSO). Columns represent means from three experiments; bars indicate SD; *, P < 0.05.