Figure 1.

Effect of Activation of RAC1^P29S on Survival of Melanocytes

(A) Schematic of the ER-RAC1^P29S fusion protein system. ER, estrogen receptor; 4OHT, 4-hydroxytamoxifen; HSP, heat shock protein.

(B) RAC1-GTP assay in MCF10A cells (left) and mouse melanocyte cell line melan-a (right) stably expressing ER-RAC1^P29S. Cells were treated with 500 nM 4OHT for 24 h and ER-RAC1^P29S binding to the PAK1 RAC1 binding domain was assessed using a pull-down assay.

(C) PAK1/2 and ERK1/2 phosphorylation by ER-RAC1^P29S in melanocytes. Cells were treated with 500 nM 4OHT for 24 h.

(D) Quantification of phospho-PAK1/2 and phospho-ERK1/2 levels detected by immunoblotting, normalized using vinculin loading control (n = 3 or more independent experiments); t test versus 0 h was used with Holm-Sidak correction for multiple testing; ^∗p < 0.05, ^∗∗∗p < 0.001; n.s., not statistically significant.

(E) Morphological changes induced by ER-RAC1^P29S activation in melan-a melanocytes treated with 500 nM 4OHT.

(F) Viability of melan-a melanocytes with ER-RAC1^P29S in growth factor-reduced medium (fetal calf serum [FCS] 0.25%, 12-O-tetradecanoylphorbol-13-acetate [TPA]-free). Cells were treated with 1 μM 4OHT and viability was quantified using CellTiter-Glo (n = 3 independent experiments); t test was used for statistical comparison.

(G) Soft agar sphere formation of melan-a melanocytes with activated ER-RAC1^P29S. Cells were grown in full medium containing 1 μM 4OHT for 3 weeks before staining, imaging, and automated counting (n = 3 biological replicates).

(H) Effect of activation of ER-RAC1^P29S on apoptosis in melan-a melanocytes. Cells were treated with 1 μM 4OHT. Cleaved caspase-3 was quantified and normalized to cell viability (n = 2 independent experiments, three biological replicates per experiment).

Bars represent means ± SD. See also Figure S1.