Extended Data Fig. 5: PAK4 inhibition disrupts WNT signalling and melanogenesis.
a, Cells were cultured with 2µM KPT-9274 for 72 hours before nuclear protein isolation. Showing immunoblots for nuclear β-catenin, nuclear phosphor-β-catenin (S675) and nuclear PAK4 protein levels. Results are representative from two independent experiments. b, Cells were cultured with 2µM KPT-9274 for 72 hours and Wnt-3a for 8 hours prior to Topflash assay (n= 3 per group). Pharmacological inhibition of PAK4 significantly decreases sensitivity to Wnt-3a stimulation (P= 0.005 for WT Wnt3a vs WT KPT-9274 + Wnt3a comparison). c, Baseline WNT activity levels assessed by Topflash assay of cell treated with 2µM KPT-9274 for 72 hours (n= 3 per group) (P > 0.05). Values were normalized to untreated B16 WT CC cells. d, RT-PCR for tyrosinase expression show that PAK4 depletion reduces the expression levels of this gene. Showing means +/- SEM. Results are normalized to B16 WT CRISPR control levels and then log2 transformed (n= 3). e, For image, cells were cultured and harvest upon reaching 80% confluency. B16 WT CRISPR Control cell line maintains melanin production over time while PAK4 KO clones lose their pigmentation. Results are representative from three independent experiments. Means +/- SEM two-tailed unpaired t-test b, c. Unprocessed blots and raw data are provided as a Source Data file a-c.
