Fig 2: PPP6C regulates ERK signaling via MEK1/2(A) shCTRL, shPPP6C-1, and shPPP6C-2-expressing 501mel cells were transfected with non-targeting control siRNA or siRNAs directed to ARAF, BRAF, or CRAF as indicated. Cells were lysed and assessed by immunoblot for phosphorylated and total MEK and ERK.(B) Quantification of the relative levels of Phospho/Total MEK and ERK from (A) was normalized to shCTRL, siCONTROL. Data are represented as mean ± SD, n = 3.(C) BRAF was immunoprecipitated from 501mel cells expressing shCTRL, shPPP6C-1, or shPPP6C and evaluated in vitro in kinase assays on MEK1 over the indicated time course. Vemurafenib (1 µM) was added to negative control reactions. Reactions were evaluated by immunoblot.(D) 501mel cells expressing shCTRL, shPPP6C-1, and shPPP6C-2 were lysed and assessed by immunoblot for MEK phosphorylation at Ser218/Ser222, Thr286, and Ser298. Non-specific cross-reacting bands in the pThr286 and pSer298 blots are indicated with an asterisk.(E) Quantification of the relative levels of Phospho/Total MEK from (D) was normalized to shCTRL. Data are represented as mean ± SD, n = 3.(F) PPP6C+/+ and PPP6C-/- 501mel cell lines were transiently transfected to express His epitope-tagged MEK1 or MEK2. Cell lysates were analyzed by immunoblot for phosphorylated and total MEK. Open arrows indicate ectopically expressed His-tagged MEK1/2, and solid arrows indicate endogenous MEK1/2. See also Figure S5.

Fig 3: PPP6C regulation of ERK signaling is prominent in ERK pathway-driven cancer cells(A) The indicated cell lines were transduced to stably express control (shCTRL) or PPP6C-targeting (shPPP6C-1 or PPP6C-2) shRNAs. Cells were lysed and assessed by immunoblot for levels of phosphorylated and total MEK and ERK. Quantification is shown in Figure S3. n = 2.(B) PPP6C CERES scores for skin cancer cell lines with WT BRAF or hotspot BRAF mutations from the Cancer Dependency Map Project. Cell lines harboring BRAF variants of unknown significance were excluded. Data are represented as mean ± SD. ***p < 0.0005, Welch’s t test.(C) Heatmaps depicting CERES scores of PPP6C, ERK-selective DUSPs, and ERK MAPK cascade components in skin cancer cell lines using data from the Cancer Dependency Map Project. Pearson’s correlation coefficients and p values from linear regression analysis of each gene with PPP6C in the DepMap portal are listed.(D) PPP6C RNA-seq mRNA expression level plotted against MEK1 phosphorylation levels for TCGA tumor samples in cBioPortal. Correlation coefficients and associated p values from linear regression analyses are indicated.(E) PPP6C mRNA levels plotted against ERK2 phosphorylation levels for TCGA tumor samples as in (D).See also Figure S4.