Figure 3: MAP3K8 is upstream of MEK and involved in regulating cyclin D1 and FAK in ovarian cancer cells. | Nature Communications

Figure 3: MAP3K8 is upstream of MEK and involved in regulating cyclin D1 and FAK in ovarian cancer cells.

From: MAP3K8/TPL-2/COT is a potential predictive marker for MEK inhibitor treatment in high-grade serous ovarian carcinomas

Figure 3

(a) Representative western blots showing the phosphorylated form of MEK (P-MEK) and total MEK protein levels in SKOV3 and IGROV-1 OCCL either treated with MAP3K8 kinase inhibitor (KI) or with KI vehicle (DMSO), for 1 h before serum (FBS) stimulation for the indicated times. Actin is used as an internal control for protein loading. (b) Bar plots showing P-MEK/MEK ratio, as assessed by densitometry analysis of western blots (as shown in a) and normalized to the DMSO t=0 h time point (n=3). (c) Western blots showing MAP3K8, P-MEK and MEK protein levels in stable cell lines (shCtrl, shMAP3K8_1 and shMAP3K8_2) derived from SKOV3 and IGROV-1 OCCL and kept without serum (−FBS) or stimulated with serum (+FBS). Actin is used as an internal control for protein loading. (d) Bar graphs showing P-MEK/MEK ratio assessed by densitometry analysis of western blots (as shown in c) and normalized to shCtrl (n⩾3). (e) Representative western blots showing P-p90RSK, p90RSK, cyclin D1, P-FAK and FAK protein levels upon serum (FBS) stimulation for the indicated times in SKOV3 and IGROV-1 OCCL pretreated either with MAP3K8 KI or with KI vehicle (DMSO). Actin is used as an internal control for protein loading. (f) Bar plots showing P-p90RSK/p90RSK ratio, cyclin D1 and P-FAK/FAK ratio, as assessed by densitometry analysis of western blots (as shown in e) and expressed as fold change compared with the DMSO t=0 h time point (n⩾3). (g) Representative western blots showing P-p90RSK, MAP3K8 and cyclin D1 upon silencing of either p90RSK or MAP3K8, or following combined inactivation of both MAP3K8 and p90RSK in SKOV3 ovarian cancer cells. Actin is used as an internal control for protein loading. (h) Bar plots showing p90RSK, MAP3K8 and cyclin D1 protein levels, as assessed by densitometry analysis of western blots (as shown in e) and expressed as fold change compared with the control (siCtrl) (n=3). For all panels, data are shown as mean±s.e.m. P values are based on the Student’s t-test. *P≤0.05; **P≤0.005 and ***P≤0.0005. n stands for the number of replicated independent experiments.

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