APEH inhibition or knockdown inhibits KRASG12V PM localization and nanoclustering. (A) Structure of G01 and ebelactone. (B) MDCK cells stably expressing mGFP–KRASG12V and mCherry–CAAX transfected with NT shRNA or APEH shRNAs were lysed and blotted for APEH. The level of APEH was normalized to the level of β-actin. Representative western blots are shown. Results are mean±s.e.m. (n=3). **P<0.01 between APEH-knockdown and control APEH levels (one-way ANOVA). (C) Cells treated as in B together with MDCK cells stably expressing mGFP–KRASG12V and mCherry–CAAX and treated with ebelactone or G01 for 48 h were imaged in a confocal microscope. Representative images are shown. Scale bar: 10 μm. (D) Upper left, the extent of KRAS mislocalization was quantified by determining the Manders’ coefficients, which evaluate the extent of colocalization of mCherry–CAAX and mGFP–KRASG12V. Results are mean±s.e.m. (n=3). ***P<0.001 between APEH-knockdown and control Manders’ coefficients (one-way ANOVA). Upper right and bottom left, estimated IC₅₀ values for ebelactone or G01 were obtained from the respective Manders’ coefficient (mean±s.e.m.; n=3) dose–response plots. (E) MDCK cells stably expressing mGFP–KRASG12V transfected with NT shRNA or APEH shRNAs were lysed and blotted for APEH. The level of APEH was normalized to the level of β-actin. Representative western blots are shown. Results are mean±s.e.m. (n=3). ***P<0.001 between APEH-knockdown and control APEH levels (one-way ANOVA). (F) Basal PM sheets were generated from cells from E and imaged by EM after incubation with anti-GFP antibody conjugated to 4.5 nm gold particles. Left, the extent of clustering was analyzed with Ripley's K-function expressed as L(r)−r functions, and normalized on the 99% confidence interval (c.i.). L_max, which is the maximum value of L(r)−r, serves as the summary statistic. Values of L_max above the c.i. indicate clustering, with the extent of clustering given by the value of L_max (mean±s.e.m.; n≥12). *P<0.05; **P<0.01 comparing the pattern for control cells with that for APEH knockdown cells (bootstrap tests). Right, gold labeling density on the PM sheets is shown as mean±s.e.m. (n≥12). *P<0.05; **P<0.01 for the difference in gold labeling density (one-way ANOVA). (G) L_max and gold labeling density calculated as in F of basal PM sheets generated from cells treated with vehicle (DMSO) or ebelactone or G01 for 48 h.

Expression of exogenous APEH rescues the KRASG12V mislocalization induced by APEH knockdown or G01 treatment. (A) Cells as in Fig. 2B were transfected with 0.25 μg APEH–Myc cDNA and imaged in a confocal microscope. Representative images are shown. (B) Parallel cultures of the cells as in A were analyzed by immunoblotting. Representative blots are shown. (C) The extent of KRAS mislocalization was quantified by determining the Manders’ coefficients. ***P<0.001 (Student's t-test). (D) MDCK cells stably expressing mGFP–KRASG12V and mCherry–CAAX were transfected with 0.5 or 2.5 μg APEH–Myc cDNA and lysed for immunoblotting. Representative blots are shown. (E) Parallel cultures of the cells as in D were imaged in a confocal microscope and representative images are shown. The extent of KRAS mislocalization was quantified by determining the Manders’ coefficients. There was no significant difference between APEH-overexpressing and control cells (one-way ANOVA). (F) Cells as in D were seeded on 12-well plates and treated with vehicle (DMSO) or G01 for 48 h. Representative images are shown. The extent of KRAS mislocalization was quantified by determining the Manders’ coefficients. **P<0.01; ***P<0.001 compared with cells without APEH overexpression (one-way ANOVA). (G) MDCK cells stably expressing mGFP–KRASG12V and mCherry–CAAX were treated with vehicle (DMSO) or bortezomib for 48 h. Cells were imaged in a confocal microscope. Representative images are shown. The extent of KRAS mislocalization was quantified determining the Manders’ coefficients. Estimated IC₅₀ values were obtained from the respective Manders’ coefficients dose–response plots. All graphs show mean±s.e.m. (n=3). Scale bars: 10 μm.

PtdSer and cholesterol are mislocalized upon APEH knockdown or inhibition. (A) MDCK cells stably expressing mGFP–LactC2 and mCherry–D4H were transfected with NT shRNA or APEH shRNAs and lysed for quantitative immunoblotting. The level of APEH was normalized to the level of β-actin. Representative western blots are shown. Results are mean±s.e.m. (n=3). ***P<0.001 between APEH-knockdown and control cells (one-way ANOVA). (B) Parallel cultures of cells as in A were imaged in a confocal microscope. Representative images are shown. (C) MDCK cells stably expressing mGFP–LactC2 and mCherry–D4H were treated with vehicle (DMSO) or staurosporine (STS) or ebelactone or G01 for 48 h and imaged in a confocal microscope. Representative images are shown. (D) Left, MDCK cells stably expressing mGFP–LactC2 and mCherry–CAAX were transfected with NT shRNA or APEH shRNAs and imaged in a confocal microscope. The extent of LactC2 mislocalization was quantified by determining the Manders’ coefficients. ***P<0.001 (one-way ANOVA). Middle and right, MDCK cells stably expressing mGFP–LactC2 and mCherry–CAAX were treated with G01 or ebelactone for 48 h and imaged in a confocal microscope. The extent of LactC2 mislocalization was quantified by determining the Manders’ coefficients. Estimated IC₅₀ values for ebelactone or G01 were obtained from the respective Manders’ coefficient dose–response plots. Results are mean±s.e.m. (n=3). Scale bars: 10 μm.

The endocytic recycling of EGFR is disrupted by APEH knockdown or inhibition. (A) CHO cells stably expressing mGFP–EGFR were transfected with NT shRNA or APEH shRNAs and lysed for quantitative immunoblotting. The level of APEH was normalized to the level of β-actin. Results are mean±s.e.m. (n=3). Representative western blots are shown. ***P<0.001 between APEH-knockdown and control APEH levels (one-way ANOVA). (B) Cells as in A were serum-starved for 2 h and incubated with 50 ng/ml EGF on ice for 20 min. Excess EGF was washed away and cells were incubated with fresh warm medium. Cells were fixed at different time points and imaged in a confocal microscope. Representative images are shown. (C) CHO cells stably expressing mGFP–EGFR were treated with vehicle (DMSO) or ebelactone. Cells were serum-starved for 2 h and incubated with 50 ng/ml EGF on ice for 20 min. Excess EGF was washed away and cells were incubated with fresh warm medium. Cells were fixed at different time points and imaged in a confocal microscope. Representative images are shown. Scale bars: 10 μm.

APEH knockdown does not alter cellular SM levels. (A) Cells as in Fig. 1D were stained with GFP–lysenin and imaged in a confocal microscope with fixed imaging parameters to compare fluorescence intensities. Representative images are shown. The GFP fluorescence intensity was quantified using ImageJ and normalized to the mean of those in NT shRNA group (set at one). Results are mean±s.e.m. (n≥12). There was no significant difference in the relative fluorescence intensity (one-way ANOVA). (B) Cells as in Fig. 1D were stained with GFP–lysenin and DAPI following by cell permeabilization. Cells were imaged in a confocal microscope with fixed imaging parameters for comparison of fluorescent intensities. Representative images are shown. (C) MDCK cells were treated with vehicle (DMSO), staurosporine (STS), ebelactone or G01 for 48 h, and imaged and analyzed as in A. *P<0.05; **P<0.01; ***P<0.001 compared with DMSO (one-way ANOVA). (D) MDCK cells were treated with vehicle (DMSO), STS, ebelactone or G01 for 48 h and permeabilized. Cells were stained with GFP–lysenin and DAPI and were imaged as in B. Representative images are shown. (E) MDCK cells were treated with vehicle (DMSO), GW4869, G01 or ebelactone for 48 h and lysed. Cell lysates were analyzed for neutral and acid SMase activity. ***P<0.001 (one-way ANOVA). (F) Cells as in Fig. 1D were lysed and analyzed for neutral and acid SMase activity. There was no significant difference between the control and knockdown cells (one-way ANOVA). Graphs in E and F show mean±s.e.m. (n=3). Scale bars: 10 μm.

MAPK signaling is reduced by APEH knockdown or inhibition. (A) MDCK cells expressing KRASG12V were treated with vehicle (DMSO) or ebelactone or G01 for 48 h. These cells, together with the cells as in Fig. 2E, were lysed for quantitative immunoblotting. Representative blots are shown. (B) Levels of ppERK were normalized to the total level of ERK. Levels of KRASG12V, APEH, and endogenous RAS were normalized to the total level of β-actin. Results are mean±s.e.m. (n=3). *P<0.05; **P<0.01; ***P<0.001 between drug-treated or shRNA-transfected and control protein levels (one-way ANOVA).