Fig 1: Palmitoylation of SCRIB is required to suppress HRasV12-induced cell invasion(a) Representative images of different MCF10A stable cell lines cultured in Matrigel/collagen mixture for 6 days. Scale bars represent 50 µm.(b) Quantification of the percentage of acini with invasive protrusions in each cell line. At least 100 acini were analyzed for each cell line in three independent experiments.(c) Representative images of control or ZDHHC7-knockout cells cultured in Matrigel/collagen mixture for 6 days. High magnification (upper, 20×) view and low magnification (lower, 10×) view are shown, respectively. Scale bars represent 50 µm, and 100 µm, respectively.(d) Quantification of the percentage of acini with invasive protrusions in vector control and ZDHHC7-knockout MCF10A cells. At least 100 acini were analyzed for each cell line in three independent experiments.All data are represented as mean ± SEM, n = 6. P values were determined by two-tailed t-test, compared with the vector control cells. *, P< 0.05; ***, P< 0.001.
Fig 2: ZDHHC7-mediated palmitoylation regulates SCRIB localization and YAP translocation(a) Knockout of ZDHHC7 in MCF10A cells led to SCRIB mislocalization. Cells were stained with anti-SCRIB antibody (red), anti-ZDHHC7 antibody (green) and DAPI (blue).(b) Quantification of the percentage of cells with mislocalized SCRIB in ZDHHC7 knockout cells.(c) Expression of WT ZDHHC7, but not the C160S mutant partially restored SCRIB membrane localization in ZDHHC7 KO cells. Cells were stained with anti-SCRIB antibody (red), anti-ZDHHC7 antibody (green) and DAPI (blue).(d) Quantification of the percentage of cells with mislocalized SCRIB in different ZDHHC7 knockout cells.(e) Knockout of ZDHHC7 in MCF10A cells led to increased YAP nuclear localization. Cells were stained with anti-SCRIB antibody (red), anti-YAP antibody (green) and DAPI (blue).(f) Quantification of the percentage of YAP nuclear localization in ZDHHC7 knockout cells.In all images, the yellow line indicates the position of the Z stack (XY or XZ). Scale bars represent 20 µm.Data are represented as mean ± SEM, n=3. P values were determined by two-tailed t-test. *, P<0.05, **, P<0.01.
Fig 3: ZDHHC7 is a major palmitoyl acyltransferase regulating SCRIB palmitoylation(a) Overexpression of ZDHHC7 significantly increased SCRIB palmitoylation levels. HEK293A cells were co-transfected with Flag-SCRIB construct and HA-ZDHHC constructs or empty vector.(b) Flag-SCRIB physically interacted with HA-ZDHHC7 in co-immunoprecipitation assays when co-transfected into cells.(c) Endogenous SCRIB interacted with HA-ZDHHC7 in co-immunoprecipitation assays.(d) The catalytic inactive mutant of ZDHHC7 (C160S) failed to induce SCRIB palmitoylation.(e) The N-terminal LRR domains of SCRIB are required and sufficient for ZDHHC7-SCRIB interaction.(f) Expression of siRNA-resistant construct of mouse WT HA-ZDHHC7 rather than the catalytically inactive mutant (C160S) restored SCRIB palmitoylation.All blots are representatives of at least three independent experiments.See Supplementary Figure 35 for full images of the blots.
Fig 4: ZDHHC6 is a palmitoyltransferase for AEG-1. Knockout of ZDHHC3 (A) or ZDHHC7 (B) in HEK293T cells did not decrease AEG-1 palmitoylation as measured by Alk14 labeling (top). Knockdown of ZDHHC6 in HEK293T cells by two different shRNA (knockdown efficiency about 50% (C), decreased the palmitoylation level of AEG-1 (D). ZDHHC6 Sh2 decreased AEG-1 palmitoylation, but not the palmitoylation level of a negative control, RalA (E).
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