Figure 6.
ARF3 controls N-cadherin turnover from the cell surface by regulating association of internalized N-cadherin with recycling endosomes. (A) Confocal images of PC3 acini expressing mNG or ARF3-mNG and either Scr or ARF3 shRNA stained with F-actin (red) and Hoechst (nuclei, blue). F-actin intensity, FIRE LUT. Scale bars, 20 μm. Magnified images of boxed regions shown. White arrows or arrowheads, presence or absence of intense F-actin staining in junctions, respectively. Scale bars, 7 μm. Images representative of phenotypes observed in 3 experimental replicates. (B–D) Representative Western blot of PC3 cells (B) expressing mNG or ARF3-mNG and Scr or ARF3 shRNA for E-cadherin, N-cadherin, and ARF3. GAPDH loading control is shown for each blot. n = 6 independent lysate preparations. Data is presented in C and D as mean fold change ± SEM normalized to Scr. P values (Student’s two-tailed t test), **P ≤ 0.01. (E)ARF3, N-cadherin, and GAPDH mRNA expression in PC3 expressing mNG or ARF3-mNG and either Scr or ARF3 shRNA was determined by RT-qPCR. n = 3 independent RNA and cDNA preparations with 4 technical replicates/condition. Data is mean fold change ± SEM normalized to GAPDH then to Scr. P values (Student’s two-tailed t test), *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, and ****P ≤ 0.0001. (F) IP was performed using an anti-ARF3 antibody or mouse IgG and samples immunoblotted for N-cadherin and ARF3. Panels shown are representative of 3 IPs from 3 independent lysate preparations. (G) Confocal image of PC3 acini stained for ARF3 (green) and N-cadherin (red). Scale bars, 20 μm. Magnified images of boxed regions shown (a–c). Scale bars, 5 μm. White arrows, co-localization in subset of puncta. Image is representative of co-localization observed in cells in 3 experimental replicates. (H and I) PC3 cells expressing (H) mNG and Scr or ARF3 shRNA or (I) mNG and ARF3-mNG with Scr were stained for N-cadherin, HCS WCS, and Hoechst. Cell area, number, and area of N-cadherin puncta was quantified (H) per cell or (I) per sub-cellular region in each cell. n = 5 (1 technical replicate) or 3 (5 technical replicates) independent experiments, respectively. 542 (Scr), 664 (ARF3 KD1), 5,889 (mNG), and 4,353 (ARF3-mNG) cells quantified in total. Data is presented as mean ± SEM normalized to Scr. P values (Student’s two-tailed t test), *P ≤ 0.05, **P ≤ 0.01, and ***P ≤ 0.001. (J) Images of PC3 cells stained with N-cadherin (green) and RAB4, RAB11, RAB11FIP4, or LAMP2 (red). Scale bars, 20 μm. Magnified images of boxed regions shown (a–c). White arrows, co-localization in subset of puncta. Scale bars, 5 μm. Images representative of phenotypes observed in 3 experimental replicates. (K and L) Quantitation of percent overlap of N-cadherin positive puncta with puncta positive for various sub-cellular markers (K) and % overlap of markers with N-cadherin puncta in Scr or ARF3 KD1 cells are shown (L). Data is presented as mean ± SEM n = 3 independent experiments. 307 and 388 (RAB4), 243 and 195 (RAB11), 309 and 369 (RAB11FIP4), and 328 and 369 (LAMP2) cells quantified for Scr and ARF3 KD1, respectively, in total. P values stated, (Student’s two-tailed t test). (M and N) Flow cytometry was performed on PC3 cells with anti-N-cadherin antibody. Representative plot (M) and geometrical mean ± SEM (N) of surface N-cadherin levels are presented. n = 4 independent experiments. P values (Student’s two-tailed t test), **P ≤ 0.01. (O) Surface proteins in PC3 cells expressing mNG or ARF3-mNG and either Scr or ARF3 shRNA were biotinylated and N-cadherin levels were analyzed by Western blot after internalization at 0 or 4 h. GAPDH was used as loading control for lysates. Data is presented as mean ± SEM with pulldowns (PD) normalized to lysates relative to control cells (0 h). n = 3 independent experiments. P values (Student’s two-tailed t test), *P ≤ 0.05, and ***P ≤ 0.001. Source data are available for this figure: SourceData F6.

ARF3 controls N-cadherin turnover from the cell surface by regulating association of internalized N-cadherin with recycling endosomes. (A) Confocal images of PC3 acini expressing mNG or ARF3-mNG and either Scr or ARF3 shRNA stained with F-actin (red) and Hoechst (nuclei, blue). F-actin intensity, FIRE LUT. Scale bars, 20 μm. Magnified images of boxed regions shown. White arrows or arrowheads, presence or absence of intense F-actin staining in junctions, respectively. Scale bars, 7 μm. Images representative of phenotypes observed in 3 experimental replicates. (B–D) Representative Western blot of PC3 cells (B) expressing mNG or ARF3-mNG and Scr or ARF3 shRNA for E-cadherin, N-cadherin, and ARF3. GAPDH loading control is shown for each blot. n = 6 independent lysate preparations. Data is presented in C and D as mean fold change ± SEM normalized to Scr. P values (Student’s two-tailed t test), **P ≤ 0.01. (E)ARF3, N-cadherin, and GAPDH mRNA expression in PC3 expressing mNG or ARF3-mNG and either Scr or ARF3 shRNA was determined by RT-qPCR. n = 3 independent RNA and cDNA preparations with 4 technical replicates/condition. Data is mean fold change ± SEM normalized to GAPDH then to Scr. P values (Student’s two-tailed t test), *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, and ****P ≤ 0.0001. (F) IP was performed using an anti-ARF3 antibody or mouse IgG and samples immunoblotted for N-cadherin and ARF3. Panels shown are representative of 3 IPs from 3 independent lysate preparations. (G) Confocal image of PC3 acini stained for ARF3 (green) and N-cadherin (red). Scale bars, 20 μm. Magnified images of boxed regions shown (a–c). Scale bars, 5 μm. White arrows, co-localization in subset of puncta. Image is representative of co-localization observed in cells in 3 experimental replicates. (H and I) PC3 cells expressing (H) mNG and Scr or ARF3 shRNA or (I) mNG and ARF3-mNG with Scr were stained for N-cadherin, HCS WCS, and Hoechst. Cell area, number, and area of N-cadherin puncta was quantified (H) per cell or (I) per sub-cellular region in each cell. n = 5 (1 technical replicate) or 3 (5 technical replicates) independent experiments, respectively. 542 (Scr), 664 (ARF3 KD1), 5,889 (mNG), and 4,353 (ARF3-mNG) cells quantified in total. Data is presented as mean ± SEM normalized to Scr. P values (Student’s two-tailed t test), *P ≤ 0.05, **P ≤ 0.01, and ***P ≤ 0.001. (J) Images of PC3 cells stained with N-cadherin (green) and RAB4, RAB11, RAB11FIP4, or LAMP2 (red). Scale bars, 20 μm. Magnified images of boxed regions shown (a–c). White arrows, co-localization in subset of puncta. Scale bars, 5 μm. Images representative of phenotypes observed in 3 experimental replicates. (K and L) Quantitation of percent overlap of N-cadherin positive puncta with puncta positive for various sub-cellular markers (K) and % overlap of markers with N-cadherin puncta in Scr or ARF3 KD1 cells are shown (L). Data is presented as mean ± SEM n = 3 independent experiments. 307 and 388 (RAB4), 243 and 195 (RAB11), 309 and 369 (RAB11FIP4), and 328 and 369 (LAMP2) cells quantified for Scr and ARF3 KD1, respectively, in total. P values stated, (Student’s two-tailed t test). (M and N) Flow cytometry was performed on PC3 cells with anti-N-cadherin antibody. Representative plot (M) and geometrical mean ± SEM (N) of surface N-cadherin levels are presented. n = 4 independent experiments. P values (Student’s two-tailed t test), **P ≤ 0.01. (O) Surface proteins in PC3 cells expressing mNG or ARF3-mNG and either Scr or ARF3 shRNA were biotinylated and N-cadherin levels were analyzed by Western blot after internalization at 0 or 4 h. GAPDH was used as loading control for lysates. Data is presented as mean ± SEM with pulldowns (PD) normalized to lysates relative to control cells (0 h). n = 3 independent experiments. P values (Student’s two-tailed t test), *P ≤ 0.05, and ***P ≤ 0.001. Source data are available for this figure: SourceData F6.

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