Figure 2.
SNX10 regulates endocytic trafficking. (A) Graphical description of the plasma membrane EGFR staining. Live cells are put on ice and (1) incubated for 20 min with the primary anti-EGFR antibody, then washed and (2) incubated with a secondary antibody for 20 min, followed by (3) incubation with EGF for 15 or 50 min at 37°C before fixation and imaging. (B) U2OS SNX10-EGFP cells were incubated with anti-EGFR antibody as described in A, then stimulated with EGF and fixed. Cells were stained with an anti-EFG antibody after fixation. Scale bar: 10 µm. Insets: 7.24 × 7.24 µm. (C) After 72 h of siRNA transfection with siCtrl (control) or two different siSNX10 oligoes (siSNX10#1 and siSNX10#2), U2OS cells were serum starved for 2 h and then incubated with 50 ng/ml EGF + 10 µg/ml cycloheximide (CHX) for the indicated times. The cells were lysed, followed by western blotting for the indicated proteins. (D) Quantification of EGFR protein levels normalized to actin in n = 3 independent experiments ± SEM. Significance was determined by two-way ANOVA followed by Tukey’s multiple comparisons test. Normality was assumed but not formally tested. (E) Cells were transfected with siCtrl, siSNX10#1 or siSNX10#2 prior to fixation and staining for endogenous EEA1. Images were taken with Zeiss Axio Observer widefield microscope (Zen Blue 2.3; Zeiss), and a 20× objective was used. Scale bar: 10 µm. (F) Quantification of the data shown in E was performed using CellProfiler software. The values were obtained from analyzing >1,000 cells per condition, and they were normalized to control siRNA (siCtrl). The graphs display the mean values ± SEM from n = 3 independent experiments. The significance was assessed by ordinary one-way ANOVA followed by Bonferroni’s post hoc test. Data distribution was assumed to be normal but was not formally tested. (G) Representative EM images of endosomes in U2OS cells (control and siSNX10 #1). Pink arrows: Protein A conjugated with 10 nm gold (PAG10)-labeling EGFR that has been taken up into endosomes. Yellow arrows: endosome not containing internalized PAG10-labeled EGFR. Scale bar: 0.5 µm. (H) Measurements of EGFR-containing endosome diameter in control versus siSNX10-treated cells from one experiment. The graph shows the endosomal diameter (nm) of a total 24 PAG10-labeled EGFR endosomes in siCtrl cells and 15 PAG10-labeled EGFR endosomes in siSNX10 cells. The graph displays the mean values ± SEM. Significance was determined by unpaired t test with Welch’s correction in all graphs, and data distribution was assumed to be normal but was not formally tested. * = P < 0.05 and ** = P < 0.01, nonsignificant differences are not depicted. Source data are available for this figure: SourceData F2.

SNX10 regulates endocytic trafficking. (A) Graphical description of the plasma membrane EGFR staining. Live cells are put on ice and (1) incubated for 20 min with the primary anti-EGFR antibody, then washed and (2) incubated with a secondary antibody for 20 min, followed by (3) incubation with EGF for 15 or 50 min at 37°C before fixation and imaging. (B) U2OS SNX10-EGFP cells were incubated with anti-EGFR antibody as described in A, then stimulated with EGF and fixed. Cells were stained with an anti-EFG antibody after fixation. Scale bar: 10 µm. Insets: 7.24 × 7.24 µm. (C) After 72 h of siRNA transfection with siCtrl (control) or two different siSNX10 oligoes (siSNX10#1 and siSNX10#2), U2OS cells were serum starved for 2 h and then incubated with 50 ng/ml EGF + 10 µg/ml cycloheximide (CHX) for the indicated times. The cells were lysed, followed by western blotting for the indicated proteins. (D) Quantification of EGFR protein levels normalized to actin in n = 3 independent experiments ± SEM. Significance was determined by two-way ANOVA followed by Tukey’s multiple comparisons test. Normality was assumed but not formally tested. (E) Cells were transfected with siCtrl, siSNX10#1 or siSNX10#2 prior to fixation and staining for endogenous EEA1. Images were taken with Zeiss Axio Observer widefield microscope (Zen Blue 2.3; Zeiss), and a 20× objective was used. Scale bar: 10 µm. (F) Quantification of the data shown in E was performed using CellProfiler software. The values were obtained from analyzing >1,000 cells per condition, and they were normalized to control siRNA (siCtrl). The graphs display the mean values ± SEM from n = 3 independent experiments. The significance was assessed by ordinary one-way ANOVA followed by Bonferroni’s post hoc test. Data distribution was assumed to be normal but was not formally tested. (G) Representative EM images of endosomes in U2OS cells (control and siSNX10 #1). Pink arrows: Protein A conjugated with 10 nm gold (PAG10)-labeling EGFR that has been taken up into endosomes. Yellow arrows: endosome not containing internalized PAG10-labeled EGFR. Scale bar: 0.5 µm. (H) Measurements of EGFR-containing endosome diameter in control versus siSNX10-treated cells from one experiment. The graph shows the endosomal diameter (nm) of a total 24 PAG10-labeled EGFR endosomes in siCtrl cells and 15 PAG10-labeled EGFR endosomes in siSNX10 cells. The graph displays the mean values ± SEM. Significance was determined by unpaired t test with Welch’s correction in all graphs, and data distribution was assumed to be normal but was not formally tested. * = P < 0.05 and ** = P < 0.01, nonsignificant differences are not depicted. Source data are available for this figure: SourceData F2.

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