Rab21 regulates caveolin-1-mediated endocytic trafficking to promote immature neurite pruning (2022)
Figure 1

Figure 1. Rab5 and Rab21 have distinct functions in vitro.

(A-B) Primary cortical neurons from E15 cerebral cortices incubated for two days in vitro and stained with the indicated antibodies. Lower panels in (A) are high magnification images indicated by white rectangles in upper panels. The images are obtained with high-resolution microscopy (Nikon). The graph in (B) shows the Pearson's correlation coefficient of Rab5 and Rab21 or Lamp1 (Lamp1 is a negative control). Each score represents the mean of the individual points. Rab5 - Rab21: n = 15 cells, Rab5 - Lamp1: n = 37 cells.

(C-D) Primary cortical neurons from E15 cerebral cortices were transfected with the indicated plasmids plus pCAG-EGFP, incubated for two days in vitro and treated with Tf-594 for 10 or 30 minutes before fixation (Green: EGFP, Red: Tf-594). White arrow indicates the perinuclear accumulation of Tf-594. The graph in (D) shows the ratio of cells with perinuclear accumulation of Tf-594 at 30 min after the treatment, which was quantified in a blinded counting. Each score represents the mean of ratios with the individual points. Control: n = 4 biological replicates, Rab21-sh115: n = 4 biological replicates, Rab5-sh232: n = 4 biological replicates.

(E-G) Primary cortical neurons from E15 cerebral cortices were transfected with the indicated plasmids, incubated for two days in vitro and treated with Tf-555 for 30 minutes before fixation. Cells were immunostained with the indicated antibodies. The images are obtained with high-resolution microscopy (Nikon). Blue alone channels are shown in black and white images. Lower panels in (E) and (F) are high magnification images indicated by white or blue rectangles in upper panels. The graph in (G) shows the Pearson's correlation coefficient of Tf-555 and Rab11 or APPL1. Each score represents the mean with the individual points. Control: n = 23 cells, Rab21-sh115: n = 18 cells, Rab5-sh232 (Tf - Rab11): n = 23 cells, Rab5-sh232 (Tf - APPL1): n = 22 cells.

Data information: (B) Significance was determined by Mann-Whitney's U test, and no significant difference was observed between control and Rab21-sh115 (P = 0.4986). n.s.: no significant differences. (D) Significance was determined by one-way ANOVA with post hoc Tukey-Kramer. **: < the critical value at 1%, *: < the critical value at 5%, n.s.: no significant differences. Using the multiple comparison, no significant difference was observed between control and Rab21-sh115, but when Mann-Whitney's U test was applied, we found a significant difference between control and Rab21-sh115 (P = 0.02092). (G) Significance of the Pearson's correlation coefficient between Tf and Rab11 was determined by one-way ANOVA with post hoc Tukey-Kramer. **: < the critical value at 1%, *: < the critical value at 5%, n.s.: no significant differences. Significance of the Pearson's correlation coefficient in Rab5-sh232-transfected cells was determined by Mann-Whitney's U test (P = 0.00000007396). *: P<0.01. Scale bars: 5 μm in (upper panels in A), 2 μm in (lower panels in A), 10 μm in (C), 10 μm in (upper panels in E, F), 1 μm in (lower panels in E, F).