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Nikon pearson s correlation coefficient

A–C Primary cortical neurons from E15 cerebral cortices incubated for 2 days in vitro and stained with the indicated antibodies. Right panels in (A) and (B) are high magnification images indicated by white rectangles. The images are obtained with high‐resolution microscopy (Nikon). The graph in (C) shows the <t>Pearson's</t> correlation coefficient between EEA1 and Rab21 or Rab5. Each score represents the mean of ratios with the individual points. Rab21: n = 29 cells, Rab5: n = 28 cells. D Primary cortical neurons from E15 cerebral cortices were transfected with EGFP‐Rab21 and incubated for 2 days in vitro . To maintain moderate expression levels, a CMV promoter was used to express EGFP‐Rab21. Cells were immunostained with anti‐EGFP (green) and anti‐Rab5 antibodies. E Primary cortical neurons from E15 cerebral cortices were transfected with the indicated plasmids, incubated for 2 days in vitro and subjected to immunoblot analyses of cell lysates with the indicated antibodies. F Immature neurons in the IZ of the cerebral cortices at E17, electroporated with the indicated plasmids plus pCAG‐EGFP at E14. Frozen sections were immunostained with the indicated antibodies. The images are obtained with A1R with a high sensitivity GaAsP detector (Nikon). Blue alone channels are shown in black and white images. G, H Primary cortical neurons from E15 cerebral cortices were transfected with the indicated plasmids, incubated for 2 days in vitro and subjected to cell surface biotinylation assay, followed by immunoblot analyses of cell lysates with anti‐TfR antibody. The upper and lower panels indicate the precipitates with streptavidin‐sepharose beads and lysates before pull down (input), respectively. The graph in (H) shows the ratio of cell surface biotinylated transferrin receptor (TfR) to total TfR. Each score represents the mean with the individual points. Control: n = 5 biological replicates, Rab21‐sh115: n = 5 biological replicates, Rab5‐sh232: n = 5 biological replicates, Cav1‐sh490: n = 5 biological replicates. Significance was determined by one‐way ANOVA with post hoc Dunnett. No significant difference was observed between control and Rab21‐sh115 or Rab5‐sh232 or Cav1‐sh490. n.s., no significant differences. Data information: (C) Significance was determined by Student's t ‐test ( P = 0.00007779). ** P < 0.01. (H) Significance was determined by one‐way ANOVA with post hoc Dunnett. No significant difference was observed between control and Rab21‐sh115 or Rab5‐sh232 or Cav1‐sh490. n.s.: no significant differences. Scale bars: 5 μm in (left panels in A, B), 1 μm in (right panels in A, B), 3 μm in (D), 4 μm in (F).

Pearson S Correlation Coefficient, supplied by Nikon, used in various techniques. Bioz Stars score: 99/100, based on 58965 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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1) Product Images from "Rab21 regulates caveolin‐1‐mediated endocytic trafficking to promote immature neurite pruning"

Article Title: Rab21 regulates caveolin‐1‐mediated endocytic trafficking to promote immature neurite pruning

Journal: EMBO Reports

doi: 10.15252/embr.202254701

Figure Legend Snippet: A–C Primary cortical neurons from E15 cerebral cortices incubated for 2 days in vitro and stained with the indicated antibodies. Right panels in (A) and (B) are high magnification images indicated by white rectangles. The images are obtained with high‐resolution microscopy (Nikon). The graph in (C) shows the Pearson's correlation coefficient between EEA1 and Rab21 or Rab5. Each score represents the mean of ratios with the individual points. Rab21: n = 29 cells, Rab5: n = 28 cells. D Primary cortical neurons from E15 cerebral cortices were transfected with EGFP‐Rab21 and incubated for 2 days in vitro . To maintain moderate expression levels, a CMV promoter was used to express EGFP‐Rab21. Cells were immunostained with anti‐EGFP (green) and anti‐Rab5 antibodies. E Primary cortical neurons from E15 cerebral cortices were transfected with the indicated plasmids, incubated for 2 days in vitro and subjected to immunoblot analyses of cell lysates with the indicated antibodies. F Immature neurons in the IZ of the cerebral cortices at E17, electroporated with the indicated plasmids plus pCAG‐EGFP at E14. Frozen sections were immunostained with the indicated antibodies. The images are obtained with A1R with a high sensitivity GaAsP detector (Nikon). Blue alone channels are shown in black and white images. G, H Primary cortical neurons from E15 cerebral cortices were transfected with the indicated plasmids, incubated for 2 days in vitro and subjected to cell surface biotinylation assay, followed by immunoblot analyses of cell lysates with anti‐TfR antibody. The upper and lower panels indicate the precipitates with streptavidin‐sepharose beads and lysates before pull down (input), respectively. The graph in (H) shows the ratio of cell surface biotinylated transferrin receptor (TfR) to total TfR. Each score represents the mean with the individual points. Control: n = 5 biological replicates, Rab21‐sh115: n = 5 biological replicates, Rab5‐sh232: n = 5 biological replicates, Cav1‐sh490: n = 5 biological replicates. Significance was determined by one‐way ANOVA with post hoc Dunnett. No significant difference was observed between control and Rab21‐sh115 or Rab5‐sh232 or Cav1‐sh490. n.s., no significant differences. Data information: (C) Significance was determined by Student's t ‐test ( P = 0.00007779). ** P < 0.01. (H) Significance was determined by one‐way ANOVA with post hoc Dunnett. No significant difference was observed between control and Rab21‐sh115 or Rab5‐sh232 or Cav1‐sh490. n.s.: no significant differences. Scale bars: 5 μm in (left panels in A, B), 1 μm in (right panels in A, B), 3 μm in (D), 4 μm in (F).

Techniques Used: Incubation, In Vitro, Staining, Microscopy, Transfection, Expressing, Western Blot, Cell Surface Biotinylation Assay

A, B Primary cortical neurons from E15 cerebral cortices incubated for 2 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 2 days in vitro and treated with Tf‐594 for 10 or 30 min 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 2 days in vitro and treated with Tf‐555 for 30 min 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. **Less than the critical value at 1%, *less than 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. **Less than the critical value at 1%, *less than 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).

Figure Legend Snippet: A, B Primary cortical neurons from E15 cerebral cortices incubated for 2 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 2 days in vitro and treated with Tf‐594 for 10 or 30 min 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 2 days in vitro and treated with Tf‐555 for 30 min 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. **Less than the critical value at 1%, *less than 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. **Less than the critical value at 1%, *less than 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).

Techniques Used: Incubation, In Vitro, Staining, Microscopy, Negative Control, Transfection, MANN-WHITNEY

A–C Primary cortical neurons from E15 cerebral cortices were transfected with pCAG‐PM‐mAG1 (green) and incubated for 2 days in vitro . Cells were immunostained with the indicated antibodies (red and blue/white). The transfected PM‐mAG1 is a marker for the plasma membrane. The images are obtained with high‐resolution microscopy (Nikon) and the lower panels are high magnification images near the plasma membrane. Blue alone channels are shown in black and white images. The graphs in (C) show the Pearson's correlation coefficient between PM‐mAG1 and Rab21 or Caveolin‐1 (left) and between caveolin‐1 and Rab5 or Rab21 in whole cells (middle) or at the plasma membrane (right). Each score represents the mean of ratios with the individual points. Rab5: n = 18 cells (middle and right), Rab21: n = 31 cells (left and middle) or 20 cells (right), Caveolin‐1: n = 43 cells (left). D–G Immature neurons in the IZ of the cerebral cortices at E17, electroporated with pCAG‐EGFP at E14. Frozen sections 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. The images in (F) are high magnification images of (E). The graph in (G) shows the Pearson's correlation coefficient between caveolin‐1 and Rab5 or Rab21. Each score represents the mean of ratios with the individual points. Rab5: n = 38 cells, Rab21: n = 28 cells. H, I HeLa cells were immunostained with the indicated antibodies. The images are obtained with high‐resolution microscopy (Nikon). The graph in (I) shows the Pearson's correlation coefficient between caveolin‐1 and Rab5 or Rab21. Each score represents the mean of ratios with the individual points. Rab5: n = 80 cells, Rab21: n = 62 cells. Data information: (C) Significance was determined by Student's t ‐test (Middle: P = 9.144E‐22, Right: P = 2.309E‐19). ** P < 0.01. (G) Significance was determined by Welch's t ‐test ( P = 1.225E‐25). ** P < 0.01. (I) Significance was determined by Welch's t ‐test ( P = 3.209E‐40). ** P < 0.01. Scale bars: 3 μm in (upper panels in A, B, D, E), 0.5 μm in (lower panels in A, B, D, E), 0.1 μm in (F), 10 μm in (left panels in H), 1 μm in (right panels in H).

Figure Legend Snippet: A–C Primary cortical neurons from E15 cerebral cortices were transfected with pCAG‐PM‐mAG1 (green) and incubated for 2 days in vitro . Cells were immunostained with the indicated antibodies (red and blue/white). The transfected PM‐mAG1 is a marker for the plasma membrane. The images are obtained with high‐resolution microscopy (Nikon) and the lower panels are high magnification images near the plasma membrane. Blue alone channels are shown in black and white images. The graphs in (C) show the Pearson's correlation coefficient between PM‐mAG1 and Rab21 or Caveolin‐1 (left) and between caveolin‐1 and Rab5 or Rab21 in whole cells (middle) or at the plasma membrane (right). Each score represents the mean of ratios with the individual points. Rab5: n = 18 cells (middle and right), Rab21: n = 31 cells (left and middle) or 20 cells (right), Caveolin‐1: n = 43 cells (left). D–G Immature neurons in the IZ of the cerebral cortices at E17, electroporated with pCAG‐EGFP at E14. Frozen sections 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. The images in (F) are high magnification images of (E). The graph in (G) shows the Pearson's correlation coefficient between caveolin‐1 and Rab5 or Rab21. Each score represents the mean of ratios with the individual points. Rab5: n = 38 cells, Rab21: n = 28 cells. H, I HeLa cells were immunostained with the indicated antibodies. The images are obtained with high‐resolution microscopy (Nikon). The graph in (I) shows the Pearson's correlation coefficient between caveolin‐1 and Rab5 or Rab21. Each score represents the mean of ratios with the individual points. Rab5: n = 80 cells, Rab21: n = 62 cells. Data information: (C) Significance was determined by Student's t ‐test (Middle: P = 9.144E‐22, Right: P = 2.309E‐19). ** P < 0.01. (G) Significance was determined by Welch's t ‐test ( P = 1.225E‐25). ** P < 0.01. (I) Significance was determined by Welch's t ‐test ( P = 3.209E‐40). ** P < 0.01. Scale bars: 3 μm in (upper panels in A, B, D, E), 0.5 μm in (lower panels in A, B, D, E), 0.1 μm in (F), 10 μm in (left panels in H), 1 μm in (right panels in H).

Techniques Used: Transfection, Incubation, In Vitro, Marker, Microscopy

A, B Primary cortical neurons from E15 cerebral cortices were transfected with EGFP‐Rab5 (upper panels in A) or EGFP‐Rab21 (lower panels in A) and incubated for 2 days in vitro . To maintain moderate expression levels, CMV promoter was used to express EGFP‐Rab5 and EGFP‐Rab21. Cells were immunostained with anti‐EGFP (green) and anti‐caveolin‐1 (red) antibodies. White arrows in (A) indicate colocalization of EGFP‐Rab21 and caveolin‐1. The images are obtained with TCS‐SP5 (Leica). The graphs in (B) show the estimation of colocalization, which was carried out by recording fluorescence intensities of EGFP‐Rab5 or EGFP‐Rab21 and caveolin‐1 staining signals along the white line in the upper panels using Leica SP5 software. Red arrows indicate the colocalization of these proteins on the same peaks. C–F Primary cortical neurons from E15 cerebral cortices were transfected with the indicated plasmids, incubated for 2 days in vitro and immunostained with the indicated antibodies. Lower panels in (C) and (E) are high magnification images indicated by white and blue rectangles in upper panels. Blue alone channels are shown in black and white images. The graphs in (D) and (F) show the Pearson's correlation coefficient between caveolin‐1 (D) or Rab21 (F) and organelle markers. Each score represents the mean with the individual points. Caveolin‐1—APPL1: n = 38 cells, Caveolin‐1—calnexin: n = 51 cells, Rab21—calnexin: n = 59 cells, Rab21—KDEL: n = 51 cells. G–J NIH3T3 or COS‐1 cells were immunostained with the indicated antibodies. The images are obtained with high‐resolution microscopy (Nikon). The graphs in (H) and (J) show the Pearson's correlation coefficient between caveolin‐1 and Rab5 or Rab21 or between Rab21 and Rab5. Each score represents the mean with the individual points. Caveolin‐1—Rab21: n = 41 cells (H) or 63 cells (J), Caveolin‐1—Rab5: n = 63 cells (H) or 69 cells (J), Rab21—Rab5: 34 cells. Data information: (D) Significance was determined by Mann–Whitney's U test ( P = 1.310E‐14, ** P < 0.01). (F) Significance was determined by one‐way ANOVA with post hoc Tukey–Kramer. No significant difference was observed between Rab21—Calnexin and Rab21—KDEL, but compared to a negative control (Rab5—Lamp1 in Fig ), a significant difference was observed (less than the critical value at 1%: Rab21—calnexin, Rab21—KDEL [compared to a negative control]). (H, J) Significance between Caveolin‐1—Rab21 and Caveolin‐1—Rab5 was determined by Welch's t‐test (H: P = 5.795E‐27, J: P = 7.367E‐24). ** P < 0.01. Scale bar: 3 μm in (A), 10 μm in (upper panels in C, E), 1 μm in (lower panels in C, E), 5 μm in (G, I).

Figure Legend Snippet: A, B Primary cortical neurons from E15 cerebral cortices were transfected with EGFP‐Rab5 (upper panels in A) or EGFP‐Rab21 (lower panels in A) and incubated for 2 days in vitro . To maintain moderate expression levels, CMV promoter was used to express EGFP‐Rab5 and EGFP‐Rab21. Cells were immunostained with anti‐EGFP (green) and anti‐caveolin‐1 (red) antibodies. White arrows in (A) indicate colocalization of EGFP‐Rab21 and caveolin‐1. The images are obtained with TCS‐SP5 (Leica). The graphs in (B) show the estimation of colocalization, which was carried out by recording fluorescence intensities of EGFP‐Rab5 or EGFP‐Rab21 and caveolin‐1 staining signals along the white line in the upper panels using Leica SP5 software. Red arrows indicate the colocalization of these proteins on the same peaks. C–F Primary cortical neurons from E15 cerebral cortices were transfected with the indicated plasmids, incubated for 2 days in vitro and immunostained with the indicated antibodies. Lower panels in (C) and (E) are high magnification images indicated by white and blue rectangles in upper panels. Blue alone channels are shown in black and white images. The graphs in (D) and (F) show the Pearson's correlation coefficient between caveolin‐1 (D) or Rab21 (F) and organelle markers. Each score represents the mean with the individual points. Caveolin‐1—APPL1: n = 38 cells, Caveolin‐1—calnexin: n = 51 cells, Rab21—calnexin: n = 59 cells, Rab21—KDEL: n = 51 cells. G–J NIH3T3 or COS‐1 cells were immunostained with the indicated antibodies. The images are obtained with high‐resolution microscopy (Nikon). The graphs in (H) and (J) show the Pearson's correlation coefficient between caveolin‐1 and Rab5 or Rab21 or between Rab21 and Rab5. Each score represents the mean with the individual points. Caveolin‐1—Rab21: n = 41 cells (H) or 63 cells (J), Caveolin‐1—Rab5: n = 63 cells (H) or 69 cells (J), Rab21—Rab5: 34 cells. Data information: (D) Significance was determined by Mann–Whitney's U test ( P = 1.310E‐14, ** P < 0.01). (F) Significance was determined by one‐way ANOVA with post hoc Tukey–Kramer. No significant difference was observed between Rab21—Calnexin and Rab21—KDEL, but compared to a negative control (Rab5—Lamp1 in Fig ), a significant difference was observed (less than the critical value at 1%: Rab21—calnexin, Rab21—KDEL [compared to a negative control]). (H, J) Significance between Caveolin‐1—Rab21 and Caveolin‐1—Rab5 was determined by Welch's t‐test (H: P = 5.795E‐27, J: P = 7.367E‐24). ** P < 0.01. Scale bar: 3 μm in (A), 10 μm in (upper panels in C, E), 1 μm in (lower panels in C, E), 5 μm in (G, I).

Techniques Used: Transfection, Incubation, In Vitro, Expressing, Fluorescence, Staining, Software, Microscopy, MANN-WHITNEY, Negative Control

A, B NIH3T3 fibroblasts were transfected with the indicated plasmids and treated with BODIPY‐LacCer (LacCer) for 30 min before fixation. The images are obtained with high‐resolution microscopy (Nikon). The graph in (B) shows the ratio of cells with perinuclear accumulation of LacCer. Each score represents the mean with the individual points. Control: n = 4 biological replicates, Rab21‐sh115: n = 4 biological replicates, Rab5‐sh232: n = 4 biological replicates. C–E Primary cortical neurons from E15 cerebral cortices were incubated for 2 days in vitro and immunostained with the indicated antibodies. The images are obtained with high‐resolution microscopy (Nikon). The graph in (E) shows the Pearson's correlation coefficient between Endophilin and Rab21 or caveolin‐1 or between Rab21 and CD44. Each score represents the mean with the individual points. Caveolin‐1—Endophilin: n = 43 cells, Rab21—Endophilin: n = 53 cells, Rab21—CD44: n = 45 cells. F–H Primary cortical neurons from E15 cerebral cortices were transfected with the indicated plasmids plus pCAG‐PM‐mAG1, incubated for 2 days in vitro and subjected to CD44 antibody feeding assay. The graphs in (G) and (H) show the number of the internalized CD44‐positive dots and its total fluorescence intensity per cell. Each score represents the mean with the individual points. Control: n = 15 cells (G and H), Rab21‐sh115: n = 12 cells (G and H). I, J Primary cortical neurons from E15 cerebral cortices were transfected with the indicated plasmids plus pCAG‐EGFP, incubated for 8 days in vitro and stained with MAP2ab, a marker for dendrites. The graphs in (J) show the dendrite length, dendrite branch number and the number of primary dendrites. Each score represents the mean with the individual points. Control: n = 31 cells, Rab21‐sh115: n = 38 cells, Rab5‐sh232: n = 31 cells, Cav1‐sh490: n = 31 cells. K Cerebral cortex at P0, electroporated with the indicated plasmids plus pCAG‐EGFP at E14. L Frozen sections of E17 cerebral cortex immunostained with anti‐Rab21 antibody and DAPI. Data information: (B) Significance was determined by one‐way ANOVA with post hoc Dunnett and Tukey–Kramer. **Less than the critical value at 1%, *less than the critical value at 5%, n.s.: no significant differences. (E) Significance was determined by one‐way ANOVA with post hoc Tukey–Kramer. **Less than the critical value at 1%. (G, H) Significance was determined by Welch's t ‐test (G: P = 0.1942) or Mann–Whitney's U test (H: P = 0.2225). n.s.: no significant differences. (J) Significance compared to control was determined by one‐way ANOVA with post hoc Dunnett. Significant difference was observed between control and Rab21‐sh115 in the number of primary dendrites. *Less than the critical value at 5%, n.s.: no significant differences. Scale bars: 5 μm in (A, C, D, F), 10 μm in (I), 150 μm in (K), 100 μm in (L).

Figure Legend Snippet: A, B NIH3T3 fibroblasts were transfected with the indicated plasmids and treated with BODIPY‐LacCer (LacCer) for 30 min before fixation. The images are obtained with high‐resolution microscopy (Nikon). The graph in (B) shows the ratio of cells with perinuclear accumulation of LacCer. Each score represents the mean with the individual points. Control: n = 4 biological replicates, Rab21‐sh115: n = 4 biological replicates, Rab5‐sh232: n = 4 biological replicates. C–E Primary cortical neurons from E15 cerebral cortices were incubated for 2 days in vitro and immunostained with the indicated antibodies. The images are obtained with high‐resolution microscopy (Nikon). The graph in (E) shows the Pearson's correlation coefficient between Endophilin and Rab21 or caveolin‐1 or between Rab21 and CD44. Each score represents the mean with the individual points. Caveolin‐1—Endophilin: n = 43 cells, Rab21—Endophilin: n = 53 cells, Rab21—CD44: n = 45 cells. F–H Primary cortical neurons from E15 cerebral cortices were transfected with the indicated plasmids plus pCAG‐PM‐mAG1, incubated for 2 days in vitro and subjected to CD44 antibody feeding assay. The graphs in (G) and (H) show the number of the internalized CD44‐positive dots and its total fluorescence intensity per cell. Each score represents the mean with the individual points. Control: n = 15 cells (G and H), Rab21‐sh115: n = 12 cells (G and H). I, J Primary cortical neurons from E15 cerebral cortices were transfected with the indicated plasmids plus pCAG‐EGFP, incubated for 8 days in vitro and stained with MAP2ab, a marker for dendrites. The graphs in (J) show the dendrite length, dendrite branch number and the number of primary dendrites. Each score represents the mean with the individual points. Control: n = 31 cells, Rab21‐sh115: n = 38 cells, Rab5‐sh232: n = 31 cells, Cav1‐sh490: n = 31 cells. K Cerebral cortex at P0, electroporated with the indicated plasmids plus pCAG‐EGFP at E14. L Frozen sections of E17 cerebral cortex immunostained with anti‐Rab21 antibody and DAPI. Data information: (B) Significance was determined by one‐way ANOVA with post hoc Dunnett and Tukey–Kramer. **Less than the critical value at 1%, *less than the critical value at 5%, n.s.: no significant differences. (E) Significance was determined by one‐way ANOVA with post hoc Tukey–Kramer. **Less than the critical value at 1%. (G, H) Significance was determined by Welch's t ‐test (G: P = 0.1942) or Mann–Whitney's U test (H: P = 0.2225). n.s.: no significant differences. (J) Significance compared to control was determined by one‐way ANOVA with post hoc Dunnett. Significant difference was observed between control and Rab21‐sh115 in the number of primary dendrites. *Less than the critical value at 5%, n.s.: no significant differences. Scale bars: 5 μm in (A, C, D, F), 10 μm in (I), 150 μm in (K), 100 μm in (L).

Techniques Used: Transfection, Microscopy, Incubation, In Vitro, Feeding Assay, Fluorescence, Staining, Marker, MANN-WHITNEY

A, B Primary cortical neurons from E15 cerebral cortices were transfected with the indicated plasmids plus pCAG‐PM‐mAG1 (green) and incubated for 2 days in vitro . Immunocytochemical analyses with anti‐caveolin‐1 (red) antibody were performed. The images are obtained with high‐resolution microscopy (Nikon). The graph in (B) indicates the ratio of the caveolin‐1 staining signals in the plasma membrane to total fluorescence intensities of caveolin‐1 in each immature neuron. Each score represents the mean of ratios with the individual points. Control: n = 20 cells, Rab21‐sh115: n = 22 cells, Rab21‐sh115 + pCMV‐wt‐Rab21: n = 23 cells. Overexpression of wt‐Rab21 resulted in neuronal cell death, which making it difficult to determine the accurate DNA concentration of pCMV‐wt‐Rab21 in the rescue experiments. C–F Primary cortical neurons from E15 cerebral cortices were transfected with the indicated plasmids and incubated for 2 days in vitro . Immunoblot analyses of cell lysates with the indicated antibodies were performed. The graphs in (D) and (F) indicate the mean ratios of immunoblot band intensities of caveolin‐1/β‐tubulin with the individual points. G Immature neurons in the IZ of the cerebral cortices at E17, electroporated with the indicated plasmids plus pCAG‐EGFP at E14. Frozen sections were immunostained with the indicated antibodies. Blue alone channels are shown in black and white images. H, I Primary cortical neurons from E15 cerebral cortices were transfected with the indicated plasmids, incubated for 2 days in vitro , treated with Bafilomycin A1 (Baf A1) for 6 h and stained with the indicated antibodies. The images are obtained with high‐resolution microscopy (Nikon) and the lower panels are high magnification images of the lysosomes, indicated by white or blue rectangles. Blue alone channels are shown in black and white images. The graph in (I) shows the Pearson's correlation coefficient between caveolin‐1 and Lamp1, a lysosomal marker, in control and Rab21‐sh115‐transfected neurons. Each score represents the mean of ratios with the individual points. Control: n = 31 cells, Rab21‐sh115: n = 22 cells. Data information: (B) Significance was determined by one‐way ANOVA with post hoc Dunnett and Tukey–Kramer. **Less than the critical value at 1%. Significant difference was observed between control and Rab21‐sh115, but not between control and the co‐expression of Rab21‐sh115 and pCMV‐wt‐Rab21, by Dunnett and Tukey–Kramer. However, no significance was observed between Rab21‐sh115 alone and co‐expression of Rab21‐sh115 and pCMV‐wt‐Rab21 by Tukey–Kramer. (D and F) Significance was determined by one‐way ANOVA with post hoc Tukey–Kramer (D: n = 9 biological replicates) and Mann–Whitney's U test (F: P = 0.02102, n = 4 biological replicates). n.s.: no significant differences, *less than the critical value at 5%, **less than the critical value at 1%. (I) Significance was determined by Mann–Whitney's U test ( P = 0.0000006290). ** P < 0.01. Scale bars: 1 μm in (A, lower panels in H), 4 μm in (G), 5 μm in (upper panels in H).

Figure Legend Snippet: A, B Primary cortical neurons from E15 cerebral cortices were transfected with the indicated plasmids plus pCAG‐PM‐mAG1 (green) and incubated for 2 days in vitro . Immunocytochemical analyses with anti‐caveolin‐1 (red) antibody were performed. The images are obtained with high‐resolution microscopy (Nikon). The graph in (B) indicates the ratio of the caveolin‐1 staining signals in the plasma membrane to total fluorescence intensities of caveolin‐1 in each immature neuron. Each score represents the mean of ratios with the individual points. Control: n = 20 cells, Rab21‐sh115: n = 22 cells, Rab21‐sh115 + pCMV‐wt‐Rab21: n = 23 cells. Overexpression of wt‐Rab21 resulted in neuronal cell death, which making it difficult to determine the accurate DNA concentration of pCMV‐wt‐Rab21 in the rescue experiments. C–F Primary cortical neurons from E15 cerebral cortices were transfected with the indicated plasmids and incubated for 2 days in vitro . Immunoblot analyses of cell lysates with the indicated antibodies were performed. The graphs in (D) and (F) indicate the mean ratios of immunoblot band intensities of caveolin‐1/β‐tubulin with the individual points. G Immature neurons in the IZ of the cerebral cortices at E17, electroporated with the indicated plasmids plus pCAG‐EGFP at E14. Frozen sections were immunostained with the indicated antibodies. Blue alone channels are shown in black and white images. H, I Primary cortical neurons from E15 cerebral cortices were transfected with the indicated plasmids, incubated for 2 days in vitro , treated with Bafilomycin A1 (Baf A1) for 6 h and stained with the indicated antibodies. The images are obtained with high‐resolution microscopy (Nikon) and the lower panels are high magnification images of the lysosomes, indicated by white or blue rectangles. Blue alone channels are shown in black and white images. The graph in (I) shows the Pearson's correlation coefficient between caveolin‐1 and Lamp1, a lysosomal marker, in control and Rab21‐sh115‐transfected neurons. Each score represents the mean of ratios with the individual points. Control: n = 31 cells, Rab21‐sh115: n = 22 cells. Data information: (B) Significance was determined by one‐way ANOVA with post hoc Dunnett and Tukey–Kramer. **Less than the critical value at 1%. Significant difference was observed between control and Rab21‐sh115, but not between control and the co‐expression of Rab21‐sh115 and pCMV‐wt‐Rab21, by Dunnett and Tukey–Kramer. However, no significance was observed between Rab21‐sh115 alone and co‐expression of Rab21‐sh115 and pCMV‐wt‐Rab21 by Tukey–Kramer. (D and F) Significance was determined by one‐way ANOVA with post hoc Tukey–Kramer (D: n = 9 biological replicates) and Mann–Whitney's U test (F: P = 0.02102, n = 4 biological replicates). n.s.: no significant differences, *less than the critical value at 5%, **less than the critical value at 1%. (I) Significance was determined by Mann–Whitney's U test ( P = 0.0000006290). ** P < 0.01. Scale bars: 1 μm in (A, lower panels in H), 4 μm in (G), 5 μm in (upper panels in H).

Techniques Used: Transfection, Incubation, In Vitro, Microscopy, Staining, Fluorescence, Over Expression, Concentration Assay, Western Blot, Marker, Expressing, MANN-WHITNEY

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Correlations with best corrected visual acuity (BCVA). A total of 78 proteins correlated with BCVA. Correlations are shown for the six proteins with the strongest positive correlations with BCVA and for the six proteins with strongest negative correlations with BCVA. Correlations were calculated as Pearson's correlation coefficient, r . Label-free quantification (LFQ) values denote the protein content measured in the proteomic analysis. ( A-F ) The proteins with the strongest positive correlations with BCVA (LogMAR) were fibrinogen alpha chain, inter-alpha-trypsin inhibitor heavy chain H4, fibrinogen beta chain, Ig lambda-6 chain C region, fibrinogen gamma chain and complement C5. ( G-L ) The strongest negative correlations with BCVA were observed for opticin, extracellular superoxide dismutase, transforming growth factor-beta-induced protein ig-h3, spondin-1, clusterin, and procollagen C-endopeptidase enhancer 1.

Journal: Investigative Ophthalmology & Visual Science

Article Title: Macular Edema in Central Retinal Vein Occlusion Correlates With Aqueous Fibrinogen Alpha Chain

doi: 10.1167/iovs.64.2.23

Figure Lengend Snippet: Correlations with best corrected visual acuity (BCVA). A total of 78 proteins correlated with BCVA. Correlations are shown for the six proteins with the strongest positive correlations with BCVA and for the six proteins with strongest negative correlations with BCVA. Correlations were calculated as Pearson's correlation coefficient, r . Label-free quantification (LFQ) values denote the protein content measured in the proteomic analysis. ( A-F ) The proteins with the strongest positive correlations with BCVA (LogMAR) were fibrinogen alpha chain, inter-alpha-trypsin inhibitor heavy chain H4, fibrinogen beta chain, Ig lambda-6 chain C region, fibrinogen gamma chain and complement C5. ( G-L ) The strongest negative correlations with BCVA were observed for opticin, extracellular superoxide dismutase, transforming growth factor-beta-induced protein ig-h3, spondin-1, clusterin, and procollagen C-endopeptidase enhancer 1.

Article Snippet: Correlations were calculated as the Pearson's correlation coefficient ( r ) in STATA version 16.0.

Techniques:

Correlations with severity of macular edema. A total of 42 proteins correlated with severity of macular edema. Correlations are shown for the six proteins with the strongest positive correlations and the six proteins with the strongest negative correlations with the severity of macular edema. Correlations were calculated as Pearson's correlation coefficient, r . ( A-F ) The strongest positive correlations with severity of macular edema were observed for Ig alpha-1 chain C region, Ig lambda-6 chain C region, Ig mu chain C region, fibrinogen alpha chain, fibrinogen beta chain, and Ig kappa chain V-I region Daudi. ( G-L ) The strongest negative correlations with severity of macular edema were observed for fibrillin-1, cadherin-2, opticin, procollagen endopeptidase enhancer 1, reelin, and dipeptidyl peptidase 2.

Journal: Investigative Ophthalmology & Visual Science

Article Title: Macular Edema in Central Retinal Vein Occlusion Correlates With Aqueous Fibrinogen Alpha Chain

doi: 10.1167/iovs.64.2.23

Figure Lengend Snippet: Correlations with severity of macular edema. A total of 42 proteins correlated with severity of macular edema. Correlations are shown for the six proteins with the strongest positive correlations and the six proteins with the strongest negative correlations with the severity of macular edema. Correlations were calculated as Pearson's correlation coefficient, r . ( A-F ) The strongest positive correlations with severity of macular edema were observed for Ig alpha-1 chain C region, Ig lambda-6 chain C region, Ig mu chain C region, fibrinogen alpha chain, fibrinogen beta chain, and Ig kappa chain V-I region Daudi. ( G-L ) The strongest negative correlations with severity of macular edema were observed for fibrillin-1, cadherin-2, opticin, procollagen endopeptidase enhancer 1, reelin, and dipeptidyl peptidase 2.

Article Snippet: Correlations were calculated as the Pearson's correlation coefficient ( r ) in STATA version 16.0.

Techniques:

Validation by ELISA. Correlations were calculated as Pearson's correlation coefficient, r . ( A ) ELISA confirmed an increased level of aqueous fibrinogen alpha chain in CRVO. ( B ) CRVO was associated with an increased level of VEGF. ( C ) ELISA confirmed a significant correlation between fibrinogen alpha chain and the severity of macular edema. ( D ) ELISA indicated a correlation between fibrinogen alpha chain and VEGF, but the correlation was not statistically significant. ( E ) The correlation between fibrinogen alpha chain and BCVA observed by proteomics was not confirmed by ELISA.

Journal: Investigative Ophthalmology & Visual Science

Article Title: Macular Edema in Central Retinal Vein Occlusion Correlates With Aqueous Fibrinogen Alpha Chain

doi: 10.1167/iovs.64.2.23

Figure Lengend Snippet: Validation by ELISA. Correlations were calculated as Pearson's correlation coefficient, r . ( A ) ELISA confirmed an increased level of aqueous fibrinogen alpha chain in CRVO. ( B ) CRVO was associated with an increased level of VEGF. ( C ) ELISA confirmed a significant correlation between fibrinogen alpha chain and the severity of macular edema. ( D ) ELISA indicated a correlation between fibrinogen alpha chain and VEGF, but the correlation was not statistically significant. ( E ) The correlation between fibrinogen alpha chain and BCVA observed by proteomics was not confirmed by ELISA.

Article Snippet: Correlations were calculated as the Pearson's correlation coefficient ( r ) in STATA version 16.0.

Techniques: Enzyme-linked Immunosorbent Assay

Journal: EMBO Reports

Article Title: Rab21 regulates caveolin‐1‐mediated endocytic trafficking to promote immature neurite pruning

doi: 10.15252/embr.202254701

Figure Lengend Snippet: A–C Primary cortical neurons from E15 cerebral cortices incubated for 2 days in vitro and stained with the indicated antibodies. Right panels in (A) and (B) are high magnification images indicated by white rectangles. The images are obtained with high‐resolution microscopy (Nikon). The graph in (C) shows the Pearson's correlation coefficient between EEA1 and Rab21 or Rab5. Each score represents the mean of ratios with the individual points. Rab21: n = 29 cells, Rab5: n = 28 cells. D Primary cortical neurons from E15 cerebral cortices were transfected with EGFP‐Rab21 and incubated for 2 days in vitro . To maintain moderate expression levels, a CMV promoter was used to express EGFP‐Rab21. Cells were immunostained with anti‐EGFP (green) and anti‐Rab5 antibodies. E Primary cortical neurons from E15 cerebral cortices were transfected with the indicated plasmids, incubated for 2 days in vitro and subjected to immunoblot analyses of cell lysates with the indicated antibodies. F Immature neurons in the IZ of the cerebral cortices at E17, electroporated with the indicated plasmids plus pCAG‐EGFP at E14. Frozen sections were immunostained with the indicated antibodies. The images are obtained with A1R with a high sensitivity GaAsP detector (Nikon). Blue alone channels are shown in black and white images. G, H Primary cortical neurons from E15 cerebral cortices were transfected with the indicated plasmids, incubated for 2 days in vitro and subjected to cell surface biotinylation assay, followed by immunoblot analyses of cell lysates with anti‐TfR antibody. The upper and lower panels indicate the precipitates with streptavidin‐sepharose beads and lysates before pull down (input), respectively. The graph in (H) shows the ratio of cell surface biotinylated transferrin receptor (TfR) to total TfR. Each score represents the mean with the individual points. Control: n = 5 biological replicates, Rab21‐sh115: n = 5 biological replicates, Rab5‐sh232: n = 5 biological replicates, Cav1‐sh490: n = 5 biological replicates. Significance was determined by one‐way ANOVA with post hoc Dunnett. No significant difference was observed between control and Rab21‐sh115 or Rab5‐sh232 or Cav1‐sh490. n.s., no significant differences. Data information: (C) Significance was determined by Student's t ‐test ( P = 0.00007779). ** P < 0.01. (H) Significance was determined by one‐way ANOVA with post hoc Dunnett. No significant difference was observed between control and Rab21‐sh115 or Rab5‐sh232 or Cav1‐sh490. n.s.: no significant differences. Scale bars: 5 μm in (left panels in A, B), 1 μm in (right panels in A, B), 3 μm in (D), 4 μm in (F).

Article Snippet: The efficiency of colocalization of two proteins in neurons was measured, and the Pearson's correlation coefficient was calculated using NIS elements software (Nikon) or Image J.

Techniques: Incubation, In Vitro, Staining, Microscopy, Transfection, Expressing, Western Blot, Cell Surface Biotinylation Assay

Journal: EMBO Reports

Article Title: Rab21 regulates caveolin‐1‐mediated endocytic trafficking to promote immature neurite pruning

doi: 10.15252/embr.202254701

Figure Lengend Snippet: A, B Primary cortical neurons from E15 cerebral cortices incubated for 2 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 2 days in vitro and treated with Tf‐594 for 10 or 30 min 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 2 days in vitro and treated with Tf‐555 for 30 min 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. **Less than the critical value at 1%, *less than 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. **Less than the critical value at 1%, *less than 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).

Article Snippet: The efficiency of colocalization of two proteins in neurons was measured, and the Pearson's correlation coefficient was calculated using NIS elements software (Nikon) or Image J.

Techniques: Incubation, In Vitro, Staining, Microscopy, Negative Control, Transfection, MANN-WHITNEY

Journal: EMBO Reports

Article Title: Rab21 regulates caveolin‐1‐mediated endocytic trafficking to promote immature neurite pruning

doi: 10.15252/embr.202254701

Figure Lengend Snippet: A–C Primary cortical neurons from E15 cerebral cortices were transfected with pCAG‐PM‐mAG1 (green) and incubated for 2 days in vitro . Cells were immunostained with the indicated antibodies (red and blue/white). The transfected PM‐mAG1 is a marker for the plasma membrane. The images are obtained with high‐resolution microscopy (Nikon) and the lower panels are high magnification images near the plasma membrane. Blue alone channels are shown in black and white images. The graphs in (C) show the Pearson's correlation coefficient between PM‐mAG1 and Rab21 or Caveolin‐1 (left) and between caveolin‐1 and Rab5 or Rab21 in whole cells (middle) or at the plasma membrane (right). Each score represents the mean of ratios with the individual points. Rab5: n = 18 cells (middle and right), Rab21: n = 31 cells (left and middle) or 20 cells (right), Caveolin‐1: n = 43 cells (left). D–G Immature neurons in the IZ of the cerebral cortices at E17, electroporated with pCAG‐EGFP at E14. Frozen sections 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. The images in (F) are high magnification images of (E). The graph in (G) shows the Pearson's correlation coefficient between caveolin‐1 and Rab5 or Rab21. Each score represents the mean of ratios with the individual points. Rab5: n = 38 cells, Rab21: n = 28 cells. H, I HeLa cells were immunostained with the indicated antibodies. The images are obtained with high‐resolution microscopy (Nikon). The graph in (I) shows the Pearson's correlation coefficient between caveolin‐1 and Rab5 or Rab21. Each score represents the mean of ratios with the individual points. Rab5: n = 80 cells, Rab21: n = 62 cells. Data information: (C) Significance was determined by Student's t ‐test (Middle: P = 9.144E‐22, Right: P = 2.309E‐19). ** P < 0.01. (G) Significance was determined by Welch's t ‐test ( P = 1.225E‐25). ** P < 0.01. (I) Significance was determined by Welch's t ‐test ( P = 3.209E‐40). ** P < 0.01. Scale bars: 3 μm in (upper panels in A, B, D, E), 0.5 μm in (lower panels in A, B, D, E), 0.1 μm in (F), 10 μm in (left panels in H), 1 μm in (right panels in H).

Article Snippet: The efficiency of colocalization of two proteins in neurons was measured, and the Pearson's correlation coefficient was calculated using NIS elements software (Nikon) or Image J.

Techniques: Transfection, Incubation, In Vitro, Marker, Microscopy

Journal: EMBO Reports

Article Title: Rab21 regulates caveolin‐1‐mediated endocytic trafficking to promote immature neurite pruning

doi: 10.15252/embr.202254701

Figure Lengend Snippet: A, B Primary cortical neurons from E15 cerebral cortices were transfected with EGFP‐Rab5 (upper panels in A) or EGFP‐Rab21 (lower panels in A) and incubated for 2 days in vitro . To maintain moderate expression levels, CMV promoter was used to express EGFP‐Rab5 and EGFP‐Rab21. Cells were immunostained with anti‐EGFP (green) and anti‐caveolin‐1 (red) antibodies. White arrows in (A) indicate colocalization of EGFP‐Rab21 and caveolin‐1. The images are obtained with TCS‐SP5 (Leica). The graphs in (B) show the estimation of colocalization, which was carried out by recording fluorescence intensities of EGFP‐Rab5 or EGFP‐Rab21 and caveolin‐1 staining signals along the white line in the upper panels using Leica SP5 software. Red arrows indicate the colocalization of these proteins on the same peaks. C–F Primary cortical neurons from E15 cerebral cortices were transfected with the indicated plasmids, incubated for 2 days in vitro and immunostained with the indicated antibodies. Lower panels in (C) and (E) are high magnification images indicated by white and blue rectangles in upper panels. Blue alone channels are shown in black and white images. The graphs in (D) and (F) show the Pearson's correlation coefficient between caveolin‐1 (D) or Rab21 (F) and organelle markers. Each score represents the mean with the individual points. Caveolin‐1—APPL1: n = 38 cells, Caveolin‐1—calnexin: n = 51 cells, Rab21—calnexin: n = 59 cells, Rab21—KDEL: n = 51 cells. G–J NIH3T3 or COS‐1 cells were immunostained with the indicated antibodies. The images are obtained with high‐resolution microscopy (Nikon). The graphs in (H) and (J) show the Pearson's correlation coefficient between caveolin‐1 and Rab5 or Rab21 or between Rab21 and Rab5. Each score represents the mean with the individual points. Caveolin‐1—Rab21: n = 41 cells (H) or 63 cells (J), Caveolin‐1—Rab5: n = 63 cells (H) or 69 cells (J), Rab21—Rab5: 34 cells. Data information: (D) Significance was determined by Mann–Whitney's U test ( P = 1.310E‐14, ** P < 0.01). (F) Significance was determined by one‐way ANOVA with post hoc Tukey–Kramer. No significant difference was observed between Rab21—Calnexin and Rab21—KDEL, but compared to a negative control (Rab5—Lamp1 in Fig ), a significant difference was observed (less than the critical value at 1%: Rab21—calnexin, Rab21—KDEL [compared to a negative control]). (H, J) Significance between Caveolin‐1—Rab21 and Caveolin‐1—Rab5 was determined by Welch's t‐test (H: P = 5.795E‐27, J: P = 7.367E‐24). ** P < 0.01. Scale bar: 3 μm in (A), 10 μm in (upper panels in C, E), 1 μm in (lower panels in C, E), 5 μm in (G, I).

Article Snippet: The efficiency of colocalization of two proteins in neurons was measured, and the Pearson's correlation coefficient was calculated using NIS elements software (Nikon) or Image J.

Techniques: Transfection, Incubation, In Vitro, Expressing, Fluorescence, Staining, Software, Microscopy, MANN-WHITNEY, Negative Control

Journal: EMBO Reports

Article Title: Rab21 regulates caveolin‐1‐mediated endocytic trafficking to promote immature neurite pruning

doi: 10.15252/embr.202254701

Figure Lengend Snippet: A, B NIH3T3 fibroblasts were transfected with the indicated plasmids and treated with BODIPY‐LacCer (LacCer) for 30 min before fixation. The images are obtained with high‐resolution microscopy (Nikon). The graph in (B) shows the ratio of cells with perinuclear accumulation of LacCer. Each score represents the mean with the individual points. Control: n = 4 biological replicates, Rab21‐sh115: n = 4 biological replicates, Rab5‐sh232: n = 4 biological replicates. C–E Primary cortical neurons from E15 cerebral cortices were incubated for 2 days in vitro and immunostained with the indicated antibodies. The images are obtained with high‐resolution microscopy (Nikon). The graph in (E) shows the Pearson's correlation coefficient between Endophilin and Rab21 or caveolin‐1 or between Rab21 and CD44. Each score represents the mean with the individual points. Caveolin‐1—Endophilin: n = 43 cells, Rab21—Endophilin: n = 53 cells, Rab21—CD44: n = 45 cells. F–H Primary cortical neurons from E15 cerebral cortices were transfected with the indicated plasmids plus pCAG‐PM‐mAG1, incubated for 2 days in vitro and subjected to CD44 antibody feeding assay. The graphs in (G) and (H) show the number of the internalized CD44‐positive dots and its total fluorescence intensity per cell. Each score represents the mean with the individual points. Control: n = 15 cells (G and H), Rab21‐sh115: n = 12 cells (G and H). I, J Primary cortical neurons from E15 cerebral cortices were transfected with the indicated plasmids plus pCAG‐EGFP, incubated for 8 days in vitro and stained with MAP2ab, a marker for dendrites. The graphs in (J) show the dendrite length, dendrite branch number and the number of primary dendrites. Each score represents the mean with the individual points. Control: n = 31 cells, Rab21‐sh115: n = 38 cells, Rab5‐sh232: n = 31 cells, Cav1‐sh490: n = 31 cells. K Cerebral cortex at P0, electroporated with the indicated plasmids plus pCAG‐EGFP at E14. L Frozen sections of E17 cerebral cortex immunostained with anti‐Rab21 antibody and DAPI. Data information: (B) Significance was determined by one‐way ANOVA with post hoc Dunnett and Tukey–Kramer. **Less than the critical value at 1%, *less than the critical value at 5%, n.s.: no significant differences. (E) Significance was determined by one‐way ANOVA with post hoc Tukey–Kramer. **Less than the critical value at 1%. (G, H) Significance was determined by Welch's t ‐test (G: P = 0.1942) or Mann–Whitney's U test (H: P = 0.2225). n.s.: no significant differences. (J) Significance compared to control was determined by one‐way ANOVA with post hoc Dunnett. Significant difference was observed between control and Rab21‐sh115 in the number of primary dendrites. *Less than the critical value at 5%, n.s.: no significant differences. Scale bars: 5 μm in (A, C, D, F), 10 μm in (I), 150 μm in (K), 100 μm in (L).

Article Snippet: The efficiency of colocalization of two proteins in neurons was measured, and the Pearson's correlation coefficient was calculated using NIS elements software (Nikon) or Image J.

Techniques: Transfection, Microscopy, Incubation, In Vitro, Feeding Assay, Fluorescence, Staining, Marker, MANN-WHITNEY

Journal: EMBO Reports

Article Title: Rab21 regulates caveolin‐1‐mediated endocytic trafficking to promote immature neurite pruning

doi: 10.15252/embr.202254701

Figure Lengend Snippet: A, B Primary cortical neurons from E15 cerebral cortices were transfected with the indicated plasmids plus pCAG‐PM‐mAG1 (green) and incubated for 2 days in vitro . Immunocytochemical analyses with anti‐caveolin‐1 (red) antibody were performed. The images are obtained with high‐resolution microscopy (Nikon). The graph in (B) indicates the ratio of the caveolin‐1 staining signals in the plasma membrane to total fluorescence intensities of caveolin‐1 in each immature neuron. Each score represents the mean of ratios with the individual points. Control: n = 20 cells, Rab21‐sh115: n = 22 cells, Rab21‐sh115 + pCMV‐wt‐Rab21: n = 23 cells. Overexpression of wt‐Rab21 resulted in neuronal cell death, which making it difficult to determine the accurate DNA concentration of pCMV‐wt‐Rab21 in the rescue experiments. C–F Primary cortical neurons from E15 cerebral cortices were transfected with the indicated plasmids and incubated for 2 days in vitro . Immunoblot analyses of cell lysates with the indicated antibodies were performed. The graphs in (D) and (F) indicate the mean ratios of immunoblot band intensities of caveolin‐1/β‐tubulin with the individual points. G Immature neurons in the IZ of the cerebral cortices at E17, electroporated with the indicated plasmids plus pCAG‐EGFP at E14. Frozen sections were immunostained with the indicated antibodies. Blue alone channels are shown in black and white images. H, I Primary cortical neurons from E15 cerebral cortices were transfected with the indicated plasmids, incubated for 2 days in vitro , treated with Bafilomycin A1 (Baf A1) for 6 h and stained with the indicated antibodies. The images are obtained with high‐resolution microscopy (Nikon) and the lower panels are high magnification images of the lysosomes, indicated by white or blue rectangles. Blue alone channels are shown in black and white images. The graph in (I) shows the Pearson's correlation coefficient between caveolin‐1 and Lamp1, a lysosomal marker, in control and Rab21‐sh115‐transfected neurons. Each score represents the mean of ratios with the individual points. Control: n = 31 cells, Rab21‐sh115: n = 22 cells. Data information: (B) Significance was determined by one‐way ANOVA with post hoc Dunnett and Tukey–Kramer. **Less than the critical value at 1%. Significant difference was observed between control and Rab21‐sh115, but not between control and the co‐expression of Rab21‐sh115 and pCMV‐wt‐Rab21, by Dunnett and Tukey–Kramer. However, no significance was observed between Rab21‐sh115 alone and co‐expression of Rab21‐sh115 and pCMV‐wt‐Rab21 by Tukey–Kramer. (D and F) Significance was determined by one‐way ANOVA with post hoc Tukey–Kramer (D: n = 9 biological replicates) and Mann–Whitney's U test (F: P = 0.02102, n = 4 biological replicates). n.s.: no significant differences, *less than the critical value at 5%, **less than the critical value at 1%. (I) Significance was determined by Mann–Whitney's U test ( P = 0.0000006290). ** P < 0.01. Scale bars: 1 μm in (A, lower panels in H), 4 μm in (G), 5 μm in (upper panels in H).

Article Snippet: The efficiency of colocalization of two proteins in neurons was measured, and the Pearson's correlation coefficient was calculated using NIS elements software (Nikon) or Image J.

Techniques: Transfection, Incubation, In Vitro, Microscopy, Staining, Fluorescence, Over Expression, Concentration Assay, Western Blot, Marker, Expressing, MANN-WHITNEY

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