Figure 4.
A subset of EEA1-positive GFP-CD63 endosomes associate preferentially with septin-coated microtubules. (A) Confocal microscopy images show single optical sections from subconfluent MDCK cells transfected with GFP-CD63 (inverted forest green), which were stained for endogenous SEPT7 (inverted blue) and EEA1 (inverted magenta). Overlap between GFP-CD63 and EEA1 is shown in black color. Insets show outlined regions (dashed rectangle) in higher magnification. Scale bars, 10 and 5 μm (insets). (B and C) Quantification of the mean percentage (±SEM, error bars) of EEA1-positive (B) and EEA1-negative (C) GFP-CD63 endosomes that exhibit overlap (SEPT7-bound) or do not overlap with SEPT7 (SEPT7-free) filaments. Analysis was performed in MDCK cells transfected with GFP-CD63 and data (n = 9 cells) were analyzed with an unpaired t test with Welch’s correction. **** P < 0.0001. (D) Mean ratios (±SEM, errors) of SEPT7-bound to SEPT7-free GFP-CD63 endosomes that were EEA1 positive or EEA1 negative in MDCK cells transfected with GFP-CD63. Data (n = 9 cells) were statistically analyzed with an unpaired t test with Welch’s correction. * P < 0.05. (E) Confocal microscopy images show single optical sections from subconfluent MDCK cells transfected with GFP-CD63 (inverted forest green), which were stained for endogenous SEPT7 (inverted blue) and LBPA (inverted magenta). Overlap between GFP-CD63 and LBPA is shown in black color. Insets show outlined regions (dashed rectangle) in higher magnification. Scale bars, 10 and 5 μm (insets). (F and G) Quantification of the mean percentage (±SEM, error bars) of LBPA-positive (F) and LBPA-negative (G) GFP-CD63 endosomes that exhibit overlap (SEPT7-bound) or do not overlap with SEPT7 (SEPT7-free) filaments. Analysis was performed in MDCK-GFP-CD63 cells (n = 11) and data were analyzed with an unpaired t test with Welch’s correction. **** P < 0.0001. (H) Mean ratios (±SEM, error bars) of SEPT7-bound to SEPT7-free GFP-CD63 endosomes that were LBPA positive or LBPA negative in MDCK cells transfected with GFP-CD63. Data (n = 9 cells) were statistically analyzed with a Mann-Whitney test. n.s., not significant. (I and J) Mean percentage (±SEM, error bars) of SEPT7-bound (I) and total (J) GFP-CD63 endosomes that were positive for EEA1 or LBPA. Analysis was performed in MDCK cells transfected with GFP-CD63 stained for endogenous EEA1 (n = 9 cells) or LBPA (n = 11 cells), and data were statistically analyzed with an unpaired t test with Welch’s correction. **** P < 0.0001. (K) Mean percentage (±SEM, error bars) of SEPT7-bound GFP-CD63 endosomes that were positive for EEA1 or negative for LBPA. Analysis was performed in MDCK cells transfected with GFP-CD63 stained for endogenous EEA1 (n = 9 cells) or LBPA (n = 11 cells), and data were statistically analyzed with an unpaired t test with Welch’s correction. n.s., not significant. (L) Mean percentage (±SEM, error bars) of SEPT7-bound GFP-CD63 endosomes that were negative for EEA1 or positive for LBPA. Analysis was performed in MDCK cells transfected with GFP-CD63 cells, and stained for endogenous EEA1 (n = 9 cells) or LBPA (n = 11 cells). Data were statistically analyzed with an unpaired t test with Welch’s correction. n.s., not significant.

A subset of EEA1-positive GFP-CD63 endosomes associate preferentially with septin-coated microtubules. (A) Confocal microscopy images show single optical sections from subconfluent MDCK cells transfected with GFP-CD63 (inverted forest green), which were stained for endogenous SEPT7 (inverted blue) and EEA1 (inverted magenta). Overlap between GFP-CD63 and EEA1 is shown in black color. Insets show outlined regions (dashed rectangle) in higher magnification. Scale bars, 10 and 5 μm (insets). (B and C) Quantification of the mean percentage (±SEM, error bars) of EEA1-positive (B) and EEA1-negative (C) GFP-CD63 endosomes that exhibit overlap (SEPT7-bound) or do not overlap with SEPT7 (SEPT7-free) filaments. Analysis was performed in MDCK cells transfected with GFP-CD63 and data (n = 9 cells) were analyzed with an unpaired t test with Welch’s correction. **** P < 0.0001. (D) Mean ratios (±SEM, errors) of SEPT7-bound to SEPT7-free GFP-CD63 endosomes that were EEA1 positive or EEA1 negative in MDCK cells transfected with GFP-CD63. Data (n = 9 cells) were statistically analyzed with an unpaired t test with Welch’s correction. * P < 0.05. (E) Confocal microscopy images show single optical sections from subconfluent MDCK cells transfected with GFP-CD63 (inverted forest green), which were stained for endogenous SEPT7 (inverted blue) and LBPA (inverted magenta). Overlap between GFP-CD63 and LBPA is shown in black color. Insets show outlined regions (dashed rectangle) in higher magnification. Scale bars, 10 and 5 μm (insets). (F and G) Quantification of the mean percentage (±SEM, error bars) of LBPA-positive (F) and LBPA-negative (G) GFP-CD63 endosomes that exhibit overlap (SEPT7-bound) or do not overlap with SEPT7 (SEPT7-free) filaments. Analysis was performed in MDCK-GFP-CD63 cells (n = 11) and data were analyzed with an unpaired t test with Welch’s correction. **** P < 0.0001. (H) Mean ratios (±SEM, error bars) of SEPT7-bound to SEPT7-free GFP-CD63 endosomes that were LBPA positive or LBPA negative in MDCK cells transfected with GFP-CD63. Data (n = 9 cells) were statistically analyzed with a Mann-Whitney test. n.s., not significant. (I and J) Mean percentage (±SEM, error bars) of SEPT7-bound (I) and total (J) GFP-CD63 endosomes that were positive for EEA1 or LBPA. Analysis was performed in MDCK cells transfected with GFP-CD63 stained for endogenous EEA1 (n = 9 cells) or LBPA (n = 11 cells), and data were statistically analyzed with an unpaired t test with Welch’s correction. **** P < 0.0001. (K) Mean percentage (±SEM, error bars) of SEPT7-bound GFP-CD63 endosomes that were positive for EEA1 or negative for LBPA. Analysis was performed in MDCK cells transfected with GFP-CD63 stained for endogenous EEA1 (n = 9 cells) or LBPA (n = 11 cells), and data were statistically analyzed with an unpaired t test with Welch’s correction. n.s., not significant. (L) Mean percentage (±SEM, error bars) of SEPT7-bound GFP-CD63 endosomes that were negative for EEA1 or positive for LBPA. Analysis was performed in MDCK cells transfected with GFP-CD63 cells, and stained for endogenous EEA1 (n = 9 cells) or LBPA (n = 11 cells). Data were statistically analyzed with an unpaired t test with Welch’s correction. n.s., not significant.

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