Figure 2.
WDR91 deficiency causes endosomal trapping of SNX-retromer components. (A) Co-immunostaining of endogenous SNX27 with EEA1 or Rab7 in Ctrl and KO-91 cells. Zoom images show magnified frames in the merged images. Bars, 5 µm. (B) Quantification of the co-localization of endogenous SNX27 with endosomal markers as shown in A. n = 64 cells (Ctrl, SNX27-EEA1); n = 42 cells (KO-91, SNX27-EEA1); n = 46 cells (Ctrl, SNX27-Rab7); n = 41 cells (KO-91, SNX27-Rab7). Statistical analyses were performed with the two-tailed unpaired t test. (C) Co-immunostaining of endogenous VPS35 with EEA1 or Rab7 in Ctrl and KO-91 cells. Zoom images show magnified frames in the merged images. Bars, 5 µm. (D) Quantification of the co-localization of endogenous VPS35 with endosomal markers as shown in C. n = 40 cells (Ctrl, VPS35-EEA1); n = 45 cells (KO-91, VPS35-EEA1); n = 45 cells (Ctrl, VPS35-Rab7); n = 42 cells (KO-91, VPS35-Rab7). Statistical analyses were performed with the two-tailed unpaired t test. (E) Co-immunostaining of endogenous SNX6 with EEA1 or Rab7 in Ctrl and KO-91 cells. Zoom images show magnified frames in the merged images. Bars, 5 µm. (F) Quantification of the co-localization of endogenous SNX6 with endosomal markers as shown in E. n = 54 cells (Ctrl, SNX6-EEA1); n = 48 cells (KO-91, SNX6-EEA1); n = 45 cells (Ctrl, SNX6-Rab7); n = 42 cells (KO-91, SNX6-Rab7). Statistical analyses were performed with the two-tailed unpaired t test. (G) Time-lapse chasing of SNX27-GFP tubulation events (indicated by arrowheads in different colors) from 2×FYVE-mCh–labeled endosomes in Ctrl and KO-91 cells. For each group, the top row shows SNX27-GFP images and the bottom row shows the merged images of SNX27-GFP and 2×FYVE-mCh. Bars, 2 µm. (H) Quantification of SNX27 tubulation events on 2×FYVE-mCh–positive endosomes per cell in 10 min. n = 15 cells (Ctrl); n = 18 cells (KO-91). Statistical analyses were performed with the Mann–Whitney U-test. (I) Time-lapse chasing of SNX27-GFP tubulation (indicated by arrowheads in different colors) from mCh-CD63–labeled endosomes in Ctrl and KO-91 cells. For each group, the top row shows SNX27-GFP images and the bottom row shows the merged images of SNX27-GFP and mCh-CD63. Bars, 2 µm. (J) Quantification of SNX27 tubulation events on mCh-CD63–positive endosomes per cell 10 min. n = 16 cells (Ctrl); n = 18 cells (KO-91). Statistical analyses were performed with the Mann–Whitney U-test. For all quantifications, error bars represent SEM. Co-localization was quantified according to Pearson’s correlation coefficient. Data are from three independent experiments. Statistical comparisons are between Ctrl and KO-91 cells. ***, P < 0.001.

WDR91 deficiency causes endosomal trapping of SNX-retromer components. (A) Co-immunostaining of endogenous SNX27 with EEA1 or Rab7 in Ctrl and KO-91 cells. Zoom images show magnified frames in the merged images. Bars, 5 µm. (B) Quantification of the co-localization of endogenous SNX27 with endosomal markers as shown in A. n = 64 cells (Ctrl, SNX27-EEA1); n = 42 cells (KO-91, SNX27-EEA1); n = 46 cells (Ctrl, SNX27-Rab7); n = 41 cells (KO-91, SNX27-Rab7). Statistical analyses were performed with the two-tailed unpaired t test. (C) Co-immunostaining of endogenous VPS35 with EEA1 or Rab7 in Ctrl and KO-91 cells. Zoom images show magnified frames in the merged images. Bars, 5 µm. (D) Quantification of the co-localization of endogenous VPS35 with endosomal markers as shown in C. n = 40 cells (Ctrl, VPS35-EEA1); n = 45 cells (KO-91, VPS35-EEA1); n = 45 cells (Ctrl, VPS35-Rab7); n = 42 cells (KO-91, VPS35-Rab7). Statistical analyses were performed with the two-tailed unpaired t test. (E) Co-immunostaining of endogenous SNX6 with EEA1 or Rab7 in Ctrl and KO-91 cells. Zoom images show magnified frames in the merged images. Bars, 5 µm. (F) Quantification of the co-localization of endogenous SNX6 with endosomal markers as shown in E. n = 54 cells (Ctrl, SNX6-EEA1); n = 48 cells (KO-91, SNX6-EEA1); n = 45 cells (Ctrl, SNX6-Rab7); n = 42 cells (KO-91, SNX6-Rab7). Statistical analyses were performed with the two-tailed unpaired t test. (G) Time-lapse chasing of SNX27-GFP tubulation events (indicated by arrowheads in different colors) from 2×FYVE-mCh–labeled endosomes in Ctrl and KO-91 cells. For each group, the top row shows SNX27-GFP images and the bottom row shows the merged images of SNX27-GFP and 2×FYVE-mCh. Bars, 2 µm. (H) Quantification of SNX27 tubulation events on 2×FYVE-mCh–positive endosomes per cell in 10 min. n = 15 cells (Ctrl); n = 18 cells (KO-91). Statistical analyses were performed with the Mann–Whitney U-test. (I) Time-lapse chasing of SNX27-GFP tubulation (indicated by arrowheads in different colors) from mCh-CD63–labeled endosomes in Ctrl and KO-91 cells. For each group, the top row shows SNX27-GFP images and the bottom row shows the merged images of SNX27-GFP and mCh-CD63. Bars, 2 µm. (J) Quantification of SNX27 tubulation events on mCh-CD63–positive endosomes per cell 10 min. n = 16 cells (Ctrl); n = 18 cells (KO-91). Statistical analyses were performed with the Mann–Whitney U-test. For all quantifications, error bars represent SEM. Co-localization was quantified according to Pearson’s correlation coefficient. Data are from three independent experiments. Statistical comparisons are between Ctrl and KO-91 cells. ***, P < 0.001.

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