Figure 3.
PtdIns3P contributes differently to endosomal trapping of SNX-retromer components in the absence of WDR91. (A) Co-immunostaining of endogenous SNX27 with EEA1 in Ctrl and KO-91 cells treated without or with VPS34-IN1 (2 μM) for 3 h. Zoom images show magnified frames in the merged images. Bars, 5 µm. (B) Quantification of SNX27-EEA1 co-localization as shown in A. n = 64 cells (Ctrl, DMSO); n = 56 cells (Ctrl, VPS34-IN1); n = 48 cells (KO-91, DMSO); n = 56 cells (KO-91, VPS34-IN1). (C) Co-immunostaining of endogenous SNX27 with Rab7 in Ctrl and KO-91 cells treated without or with VPS34-IN1 (2 μM) for 3 h. Zoom images show magnified frames in the merged images. Bars, 5 µm. (D) Quantification of SNX27-Rab7 co-localization as shown in C. n = 57 cells (Ctrl, DMSO); n = 57 cells (Ctrl, VPS34-IN1); n = 58 cells (KO-91, DMSO); n = 72 cells (KO-91, VPS34-IN1). (E) Co-immunostaining of endogenous VPS35 with EEA1 in Ctrl and KO-91 cells treated without or with VPS34-IN1 (2 μM) for 3 h. Zoom images show magnified frames in the merged images. Bars, 5 µm. (F) Quantification of VPS35-EEA1 co-localization as shown in E. n = 60 cells (Ctrl, DMSO); n = 83 cells (Ctrl, VPS34-IN1); n = 45 cells (KO-91, DMSO); n = 55 cells (KO-91, VPS34-IN1). (G) Co-immunostaining of endogenous VPS35 with Rab7 in Ctrl and KO-91 cells treated without or with VPS34-IN1 (2 μM) for 3 h. Zoom images show magnified frames in the merged images. Bars, 5 µm. (H) Quantification of VPS35-Rab7 co-localization as shown in G. n = 42 cells (Ctrl, DMSO); n = 52 cells (Ctrl, VPS34-IN1); n = 44 cells (KO-91, DMSO); n = 48 cells (KO-91, VPS34-IN1). For all quantifications, error bars represent SEM. The y-axis shows the value of Pearson’s correlation coefficient. Data are from three independent experiments. Statistical comparisons are between DMSO and VPS34-IN1 treatments. Statistical analyses were performed with the Kruskal–Wallis test. ***, P < 0.001. NS, P > 0.05.

PtdIns3P contributes differently to endosomal trapping of SNX-retromer components in the absence of WDR91. (A) Co-immunostaining of endogenous SNX27 with EEA1 in Ctrl and KO-91 cells treated without or with VPS34-IN1 (2 μM) for 3 h. Zoom images show magnified frames in the merged images. Bars, 5 µm. (B) Quantification of SNX27-EEA1 co-localization as shown in A. n = 64 cells (Ctrl, DMSO); n = 56 cells (Ctrl, VPS34-IN1); n = 48 cells (KO-91, DMSO); n = 56 cells (KO-91, VPS34-IN1). (C) Co-immunostaining of endogenous SNX27 with Rab7 in Ctrl and KO-91 cells treated without or with VPS34-IN1 (2 μM) for 3 h. Zoom images show magnified frames in the merged images. Bars, 5 µm. (D) Quantification of SNX27-Rab7 co-localization as shown in C. n = 57 cells (Ctrl, DMSO); n = 57 cells (Ctrl, VPS34-IN1); n = 58 cells (KO-91, DMSO); n = 72 cells (KO-91, VPS34-IN1). (E) Co-immunostaining of endogenous VPS35 with EEA1 in Ctrl and KO-91 cells treated without or with VPS34-IN1 (2 μM) for 3 h. Zoom images show magnified frames in the merged images. Bars, 5 µm. (F) Quantification of VPS35-EEA1 co-localization as shown in E. n = 60 cells (Ctrl, DMSO); n = 83 cells (Ctrl, VPS34-IN1); n = 45 cells (KO-91, DMSO); n = 55 cells (KO-91, VPS34-IN1). (G) Co-immunostaining of endogenous VPS35 with Rab7 in Ctrl and KO-91 cells treated without or with VPS34-IN1 (2 μM) for 3 h. Zoom images show magnified frames in the merged images. Bars, 5 µm. (H) Quantification of VPS35-Rab7 co-localization as shown in G. n = 42 cells (Ctrl, DMSO); n = 52 cells (Ctrl, VPS34-IN1); n = 44 cells (KO-91, DMSO); n = 48 cells (KO-91, VPS34-IN1). For all quantifications, error bars represent SEM. The y-axis shows the value of Pearson’s correlation coefficient. Data are from three independent experiments. Statistical comparisons are between DMSO and VPS34-IN1 treatments. Statistical analyses were performed with the Kruskal–Wallis test. ***, P < 0.001. NS, P > 0.05.

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