Figure S2.
Endosomal localization of SNX6, SNX2, and SNX3 depends on PtdIns3P. (A–D) Co-localization of endogenous SNX6 with EEA1 (A and B) or Rab7 (C and D) without or with VPS34-IN1 treatment (2 μM, 3 h) in Ctrl and KO-91 cells. Zoom images show magnified frames in the merged images. Bars, 5 µm. Quantifications of SNX6-EEA1 and SNX6-Rab7 co-localization are shown in B and D. SNX6-EEA1 co-localization (B): n = 83 cells (Ctrl, DMSO); n = 70 cells (Ctrl, VPS34-IN1); n = 73 cells (KO-91, DMSO); n = 66 cells (KO-91, VPS34-IN1). SNX6-Rab7 co-localization (D): n = 71 cells (Ctrl, DMSO); n = 62 cells (Ctrl, VPS34-IN1); n = 61 cells (KO-91, DMSO); n = 74 cells (KO-91, VPS34-IN1). (E–H) Co-localization of endogenous SNX2 with EEA1 (E and F) or Rab7 (G and H) without or with VPS34-IN1 treatment (2 μM, 3 h) in Ctrl and KO-91 cells. Zoom images show magnified frames in the merged images. Bars, 5 µm. Quantifications of SNX2-EEA1 and SNX2-Rab7 co-localization are shown in F and H. SNX2-EEA1 co-localization (F): n = 53 cells (Ctrl, DMSO); n = 51 cells (Ctrl, VPS34-IN1); n = 46 cells (KO-91, DMSO); n = 52 cells (KO-91, VPS34-IN1). SNX2-Rab7 co-localization (H): n = 53 cells (Ctrl, DMSO); n = 49 cells (Ctrl, VPS34-IN1); n = 59 cells (KO-91, DMSO); n = 47 cells (KO-91, VPS34-IN1). (I–L) Co-localization of endogenous SNX3 with EEA1 (I and J) or Rab7 (K and L) without or with VPS34-IN1 treatment (2 μM, 3 h) in Ctrl and KO-91 cells. Zoom images show magnified frames in the merged images. Bars, 5 µm. Quantifications of SNX3-EEA1 and SNX3-Rab7 co-localization are shown in J and L. SNX3-EEA1 co-localization (J): n = 49 cells (Ctrl, DMSO); n = 49 cells (Ctrl, VPS34-IN1); n = 47 cells (KO-91, DMSO); n = 44 cells (KO-91, VPS34-IN1). SNX3-Rab7 co-localization (L): n = 51 cells (Ctrl, DMSO); n = 48 cells (Ctrl, VPS34-IN1); n = 47 cells (KO-91, DMSO); n = 45 cells (KO-91, VPS34-IN1). For all quantifications, the y-axis shows the value of Pearson’s correlation coefficient. Error bars represent SEM. Data are from three independent experiments. Statistical analyses were performed with the Kruskal–Wallis test. ***, P < 0.001.

Endosomal localization of SNX6, SNX2, and SNX3 depends on PtdIns3P. (A–D) Co-localization of endogenous SNX6 with EEA1 (A and B) or Rab7 (C and D) without or with VPS34-IN1 treatment (2 μM, 3 h) in Ctrl and KO-91 cells. Zoom images show magnified frames in the merged images. Bars, 5 µm. Quantifications of SNX6-EEA1 and SNX6-Rab7 co-localization are shown in B and D. SNX6-EEA1 co-localization (B): n = 83 cells (Ctrl, DMSO); n = 70 cells (Ctrl, VPS34-IN1); n = 73 cells (KO-91, DMSO); n = 66 cells (KO-91, VPS34-IN1). SNX6-Rab7 co-localization (D): n = 71 cells (Ctrl, DMSO); n = 62 cells (Ctrl, VPS34-IN1); n = 61 cells (KO-91, DMSO); n = 74 cells (KO-91, VPS34-IN1). (E–H) Co-localization of endogenous SNX2 with EEA1 (E and F) or Rab7 (G and H) without or with VPS34-IN1 treatment (2 μM, 3 h) in Ctrl and KO-91 cells. Zoom images show magnified frames in the merged images. Bars, 5 µm. Quantifications of SNX2-EEA1 and SNX2-Rab7 co-localization are shown in F and H. SNX2-EEA1 co-localization (F): n = 53 cells (Ctrl, DMSO); n = 51 cells (Ctrl, VPS34-IN1); n = 46 cells (KO-91, DMSO); n = 52 cells (KO-91, VPS34-IN1). SNX2-Rab7 co-localization (H): n = 53 cells (Ctrl, DMSO); n = 49 cells (Ctrl, VPS34-IN1); n = 59 cells (KO-91, DMSO); n = 47 cells (KO-91, VPS34-IN1). (I–L) Co-localization of endogenous SNX3 with EEA1 (I and J) or Rab7 (K and L) without or with VPS34-IN1 treatment (2 μM, 3 h) in Ctrl and KO-91 cells. Zoom images show magnified frames in the merged images. Bars, 5 µm. Quantifications of SNX3-EEA1 and SNX3-Rab7 co-localization are shown in J and L. SNX3-EEA1 co-localization (J): n = 49 cells (Ctrl, DMSO); n = 49 cells (Ctrl, VPS34-IN1); n = 47 cells (KO-91, DMSO); n = 44 cells (KO-91, VPS34-IN1). SNX3-Rab7 co-localization (L): n = 51 cells (Ctrl, DMSO); n = 48 cells (Ctrl, VPS34-IN1); n = 47 cells (KO-91, DMSO); n = 45 cells (KO-91, VPS34-IN1). For all quantifications, the y-axis shows the value of Pearson’s correlation coefficient. Error bars represent SEM. Data are from three independent experiments. Statistical analyses were performed with the Kruskal–Wallis test. ***, P < 0.001.

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