CTLA4 accumulates in enlarged endosomes following USP8 depletion. (A and B) Representative confocal images of CTLA4-HA co-stained for EEA1 (A) or LAMP1 (B) in HeLa S3 Flp-In CTLA4-HA cells transfected for 72 h with non-targeting (NT1) or USP8 siRNA. Scale bar = 15 µm (main figure) or 5 µm (inset). (C and D) Colocalization analysis of CTLA4-HA with EEA1 and LAMP1 in HeLa S3 Flp-In CTLA4-HA for data represented in A and B. Shown are the Pearson’s (C) or Mander’s coefficients (D). Error bars show SD from three independent, color-coded experiments. Opaque circles with black outlines correspond to the mean value from each experiment. Unpaired t test. *P < 0.05, ***P < 0.001. (E and F) Representative confocal images of CTLA4 co-stained for EEA1 (E) or LAMP1 (F) in A2058 cells transfected for 72 h with NT1 or USP8 siRNA. Scale bar = 15 µm (main figure) or 5 µm (inset). (G and H) Colocalization analysis of CTLA4 with EEA1 and LAMP1 in A2058 cells for data represented in E and F. Graphs show Pearson’s (G) or Mander’s coefficients (H) between CTLA4 and EEA1 or LAMP1. Error bars show SD from three independent, color-coded experiments. Opaque circles with black outlines correspond to the mean value from each experiment. Unpaired t test. *P < 0.05, **P < 0.01.
Figure 6.

CTLA4 accumulates in enlarged endosomes following USP8 depletion. (A and B) Representative confocal images of CTLA4-HA co-stained for EEA1 (A) or LAMP1 (B) in HeLa S3 Flp-In CTLA4-HA cells transfected for 72 h with non-targeting (NT1) or USP8 siRNA. Scale bar = 15 µm (main figure) or 5 µm (inset). (C and D) Colocalization analysis of CTLA4-HA with EEA1 and LAMP1 in HeLa S3 Flp-In CTLA4-HA for data represented in A and B. Shown are the Pearson’s (C) or Mander’s coefficients (D). Error bars show SD from three independent, color-coded experiments. Opaque circles with black outlines correspond to the mean value from each experiment. Unpaired t test. *P < 0.05, ***P < 0.001. (E and F) Representative confocal images of CTLA4 co-stained for EEA1 (E) or LAMP1 (F) in A2058 cells transfected for 72 h with NT1 or USP8 siRNA. Scale bar = 15 µm (main figure) or 5 µm (inset). (G and H) Colocalization analysis of CTLA4 with EEA1 and LAMP1 in A2058 cells for data represented in E and F. Graphs show Pearson’s (G) or Mander’s coefficients (H) between CTLA4 and EEA1 or LAMP1. Error bars show SD from three independent, color-coded experiments. Opaque circles with black outlines correspond to the mean value from each experiment. Unpaired t test. *P < 0.05, **P < 0.01.

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