![Figure 6. Silencing of Rab27a reduces PI(4,5)P2 levels in the PM of CD4+ T cells and in VCCs of macrophages. (A) LSCM visualization of PH-GFP in control and Rab27a-silenced Jurkat cells. Cell surface CD81 was labeled in nonpermeabilized cells and used as a reference of PM localization. Boxes indicate areas magnified on the right. (B) Representative single-cell intensity profile plots of the fluorescence of PH-GFP and CD81 at the PM quantified in a confocal slice along a line located on a representative segment of the cell (indicated with a scattered white line in the zoom shown in A). (C) Quantification of the localization of the PH-GFP signal predominantly at the PM or the cytosol in control and Rab27a-silenced cells was performed by blinded observers on a per-cell basis, in 200 cells of each condition. Data are expressed as percentages of cells in each category. (D) Incorporation of 32P in phosphoinositides was evaluated by TLC separation of lipid extracts from cells pulsed with [32P]orthophosphate. Shown is a representative autoradiograph. Positions of (phosphatidylinositol monophosphates [PIP]) and PI(4,5)P2 standards are indicated. (E) Quantification of the relative amounts of different phosphoinositides (n = 3). Error bars show SDs. (F) LSCM images of PH-GFP distribution in Rab27a-silenced primary CD4+ T cells isolated from blood. (G) Quantification of the localization of the PH-GFP signal predominantly at the PM or the cytosol of control and Rab27a-silenced CD4+ T cells. 200 cells were evaluated for each condition. (H) Representative LSCM images showing PH-GFP and CD81 distribution in control and Rab27a-silenced MDMs. (I) Quantification of the percentages of cells containing PH-GFP/CD81 double-positive structures in VCCs or at the peripheral PM. 90 cells were evaluated for each condition. (J and K) Restoring Rab27a expression in Jurkat cells expressing the 3′UTR Rab27a shRNA rescues PI(4,5)P2 levels at the PM. (J) Representative images of PH-mRFP distribution in Rab27a-silenced cells (using the 3′UTR shRNA) that were transduced (green cell) or not transduced with the Rab27a-encoding lentivirus. (K) Quantifications were performed by blinded observers on a per-cell basis, in ≥30 cells of each condition. Data are expressed as percentages of cells in each category. *, P < 0.01; ***, P < 0.001. KD, knockdown. Bars: (A [left], F, H, and J) 2 µm; (A, right) 1 µm.](https://cdn.rupress.org/rup/content_public/journal/jcb/209/3/10.1083_jcb.201409082/7/jcb_201409082_fig6.jpeg?Expires=1771655423&Signature=yF08N4QhUnKP07-ZjqL~whL50KgcIJhk6oIAeKYCHtkdIThtvyM6e6zIbn7cU1m2SqToqhwmjCoQ~ux5RsFYsCC3j2QwT5flR5lPdng4M-nHX~0EWhWGmhMqFaSwFrdU0amiqQ-9V921IV5haAmO73OusIF~PLlRtt7yuQJ31mT5oOy9v2VcjUWxV6UlIjZxy2htbdx4BSlQBT8CKnNbxkpgVzarVAQ7RQgP3EBqKnPrLpKyGPMZnhfHpFOO7HG-CHfouF5VHk8rLUBv54VFekBYu3xPBa~HQ8CiM3JN0xGKcncu3ktUt8cRdVGpLM3m4~kwsdLg1I0k2BvWYR5uNg__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
Silencing of Rab27a reduces PI(4,5)P2 levels in the PM of CD4+ T cells and in VCCs of macrophages. (A) LSCM visualization of PH-GFP in control and Rab27a-silenced Jurkat cells. Cell surface CD81 was labeled in nonpermeabilized cells and used as a reference of PM localization. Boxes indicate areas magnified on the right. (B) Representative single-cell intensity profile plots of the fluorescence of PH-GFP and CD81 at the PM quantified in a confocal slice along a line located on a representative segment of the cell (indicated with a scattered white line in the zoom shown in A). (C) Quantification of the localization of the PH-GFP signal predominantly at the PM or the cytosol in control and Rab27a-silenced cells was performed by blinded observers on a per-cell basis, in 200 cells of each condition. Data are expressed as percentages of cells in each category. (D) Incorporation of 32P in phosphoinositides was evaluated by TLC separation of lipid extracts from cells pulsed with [32P]orthophosphate. Shown is a representative autoradiograph. Positions of (phosphatidylinositol monophosphates [PIP]) and PI(4,5)P2 standards are indicated. (E) Quantification of the relative amounts of different phosphoinositides (n = 3). Error bars show SDs. (F) LSCM images of PH-GFP distribution in Rab27a-silenced primary CD4+ T cells isolated from blood. (G) Quantification of the localization of the PH-GFP signal predominantly at the PM or the cytosol of control and Rab27a-silenced CD4+ T cells. 200 cells were evaluated for each condition. (H) Representative LSCM images showing PH-GFP and CD81 distribution in control and Rab27a-silenced MDMs. (I) Quantification of the percentages of cells containing PH-GFP/CD81 double-positive structures in VCCs or at the peripheral PM. 90 cells were evaluated for each condition. (J and K) Restoring Rab27a expression in Jurkat cells expressing the 3′UTR Rab27a shRNA rescues PI(4,5)P2 levels at the PM. (J) Representative images of PH-mRFP distribution in Rab27a-silenced cells (using the 3′UTR shRNA) that were transduced (green cell) or not transduced with the Rab27a-encoding lentivirus. (K) Quantifications were performed by blinded observers on a per-cell basis, in ≥30 cells of each condition. Data are expressed as percentages of cells in each category. *, P < 0.01; ***, P < 0.001. KD, knockdown. Bars: (A [left], F, H, and J) 2 µm; (A, right) 1 µm.
