Figure 2.
Figure 2. Intracellular calcium increase and Syt7 regulate the release of LAT and TCRζ from their vesicular compartments to the plasma membrane, whereas MAL specifically regulates Lck traffic. (A–G) Primary CD4 T cells were transfected with siRNA against Syt7, a nonrelevant sequence (siCtr), or left untransfected (Med) and then treated with 5 µM thapsigargin (TPS) for 30 min. Cells were stained for LAT (A and B), TGN38 (C), TCRζ (D and E), and Lck (F and G) and analyzed by 3D confocal microscopy. (H–N) Population analysis quantifying the 3D fluorescence intensity in the vesicular compartment (see Materials and methods) relative to total fluorescence of TGN38 (H), LAT (I and J), TCRζ (K and L), and Lck (M and N) of at least 20 cells per group processed as in A–G. (O) Population analysis of n = 20 cells per group quantifying Lck, TCRζ, and LAT localized at the plasma membrane in cells untreated (−), or treated with TPS (+) for 30 min and processed as in A–G. (P) Jurkat cells were transfected with siRNA against MAL or a nonrelevant sequence (siCtr). Cells were stained for LAT (middle), TCRζ (right), and Lck (left) and analyzed by 3D confocal microscopy. (Q) Population analysis of n = 20 cells per group, quantifying Lck, LAT, and TCRζ in the vesicular compartment relative to total cellular fluorescence (see Materials and methods) in MAL-silenced cells (siMAL+) compared with control (siMAL−), treated as in P. (R and S) Syt7, MAL, and actin (control) levels in primary CD4 T cells transfected with siRNA control (siCtr), siRNA Syt7 (siSyt7), or siRNA MAL (siMAL). Cell lysates were analyzed by Western blot. Confocal images were post-treated by deconvolution. A 1-µm-thick medial stack is shown. Each dot in plots represents one cell. ***, P ≤ 0.0001; **, P ≤ 0.01; *, P ≤ 0.05; Mann-Whitney test. Images representative of three experiments. Bar, 5 µm.

Intracellular calcium increase and Syt7 regulate the release of LAT and TCRζ from their vesicular compartments to the plasma membrane, whereas MAL specifically regulates Lck traffic. (A–G) Primary CD4 T cells were transfected with siRNA against Syt7, a nonrelevant sequence (siCtr), or left untransfected (Med) and then treated with 5 µM thapsigargin (TPS) for 30 min. Cells were stained for LAT (A and B), TGN38 (C), TCRζ (D and E), and Lck (F and G) and analyzed by 3D confocal microscopy. (H–N) Population analysis quantifying the 3D fluorescence intensity in the vesicular compartment (see Materials and methods) relative to total fluorescence of TGN38 (H), LAT (I and J), TCRζ (K and L), and Lck (M and N) of at least 20 cells per group processed as in A–G. (O) Population analysis of n = 20 cells per group quantifying Lck, TCRζ, and LAT localized at the plasma membrane in cells untreated (−), or treated with TPS (+) for 30 min and processed as in A–G. (P) Jurkat cells were transfected with siRNA against MAL or a nonrelevant sequence (siCtr). Cells were stained for LAT (middle), TCRζ (right), and Lck (left) and analyzed by 3D confocal microscopy. (Q) Population analysis of n = 20 cells per group, quantifying Lck, LAT, and TCRζ in the vesicular compartment relative to total cellular fluorescence (see Materials and methods) in MAL-silenced cells (siMAL+) compared with control (siMAL−), treated as in P. (R and S) Syt7, MAL, and actin (control) levels in primary CD4 T cells transfected with siRNA control (siCtr), siRNA Syt7 (siSyt7), or siRNA MAL (siMAL). Cell lysates were analyzed by Western blot. Confocal images were post-treated by deconvolution. A 1-µm-thick medial stack is shown. Each dot in plots represents one cell. ***, P ≤ 0.0001; **, P ≤ 0.01; *, P ≤ 0.05; Mann-Whitney test. Images representative of three experiments. Bar, 5 µm.

This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal