Fig 5. Dlg1 regulates TCR-triggered, p38-independent degranulation and actin polymerization.

(A-C) OT-1 hybridoma CD8+ T cells infected with re-expression and/or knockdown constructs were stimulated with EG.7 cells in the presence of anti-CD107a. (A) Representative unstimulated (filled) and stimulated (line) histograms for each condition are shown. The percentage of CD107+ cells in each stimulated condition and the change in gMFI (stimulated gMFI- unstimulated gMFI) of the entire population are shown within each panel. (B) The percentage of degranulation relative to control is quantified as Degranulation(%), where Degranulation (%) = stimulated CD107+(%)–unstimulated CD107+(%), and where the control condition is set to 100%. Error bars represent SD of means from six independent experiments. (C-D) OT-1 hybridomas (C) or primary OT-1 CD8+ T cells (D) pretreated with DMSO, cytochalasin D (0.1, 1.0, 10 μM) or SB203580 (0.1, 1.0, 10 μM) were stimulated with EG.7 cells. Degranulation was quantified as Degranulation (%). Error bars represent SD of samples analyzed in triplicate. (E) Primary OT-1 CD8+ CTLs pretreated with DMSO or SB203580 (0.625, 1.25, 2.5, 5, 10 μM) were stimulated with anti-CD3/anti-CD28 for 48hrs. Supernatants were analyzed via ELISA. Error bars represent SD of samples analyzed in triplicate. (F-H) Primary OT-1 CD8+ T cells infected with miR-based viruses (F-G) or pretreated with 10μM SB203580 or DMSO for 30mins (H) were stimulated with MEF.B7.OVA cells for 15mins. Actin polymerization was assessed by phalloidin staining. (F) Histograms of phalloidin for CD8+GFP+ cells. (G-H) Change in actin polymerization was quantified as, ΔMFI = stimulated MFI—unstimulated MFI, and miR-control is set to 100%. Error bars represent SD of means from four independent experiments. *p < 0.05.