Article Figures & Data
Figures
- Fig. 1.
Ad interaction with cells induces morphologic changes and reorganization of the actin cytoskeleton. Serum-starved A549 epithelial cells were incubated for various times with different ligands at 37°C, permeabilized, and stained for polymerized actin with rhodamine-labeled phalloidin. (A and G) Unstimulated control cells. (B and C) Cells incubated at 37°C for 5 to 10 min with 100 Ad2 particles/cell. (D) Cells incubated with Ad2 for 25 min at 37°C. (E and F) Cells incubated with 100 ng of EGF/ml for 5 min at 37°C. (H and I) Cells treated with 100 nM wortmannin followed by incubation with 100 Ad2 particles/cell for 10 min. Arrowheads indicate sites of membrane filopodia, while the arrows indicate sites of lamellipodia.
- Fig. 2.
Disruption of cortical actin filaments with cytochalasin D inhibits Ad internalization. A549 cells were incubated in the presence of 0.5 μM (squares) or 2 μM (open circles) cytochalasin D at 37°C for 90 min or in medium containing 0.1% dimethyl sulfoxide (closed circles). Internalization of 125I-labeled Ad2 particles was subsequently measured by resistance to trypsin treatment as described in Materials and Methods.
- Fig. 3.
Inhibition of Ad internalization by C. difficile toxin B. A549 cells were incubated for 5 h in serum-free medium containing various amounts of recombinant toxin B, a specific inhibitor of Rho family GTPases, prior to measuring Ad internalization by resistance to trypsin treatment at 10 min postwarming.
- Fig. 4.
Ad internalization is specifically inhibited by overexpression of dominant-negative Rac mutant protein. SW480 cells were transiently transfected with a control plasmid lacking a transgene or with plasmids encoding a wild-type (solid bars), a constitutively active (Q61L) (shaded bars), or a dominant-negative (17N) (cross-hatched bars) form of Rac or H-Ras. Ad internalization was assayed 45 h posttransfection. The lower panels show Western blots of transfected cell lysates probed for Rac or Ras expression. WT, wild type.
- Fig. 5.
Overexpression of a constitutively active Rac protein restores Ad internalization into wortmannin-treated cells. SW480 cells were transfected with a control plasmid or a plasmid encoding a wild-type (WT) or constitutively active (Q61L) Rac. Forty-eight hours posttransfection, the cells were incubated in the presence (cross-hatched bars) or absence (solid bars) of wortmannin for 10 min at 4°C prior to measuring Ad internalization. The inset shows a Western blot of transfected cell lysates probed for Rac expression.
- Fig. 6.
Expression of an effector domain mutant of Rac that impairs cytoskeletal function but not JNK/MAP kinase activation inhibits Ad internalization. (A) SW480 cells were transiently transfected with a control plasmid, a wild-type Rac, or Rac mutants as indicated. Ad internalization was measured 45 h posttransfection. (B) Western blot of transfected cell lysates with an anti-c-Myc antibody. (C) JNK activation by transfected cells. Aniso, anisomycin.
- Fig. 7.
Expression of an effector domain mutant of CDC42 (Q61,F37A) inhibits Ad internalization and gene delivery. (A) A stably transfected cell line expressing CDC42 (Q61L,F37A) was assayed for its ability to support Ad internalization 40 h after the removal of tetracycline (open squares) or in the continued presence of tetracycline (filled circles). (B) Transfected cells cultured in the absence (cross-hatched bars) or presence (solid bars) of tetracycline were also assayed for susceptibility to Ad-mediated delivery of a reporter gene (β-galactosidase). The inset shows Western blot analysis of the CDC42 mutant protein and actin expressed in cells cultured in the presence or absence of tetracycline.
- Fig. 8.
Ad internalization does not involve activation of p65PAK or JNK/MAP kinase. Serum-starved SW480 cell lysates derived from cells incubated with Ad were probed for p65PAK (A) or JNK activation (B) as described in Materials and Methods. Cells were treated with insulin or anisomycin (Aniso) or transfected with a dominant active PAK1 (T432E) as positive controls for kinase activation.
