Figure 7. Lem8 contributes to cell migration inhibition by L. pneumophila.

(A) Establishment of cell lines stably expressing Lem8 or its enzymatically inactive mutant. HEK293T cells were transduced with lentiviral particles harboring the indicated plasmid at an multiplicity of infection (MOI) of 10 for 2 days, and the GFP-positive cells were isolated by a BD Influx cell sorter. Lysates of each cell line were probed by immunoblotting with antibodies specific for Phldb2 or Lem8. Tubulin was used as a loading control. (B) Wound-healing scratch assay of the three stable cell lines. The three cell lines were individually seeded into 6-well plates. When reached confluency, cell monolayer of each cell line was scratched using a pipette tip. Images of the wounds were captured at 2, 24, and 48 hr after making the scratches using an Olympus IX-83 fluorescence microscope. Images of a representative experiment were shown (left panel). The wound-healing rates from three independent experiments were quantitated by Image J (right panel). (C, D) Evaluation of the impact of Lem8 on cell migration in cells infected with L. pneumophila. HEK293T cells expressing the FcγII receptor or Raw264.7 cells were infected with opsonized bacteria of the indicated L. pneumophila strains at an MOI of 50 for 2 hr. After washes, the wound-healing scratch assay was performed to evaluate the impact of infection on cell migration. Images of a representative experiment were shown (C, left panel) and the wound-healing rate was analyzed by Image J (C, right panel).

Figure 7—figure supplement 1. Overexpression of Phldb2 suppressed the inhibitory effects of Lem8 on cell migration.

(A) Overexpression of Phldb2 in 293T cells stably expressing Lem8. A HEK293T-derived cell line stably expressing Lem8 was transfected with empty vector or HA-Phldb2 for 18 hr, and the cell lysates were probed by immunoblotting with antibodies specific for Phldb2 or Lem8. Tubulin was used as a loading control. Results shown were one representative from three independent experiments with similar results. (B) Wound-healing scratch assay of cells expressing Phldb2. Eighteen hours after transfection, monolayers were scratched using a pipette tip. Images of the wounds were captured at 2, 24, and 48 hr, respectively. Results shown were from a representative of three independent experiments from which the quantitation of wound healing was obtained by Image J (right panel).