Figure 6.

HAS2 and CD44 are required for human lung fibroblast invasion. (A) Invasive capacity of human fibroblasts from normal subjects (NHF; n = 5) and IPF patients (n = 9). Results are for five separate experiments and are expressed as the invasion index of the IPF fibroblasts over the normal fibroblasts (*, P < 0.05). (B) Representative images of invasive IPF fibroblasts and normal fibroblasts. (C) Relative HAS2 mRNA levels of invasive and noninvasive IPF fibroblasts were determined using real-time PCR (n = 7; *, P < 0.05). The experiments were repeated two times. (D) 48 h after transfection with HAS2 siRNA (HAS2 si) and control siRNA (control si), photomicrographs demonstrating the effects of HAS2 si on fibroblast cellular surface HA, photomicrographs demonstrating the effects of HAS2 si on HA coat formation, and images of invasive HAS2 siRNA– and control siRNA–transfected fibroblasts are shown. (E) 48 h after HAS2 and control siRNA transfection, equal numbers of fibroblasts from normal donors (n = 2) and IPF patients (n = 3) were loaded into invasion chambers and incubated for another 24 h. Invasive cells were counted. Data are shown as the invasion index of HAS2 siRNA–transfected normal, IPF fibroblasts and control siRNA–transfected IPF fibroblasts over control siRNA–transfected normal fibroblasts (**, P < 0.01). (F) After 20 min of incubation with anti-CD44 neutralizing or isotype-matched control IgG antibody, fibroblasts from normal donors (n = 3) and IPF patients (n = 6) were subjected to the invasion assay. Data are depicted as the invasion index (**, P < 0.01). (D–F) The experiments were repeated three times. (A, C, E, and F) Error bars indicate mean ± SEM. Bars: (B and D [top and bottom]) 200 µm; (D, middle) 100 µm.