Fig. 2

RASSF1A expression decreases overall TNT number and length. (a) Quantitative description of the average number of TNTs established for each cell line. TNT’s number was classified as low (+), moderate (++) or high (+++) if there were observed ≤1, 1 ≤ 3 or > 3 TNT per cell respectively. (b) Representative images of TNT in HBEC-3, A549 and H28 cell lines fixed and stained with tubulin. (c) Quantification of the TNT number and (d) length (μm) in HBEC-3 cell line along with (e) representative images (ei, eii and eiii are zoom of the corresponding arrowheads). The HBEC-3 cells were treated with control or RASSF1A RNAi as indicated. Western blot showing the efficiency of RASSF1A depletion in HBEC-3 cells 72 h after RNAi treatment. Arrowheads indicate the TNTs. Roman numerals mark the examples of the TNT in the zoomed images. (f) Representative image of TNT between H2452 expressing or not (siRASSF1A) RASSF1A using scanning electron microscopy (g) Quantification and (h) representative images of HBEC-3 cells contained both green and red MitoTracker dyes after RNAi treatment as indicated. (i) Representative images of the double labeled MitoTracker dyes in co-culture of RASSF1A-depleted HBEC-3 cells with BEAS-2B, H28 or healthy lung fibroblast cells (j) Quantification of the TNT number and (k) length (μm) in H28 cell line along with (l) representative images (li, lii and liii are zoom of the corresponding arrowheads). The H28 cells were transfected with construct encoding wild-type RASSF1A as indicated. The TNTs are show with arrowheads. Western blot showing the efficiency of RASSF1A transfection in H28 cells 24 h after treatment. (m) Quantification and (n) representative images of H28 cells contained both green and red MitoTracker dyes after pcDNA transfection as indicated. Values are the mean ± SEM of three independent experiments in almost 200 cells. Statistical significance was calculated and p value are indicated by asterisks: *p < 0,05. See Methods for quantification details of each experiments