Next, slides were washed, incubated with secondary antibodies (1:500; Alexa Fluor 488-conjugated) for 1 h at room temperature, washed, and mounted in DAPI-containing ProLong Gold mounting medium (Invitrogen). and and = 3). *** 0.0001. (and = 5 patients). *= 0.01; **= 0.0048; ***= 0.0004. Increased Tumor Cell Motility Mediated by PI3K Therapy. Consistent with these data, PI3K inhibitors vigorously stimulated tumor cell invasion across Matrigel-coated Transwell inserts (Fig. 2 and and Fig. S1 and and and Fig. S1 and = 0.02; *** 0.0001. (= 15). *** 0.0001. (= 15). **= 0.0047; *** 0.0001. (and Fig. S2and Fig. S2and Movie S1), potentially associated with random cell motility (16). These lateral ruffles were larger and persisted for a longer time in response to PI3K therapy compared with untreated cells (Fig. 2= 15) as in were analyzed for membrane ruffle dynamics by SACED with quantification of average ruffle size (= 4). Open in a separate window Fig. S3. Adaptation-induced random tumor cell migration. ( 103. A circular region that splits the Euclidean distances 50:50 is indicated (radius). (and 0.0001. Mitochondrial Repositioning to the Cortical Cytoskeleton Supports RGS2 Adaptive Tumor Cell Invasion. When analyzed by fluorescence microscopy, PI3K therapy induced profound changes in the morphology and distribution of mitochondria. Whereas untreated cells exhibited mitochondria that were polarized and mostly clustered around the nucleus (Fig. S4 and and Fig. S4and Fig. S4and Fig. S4 and = 0.0047), thus preventing additional studies of mitochondrial relocalization or tumor cell invasion. Open in a separate window Fig. 3. Mitochondria fuel focal adhesion dynamics. (and quantified for mitochondrial infiltration into lamellipodia. At least 18 cells were analyzed at two independent lamellipodia, and data were normalized to total lamellipodia length. Mean SEM (= 36). *** 0.0001. (and scored for mitochondrial infiltration into membrane lamellipodia by fluorescence microscopy. Mean SEM. **= 0.0056; *** 0.0001. ( 0.0001. (= 0.0002. (= 631. See also Movie S2. Open in a separate window Fig. S4. Mitochondrial infiltration to the cortical cytoskeleton. (and were AMD-070 HCl analyzed for cell viability by AMD-070 HCl Trypan blue exclusion (and analyzed by confocal microscopy for changes in mitochondrial redistribution. Magnification, 63. were zoomed digitally at 20. (Scale bar, 5 m.) Full cell stacks were postprocessed for noise reduction using a median filter in LAS AF. (and Fig. S5and Movie S2), increasing both the assembly and decay of FA complexes (Fig. S5were manually tracked to determine FA dynamics. (= 10). *= 0.0353; *** 0.0001. (= 10). *= 0.0235; *** 0.0001. (= 10). *= 0.0393. Mitochondria are a primary source of reactive oxygen species (ROS), and these moieties have been implicated in tumor cell motility. PI3K antagonists increased the production of mitochondrial superoxide in tumor cells compared with untreated cultures (Fig. S6 and and Fig. S6 and (ANOVA) 0.0001. (and = 48. Mean SEM. **= 0.0044; *** 0.0009. (= 2). *** 0.0001. ns, not significant. (= 0.006. (= 44. Mean SEM. *** 0.0001. (were treated with PX-866 (5 M) and analyzed for mitochondrial morphology (polarized, perinuclear, infiltrating) by fluorescence microscopy; = 21. (= 4). *** 0.0001. Open in a separate window Fig. S6. Role of mitochondrial ROS in organelle trafficking and tumor cell invasion. (and 0.001; (ANOVA) = 0.0002. AU, arbitrary units of AMD-070 HCl fluorescence intensity. ((ANOVA) = 0.0015. ns, not significant. AU, arbitrary units of fluorescence intensity. (and (ANOVA) 0.0001. U, units of fluorescence intensity in the cortical area, normalized to total cell intensity. ((ANOVA) 0.0001. Role of Bioenergetics in Mitochondrial Trafficking and Tumor Cell Invasion. Next, we asked whether mitochondrial bioenergetics was important for this pathway, and generated LN229 cells.
Next, slides were washed, incubated with secondary antibodies (1:500; Alexa Fluor 488-conjugated) for 1 h at room temperature, washed, and mounted in DAPI-containing ProLong Gold mounting medium (Invitrogen)
