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Fig. 6 | Cell Communication and Signaling

Fig. 6

From: Differential regulation of rho GTPases during lung adenocarcinoma migration and invasion reveals a novel role of the tumor suppressor StarD13 in invadopodia regulation

Fig. 6

StarD13 silencing increases Cdc42-mediated invadopodia formation in lung adenocarcinoma cells. a Representative micrographs of A549 cells cells transfected with luciferase siRNA + vector alone, StarD13 siRNA, Cdc42-CA (constitutively active Cdc42), or StarD13 siRNA +Cdc42 siRNA for 72 h, fixed and immunostained for TKS4. Scale bar is 10 μm. The graph is a quantitation of the number of invadopodia per cell (described in materials and methods) expressed as values in every condition (15 cells/condition/experiment). Data are the mean of +/− SEM of 3 independent experiments and *p < 0.05. b Representative micrographs of WI38 cells transfected with luciferase or starD13 siRNA, fixed and immunostained for TKS4 and stained with Rhodamin-Phalloidin. Scale bar is 10 μm. The graph is a quantitation of the number of invadopodia per cell expressed as values in every condition. Data are the mean of +/− SEM of 3 independent experiments and *p < 0.05. c Representative micrographs of control A549 cells fixed and immunostained for TKS4 and StarD13. Scale bar is 10 μm. d Representative histogram of fluorescence intensity across the indicated lines within the cell (across invadopodia) stained for TKS4 and StarD13. The intensity is plotted as a function of distance (in pixels). e Representative micrographs of control A549 cells plated on Alexa568-labeled matrix for 8 h. Cells were then fixed and stained for TKS4 and Cdc42 as well as the Rhodamin channel for matrix degradation. Scale bar is 10 μm. f Representative micrographs of control A549 cells untreated and transfected with the Cdc42 FRET biosensor for 24 h then plated on Alexa568-labeled matrix for 8 h. Cells were then fixed and imaged in CFP, YFP and FRET channels to obtain the Cdc42 FRET signal as well as the Rhodamin channel for matrix degradation. Scale bar is 10 μm. g Representative micrographs of cells transfected with luciferase or StarD13 or StarD13+ Cdc42 siRNA along with a GFP vector, or with GFP-Cdc42-Q61L, or with StarD13 siRNA + GFP-Cdc42-Q61L. siRNA + GFP vector alone, StarD13 siRNA Cdc42-DA, or StarD13 siRNA +Cdc42 siRNA and plated on Alexa568-labeled matrix for 48 h. Cells were then fixed and immunostained for TKS4. Scale bar is 10 μm. The graph is a quantitation matrix degradation index expressed as ratio of mean fluorescent intensity in an ROI in the background to mean fluorescent intensity in the cell trace (as described in materials and methods). Data are the mean −/+ SEM from 3 experiments and *p < 0.05

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