Fig. 5

StarD13 silencing increases lung adenocarcinoma cell invasion through an increase in Cdc42 activation. a A549 cells were transfected with luciferase siRNA or StarD13 siRNA (2 oligos) for 72 h. Cells were then allowed to invade towards 10% FBS for 24 h. 1 × 106 cells/ml were used in each assay. The micrographs are representatives of invaded cells on the bottom side of the membrane stained with cell stain according to assay instructions. Cell stain was then extracted and colorimetric measurements were taken at 560 μm. The graph shows the measurements. Data are the mean −/+ SEM from 3 experiments and *p < 0.05. b A549 cells were transfected with vector alone or with RhoA-CA. The micrographs are representatives of invaded cells on the bottom side of the membrane stained with cell stain according to assay instructions. Cell stain was then extracted and colorimetric measurements were taken. Data are the mean −/+ SEM from 3 experiments and *p < 0.05. c A549 cells were transfected with either luciferase or StarD13 siRNA. After 72 h, cells were lysed and incubated with GST-CRIB (Cdc42 and Rac interactive binding protein) to pull down active Cdc42. Samples were then blotted with Cdc42 antibody. The lower gel is a western blot for the total cell lysate, used as a loading control. The graph is a quantitation of the active Cdc42 bands using the ImageJ software. The bands were normalized to the amount of total proteins and the data presented as fold change to luciferase control. Data are the mean from 3 experiments −/+ SEM and *p < 0.05. d A549 cells were transfected with luciferase or StarD13 siRNA and with the Cdc42 FRET biosensor. The cells were then imaged in CFP, YFP and FRET channels and image analysis performed as described in materials and methods to obtain the FRET ratios (Cdc42 activation). The graph is a quantitation of the FRET signal (Ratio FRET/CFP signal) in the total cell area presented as fold difference to luciferase control. Data are the mean −/+ SEM from 15 cells (from 3 independent experiments) and *p < 0.05. Scale bar is 10 μm. e A549 cells were transfected with either vector alone control or with Cdc42-CA (constitutively active). Cells were then grown in a monolayer, wounded and left to recover the wound then imaged at the same frame after 48 h. The graph is a quantitation of the wounds obtained. Wound widths were measured at 11 different points for each condition and the average rate of wound closure was calculated in μm/hr. Data are the mean −/+ SEM from 3 wound closure assays and *p < 0.05. f A549 cells were transfected with luciferase control siRNA or Cdc42 siRNA for 72 h. The cells were then lysed and immunoblotted by western blot analysis for Cdc42 (upper gel) or for β-actin (lower gel) for loading control. The graph is a quantitation of the bands from the Cdc42 gel, using the imageJ software, normalized to the corresponding bands in the actin gel. Data are the mean −/+ SEM from 3 different experiments and *p < 0.05. g A549 cells were transfected with luciferase siRNA + vector alone, StarD13 siRNA Cdc42-DA, or StarD13 siRNA +Cdc42 siRNA for 72 h. Cells were then allowed to invade towards 10% FBS for 24 h. Cells are then stained, stains extracted and colorimetric measurements taken at 560 μm. The graph shows the measurements. Data are the mean −/+ SEM from 3 experiments and *p < 0.05