Fig. 3

StarD13 silencing inhibits lung adenocarcinoma motility through the constitutive activation of RhoA and inhibition of Rac1. a A549 cells were transfected with either luciferase as control or StarD13 siRNA. After 72 h, cells were lysed and incubated with GST-RBD (Rhotekin binding domain) to pull down active RhoA. Samples were then blotted with RhoA 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 RhoA 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. b Representative micrographs of A549 cells transfected with luciferase or StarD13 siRNA and with the RhoA FRET biosensor and fixed. 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 (RhoA 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. c A549 cells were transfected with luciferase control siRNA or with RhoA siRNA for 72 h. The cells were then lysed and immunoblotted for RhoA or β-actin (lower gel) for loading control. The graph is a quantitation of the bands from the RhoA 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. d A549 cells were transfected with either luciferase, StarD13, RhoA, or StarD13 + RhoA siRNA. After 72 h, cells were lysed and incubated with GST-CRIB (Cdc42 and Rac interactive binding protein) to pull down active Rac1. Samples were then blotted with Rac1 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 Rac1 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. e A549 cells were transfected with luciferase or Rac1 siRNA for 72 h. The cells were then lysed and immunoblotted for Rac1 or β-actin (lower gel) for loading control. The graph is a quantitation of the bands from the Rac1 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. f A549 cells were transfected with luciferase siRNA + pcDNA3.1 vector, RhoA-CA (constitutively active), Rac1 siRNA, StarD13 siRNA + Rac1-CA (constitutively active), RhoA siRNA, and StarD13 + RhoA siRNA. Cells were then grown in a monolayer, wounded and left to recover the wound then imaged at the same frame after 48 h (lower micrographs). The graph is a quantitation of the wounds in F. 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. Scale bar is 100 μm