Figure 1.
Increased expression of IQGAP3 in lung cancer tissues.
(A) EST expression analysis of the IQGAP3 gene in normal and cancerous tissues based on the ECgene database. (B) Quantitative real-time PCR for IQGAP3 expression in 25 pairs of lung cancer versus adjacent non-cancerous tissues. IQGAP3 expression was normalized against GAPDH. Relative levels were calculated for each sample, and a value of 1 was assigned for adjacent non-cancerous tissue of patient 13. Experiments were repeated in triplicate. Data from one representative experiment are presented as mean±SD. (C) Immunohistochemical analysis of IQGAP3 protein expression in lung cancer versus adjacent non-cancerous tissues. Representative images are shown for a squamous cell carcinoma and an adenocarcinoma. Ca, Cancer tissue; Adj, adjacent non-cancerous tissues. Magnification, ×200.
Figure 2.
Enhanced cell growth, migration and invasion with enforced expression of IQGAP3.
Myc-IQGAP3 or control vectors were transfected into Hela cells and then cells were harvested at 24 h after transfection for migration and invasion assay. Cells for proliferation assay was harvested at indicated time point. (A) IQGAP3 protein expression of transfectants was verified by Western blotting. Cell proliferation was assayed using the Cell Counting Kit-8. (B, C) Cell migration and invasion assays were performed using Chemotaxis chambers with or without coating with Matrigel. Each assay was repeated at least 3 times. Data from one representative experiment are presented as mean±SD. *, P<0.05; ***, P<0.001.
Figure 3.
Reduced cell growth, migration and invasion upon inhibition of IQGAP3 expression.
A549 cells were transfected with shRNA constructs to knockdown endogenous IQGAP3 expression. (A) IQGAP3 protein levels following infection with control (NC) or two different shIQGAP3 lentiviruses (KD1 and KD2). (B) Proliferation was assayed using the Cell Counting Kit-8 for lentivirus-infected A549 cells. (C, E) Migration of lentivirus-infected A549 cells in the chemotaxis chamber assay in the absence (C) or presence (E) of human EGF (100 ng/ml). (D, F) Invasion of lentivirus-infected A549 cells across Matrigel in the absence (D) or presence (F) of human EGF. Each assay was repeated at least 3 times. Data from one representative experiment are presented as mean±SD. *, P<0.05;**, P<0.01;***, P<0.001.
Figure 4.
Decreased tumorigenicity of A549 cells in vivo with reduced IQGAP3 expression.
NOD/SCID mice received an intravenous injection of 1.5×106 A549 cells stably transfected with shIQGAP3 (IQGAP3-KD2) or control vectors (NC), and were sacrificed at day 42 after tumor inoculation (n = 7 for each group). (A) Gross appearance of lung tissues. (B) Lung weight. The horizontal and error bars show the average weight and standard deviation. (C) H&E staining of lung tissues. Four representative fields (2 for each group) are shown. Magnification, ×40. **, P<0.01.
Figure 5.
Interaction of IQGAP3 with ERK1.
(A) Myc-IQGAP3 and HA-ERK1 or HA-ERK2 constructs were transfected into HEK293T cells. Cell lysate was prepared and immunoprecipitated with mouse anti-HA mAb, anti-Myc mAb or control antibodies (mIgG). The resultant precipitate, together with the unprocessed lysate (Input) was resolved on SDS-PAGE, transferred onto blots and probed with anti-Myc and anti-HA. (B) Interaction of IQGAP3 with ERK1 was also examined in A549 cells with endogenously expressed proteins. Cell lysate was prepared from A549 cells and immunoprecipitated with rabbit anti-ERK1 mAb. The presence of IQGAP3 in the precipitate was evaluated anti-IQGAP3 antibodies. All experiments were repeated at least three times and results from one representative experiment are shown.
Figure 6.
Modulation of EGFR-ERK signaling by IQGAP3.
(A) Hela cells transiently transfected with Myc-IQGAP3 or control vector (mock) were serum deprived (0.5% fetal bovine serum) for 24 h before being stimulated with 100 ng/ml EGF. Cells were harvested at different time points and examined for ERK phosphorylation. (B) A549 cells were infected with shIQGAP3 lentivirus (IQGAP3-KD1 or KD2) or control virus (NC) and serum deprived (0.5% fetal bovine serum) prior to stimulation with 100 ng/ml EGF. Levels of phosphorylated ERK, total ERK, phosphorylated p38 and phosphorylated AKT were evaluated with western blot at different time points after EGF stimulation. (C) Luciferase reporter assays were performed to measure Elk1 activity downstream of the cascade of EGFR-ERK signaling in A549 cells transfected with siRNA oligos. (D) Proliferation of Hela cells harboring IQGAP3-expressing or control vectors was assayed using the Cell Counting Kit-8 with addition of 20 µM U0126 or DMSO. Each assay was repeated at least 3 times. The data from one representative experiment are presented as mean±SD. *, P<0.05; **, P<0.01.