Table 1.
Primers used in Q-RT-Quantitative Real-time PCR and ChIP assay.
Figure 1.
Trim28 deficiency alters the expression of EMT markers.
A, control and Trim28 knockdown A549 cells were stained (as described under “Experimental Procedures”) and examined using confocal immunofluorescence microscopy, as follows: E-cadherin (green, top panel), β-tubulin (green, bottom panel), and DAPI (blue). Merged images are in the bottom of each panel. B, RNA extracts of control and Trim28 knockdown A549 and H358 cells were subjected to real-time PCR to determine the expression of target genes as indicated. Asterisks represent significant p values, *: p<0.05. C, the expression of EMT markers E-cadherin and N-cadherin were measured in control and Trim28 knockdown A549 and H358 cells by western blotting.
Figure 2.
Trim28 proficiency alters the expression of EMT markers.
A, A549 and H358 were transfected in triplicate with E-cadherin promoter luciferase reporter, pRL-TK reporter, and expression plasmids indicated in the figure. Cells were harvested 48 hrs after transfection for determination of luciferase levels. B, control (pcDNA3) and Flag-Trim28 stably expressed A549 and H358 cells were analyzed for E-cadherin and N-cadherin levels by western blotting. C, RNA extracts of control and Trim28 stably expressed A549 and H358 cells were subjected to real-time PCR. The expression of target genes was determined. Asterisks represent significant p values, *: p<0.05.
Figure 3.
Trim28 expression is induced by TGF-β.
A and B, A549 and H358 stably expressing non-silencing shRNA and Trim28 shRNA were treated with TGF-β (2 ng/ml) for 3 to 10 days. Whole cell lysates and RNA extracts were subjected to western blotting using the indicated antibodies (A) and real-time PCR (B). Asterisks represent significant p values, *: p<0.05.
Figure 4.
Trim28 is required for TGF-β-induced EMT.
A, control and Trim28 knockdown cells were treated with TGF-β or left untreated. Cells were photographed at 40× magnification. B, RNA was extracted from control and Trim28 knockdown A549 cells treated with TGF-β as above or left untreated. Real-time PCR was performed and the expression of target genes was determined. C, control and Trim28 knockdown A549 and H358 cells were treated with TGF-β (2 ng/ml) until morphology changes were observed or left untreated. Whole cell lysates were subjected to western blotting using antibodies as indicated.
Figure 5.
Trim28 knockdown attenuates cell migration.
A, control and Trim28 knockdown H358 cells were subjected to wound-healing assay. Cells were photographed right after scratch, and 24 hrs and 48 hrs after scratch. B, control and Trim28 knockdown A549 cells were subjected to wound healing assay in the absence or presence of serum. Cells were photographed right after scratch and 24 hrs later. C, control and Trim28 knockdown cells were plated in triplicate on the top wells of Matrigel-coated or non-coated chambers. After 18 hours, cells that had invaded through the Matrigel layer were fixed and stained. The percentage of invasive cells is shown in the bar graph. Asterisks represent significant p values, *: p<0.05. D, control and Trim28 knockdown A549 cells were cultured in a three-dimensional model and treated with TGF-β (5 ng/ml) for 72 hrs. RNA was extracted from the cells and subjected to real-time PCR. The expression of target genes was determined.
Figure 6.
Trim28 regulates histone modifications of E-cadherin and N-cadherin promoters.
A, ChIP assays were conducted in control and Trim28 knockdown A549 cells. Quantitative PCR was performed to examine the occupancy of IgG, acetylated histone 3 K9, dimethylated histone 3 K9, and trimethylated histone 3 K9 on E-cadherin and N-cadherin promoters. B, ChIP assays were conducted in control and Trim28 stably expressed A549 cells. Quantitative PCR was performed to examine the occupancy of IgG, acetylated histone 3 K9, dimethylated histone 3 K9, and trimethylated histone 3 K9 on E-cadherin and N-cadherin promoters.
Figure 7.
Hypothetical model to explain the role of Trim28 in the stages of lung adenocarcinoma.
This model explains this work and our previous work demonstrating a tumor suppressor role for Trim28 [9].