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. 2014 Apr 24;124(6):2696–2708. doi: 10.1172/JCI72171

Figure 5. p110α and RHEB are downstream mediators of ZEB1.

Figure 5

(A) Invasion assays. Invasive 344SQ_Rheb shRNA cells (#3 and #5) and 344SQ_scr shRNA cells were photographed (images) and quantified (bar graph) after 24 hours of incubation. Mean ± SD from triplicate samples. Scale bars: 100 μm. (B) Scatter plots of primary tumor weight and numbers of visible lung metastases in syngeneic mice injected with 344SQ_Rheb shRNA or 344SQ_scr cells. Mean ± SD for each cohort. (C) RNA polymerase II ChIP assays of Pik3ca (top) and Rheb (bottom) promoters. (D and E) Luciferase assays of PIK3CA (D) and Rheb (E) promoter activity. 344SQ cells were cotransfected with GATA3 expression and reporter plasmids. Results were normalized using a dual firefly/Renilla luciferase system. Mean ± SD from triplicate samples. Results are expressed relative to the normalized luciferase activity in pGL3 basic vector–transfected cells. (F and G) GATA3 ChIP assays on the PIK3CA (F) and RHEB (G) promoters in 293T cells and H322 cells, respectively. Controls include whole-cell lysates (input), ChIP using IgG, and amplification of an upstream region in the PIK3CA promoter (PCR #1) and intron 7 of RHEB (PCR #2) containing no predicted GATA-binding sites. Sizes of the PCR products generated from regions containing predicted GATA-binding sites in PIK3CA promoter (PCR #2 and PCR #3) and RHEB promoter (PCR #1) are indicated.