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. 2014 Oct 18;5(22):11588–11603. doi: 10.18632/oncotarget.2597

Figure-4. AMACR overexpression confers tumor aggressiveness by promoting in vitro growth of GIST cell lines.

Figure-4

(A) Compared to primary HCSMC cells, endogenous AMACR mRNA (upper) and protein (lower) expression is higher in GIST882 and GIST48 cell lines (left panel). The two cell lines are stably silenced against endogenous AMACR expression by a lentiviral vector bearing one of the two AMACR shRNAs with different sequences for both GIST882 (middle panel) and GIST48 (right panel) cells. The efficiency of RNA silencing is confirmed by both quantitative RT-PCR (upper row) and western blotting (lower row) assays. The shLacZ plasmid, POLR2A transcript, and GADPH protein are utilized as controls in RNA interference, quantitative RT-PCR, and western blotting assays, respectively. (B) Using an ELISA-based, colorimetric assay to assess the rate of BrdUrd uptake, cell proliferation is significantly reduced in stable AMACR-knockdown GIST882 (left) and GIST48 (right) cell lines, compared to the corresponding shLacZ controls. (C) Western blotting assay validates that Cyclin D1, CDK4, and cyclin E are consistently downregulated in protein abundance in both AMACR-knockdown GIST882 and GIST48 cell lines. (D) Representative flow cytometric experiments show the induction of G1 cell cycle arrests by shAMACR in GIST cells. Two stable clones each of AMACR-knockdown GIST882 (upper) and GIST48 (lower) cells display a cell cycle arrest primarily occurring in the G1 phase.