Skip to main content
. 2017 Jun 22;12(6):e0179510. doi: 10.1371/journal.pone.0179510

Fig 1. Inducible and reversible knockdown of mutant and wildtype KRAS in vitro.

Fig 1

A. The pINDUCER lentiviral vector was used for targeted knockdown KRAS in MCA cells in vitro. Addition (+) or withdrawal (-) of DOX induced or extinguished respectively, the expression of KRAS shRNA (Fig adapted from Meerbrey et al. 2011). B. Western blots showing reduction of KRAS protein expression after induction of shRNA targeting wildtype and mutant KRAS in LS174T cells (red arrow) and restoration of KRAS protein expression within 2 days after DOX withdrawal (green arrow). Note that the KRAS western blot shown in panel B was probed with an anti-KRAS antibody that binds to both wildtype and mutant KRAS. C-E. KRAS knockdown reduces MUC2 protein expression in MCA in vitro. Knockdown of wildtype and mutant KRAS in LS174T (B) and RW7213 (D) cells reduces MUC2 protein expression (C, E). Note that the KRAS western blot shown in panel D was probed with an anti-KRAS antibody that binds to both wildtype and mutant KRAS. F, G. Knockdown of mutant but not wild type KRAS reduces MUC2. Western blots of mutant KRAS (F), wildtype KRAS (G) and MUC2 protein (F, G, bottom panels) expression in LS174T cells. Mutant KRAS knockdown in LS174T cells (F, top panel, arrows) substantially reduces MUC2 protein levels (F, bottom panel, arrows). In contrast, knockdown of wildtype KRAS (G, top panel, arrow) did not reduce MUC2 protein expression (G, bottom panel, arrow). Note that the KRAS western blot presented in panel F was probed with an anti-KRASG12D specific antibody that binds only to mutant KRAS protein while the western blot in panel G was probed with an antibody that binds both wildtype and mutant KRAS proteins. Knockdown of wildtype KRAS did not alter mutant KRAS protein levels (see S3 Fig). H, I. HRAS and NRAS are dispensable for MUC2 expression. Quantitative RTPCR results show reduction of HRAS and NRAS in LS174T cells (H). Error bars indicate standard error of the mean. Knockdown of HRAS or NRAS did not reduce MUC2 protein expression (I, green arrows) in contrast to KRAS knockdown (I, red arrow). Expression levels of proteins on western blots were quantified by densitometry and percent protein (MUC2 or KRAS) expression in DOX-treated (+) samples relative to untreated (-) controls (normalized to actin) are shown below the blots. Abbreviations: d, days; 5d+/2d- indicates that cells were cultured for 5 days in the presence of DOX and for 2 days after the removal of DOX.