Journal: Scientific Reports

Article Title: Ductal activation of oncogenic KRAS alone induces sarcomatoid phenotype

doi: 10.1038/srep13347

Figure Lengend Snippet: ( A ) The diagram of the Ela-CreERT and LGL-KRas G12V transgenes . Expression of the TAM-inducible Cre recombinase (CreERT) is driven by elastase I promoter/enhancer. KRAS G12V expression is blocked by a loxP-GFP-Stop-loxP cassette (LGL). A mouse BAC of 222-kb carrying the intact elastase I gene was used to provide the endogenous native context of elastase I gene . CreERT removes STOP cassette (LGL) through recombination, allowing the KRAS G12V expression. ( B ) Elastase I -driven recombination in mTmG and Rosa26R reporter mice ascertains ductal original. Green fluorescence was exclusively detected in the ductal cells of mTmG;Ela-CreERT reporter mice after vehicle ( panel a ) or TAM-feeding ( panel b ). Scale bar: 50 μm ( panel a ). β-galactosidase activity was observed in the SMG ductal cells of Rosa26R;Ela-CreERT mice after TAM-feeding ( panel d ) compared to control mice ( panel c ). Scale bar: 200 μm. ( C ) Characterization of SMGs of LGL-KRas G12V ;Ela-CreERT mice on day-24 post TAM-feeding. Gross anatomy revealed large ventrolateral cervical masses in SMGs of LGL-KRas G12V ;Ela-CreERT mice on day-24 ( panel b ). H&E and immunohistochemical (IHC) staining showed the microscopic abnormalities in the SMGs of TAM-fed LGL-KRas G12V ;Ela-CreERT mice on day-24 ( panels d, f, h) . Scale bars: 800 μm (in panels c and d ); 100 μm ( panels e–h ). ( D ) Wet weight of SMG from LGL-KRas G12V ;Ela-CreERT mice at 15-days post TAM-feeding (n = 13) or not (n = 7). *** p < 0.001. ( E ) Overall survival of mice after KRAS G12V induction is drastically reduced. Percent survival of Ela-CreERT versus LGL-KRas G12V ;Ela-CreERT mice after TAM administration. Median survival of LGL-KRasG12V;Ela-CreERT mice was 28-days. n = 13 per group; p < 0.0001 by Log-rank (Mantel-Cox) test. ( F ) KRAS G12V is activated in the SMG tumors of LGL-KRas G12V ;Ela-CreERT mice following TAM-gavage. Transgenic mice of indicated genotypes were gavaged with TAM. SMG samples were harvested on day-24 and whole tissue lysates (1 mg) from each sample were pulled down with Raf-1 RBD agarose beads. Pulldown reactions were resolved by SDS-PAGE (12%) and Western blotting was performed with an anti-KRAS antibody to detect active (GTP-bound) KRAS G12V .

Article Snippet: Equal amount of whole tissue lysates (1 mg) were pre-cleaned with glutathione S-transferase beads, then mixed with 20 μl of Raf1 RBD agarose beads, rotated at 4 °C for 1-hour, washed three times with lysis/wash buffer, boiled for 5-minutes in Laemmli buffer under reducing conditions, and resolved by 12% SDS-PAGE, followed by Western blotting with anti-KRAS antibody (ABD Serotec, Cat. #MCA3223Z).

Techniques: Expressing, Fluorescence, Activity Assay, Control, Immunohistochemical staining, Immunohistochemistry, Transgenic Assay, SDS Page, Western Blot