Figure 3. Effects of suppressing CRKL on NSCLC tumorigenicity.

(A) Generation of NSCLC cell lines expressing a doxycycline (DOX)-inducible CRKL-specific shRNA. A scrambled control shRNA or a validated CRKL-specific shRNA (shCRKL#1) under the control of DOX-inducible promoter were introduced into HCC515 cells. Immunoblot of CRKL proteins in cells in the absence or presence of 200 ng/ml DOX for 96 h.

(B) Experimental scheme. Each HCC515 cell line expressing a DOX-inducible vectors encoding either shCRKL#1 or scrambled control shRNA was implanted subcutaneously into the opposite flanks of the same mouse (n=10). Tumors were allowed to grow for 11 d to attain a volume of ~65 mm³. Mice were then fed on normal chow (n=5) or DOX-containing chow (n=5) to induce shRNA expression. Tumor growth was measured by caliper and bioluminescent imaging.

(C) Effect of CRKL suppression on the growth of HCC515 xenografts. DOX-induced CRKL suppression induced growth arrest of tumors derived from cell lines as described in (A) and (B). Data represent mean ± s.d.

(D) Representative bioluminescent images of the growth of HCC515 xenografts. DOX-induced CRKL suppression induced growth arrest of tumors derived from cell lines as described in (A) and (B). Images shown are the day 11 images (before DOX) and day 32 images (with or without DOX).

(E) TUNEL staining of xenografts. Left, DOX-treated tumors expressing a control shRNA or shCRKL#1 were harvested on day 32 as described in (B) and stained for TUNEL (green) and DAPI (blue). Right, Quantification of percent of TUNEL-positive cells. Mean + s.d. are shown.