(A) SNP array colorgram showing genomic amplification of chromosome 22q11.21 in NSCLC cell lines. Regions of genomic amplification and deletion are denoted in red and blue, respectively.
(B) FISH of NSCLC cells using a CRKL-specific probe (green, G) and a reference probe (red, R). HSR denotes a homogenously staining region.
(C) Quantitative PCR analysis of CRKL copy number in NSCLC cells. CRKL copy number in NSCLC cells was normalized to LINE-1 and immortalized AALE cells using the standard curve method. Data represent mean + s.d. of triplicate measurements.
(D) Quantitative RT-PCR analysis of CRKL mRNA levels in NSCLC cell lines and immortalized AALE cells. Data represent mean + s.d. of triplicate measurements.
(E) Immunoblot of CRKL in NSCLC cell lines and immortalized AALE cells. β-actin was used as a loading control. Relative intensity of CRKL levels is determined.

(A) Effect of CRKL suppression on relative proliferation of NSCLC cell lines and AALE cells. Top, Immunoblot of CRKL proteins in each cell line expressing control shRNAs or two distinct CRKL-specific shRNAs. Bottom, Cell proliferation 6 d after infection with indicated shRNAs for each cell line. Data represent mean + s.d of six replicate measurements.
(B) Effect of expression of shRNA-resistant CRKL on the proliferation of H1755 cells expressing CRKL-specific shRNAs. Top, Generation of a shRNA-resistant CRKL cDNA (CRKL^{silent mutant}). The 21-mer sequence targeted by shCRKL#1 is marked in red color. Middle, Immunoblot of CRKL 4 d after infection with indicated shRNAs in H1755 cells expressing a control vector or V5-epitope tagged CRKL^{silent mutant}. Bottom, Effect of CRKL suppression by CRKL-targeting shRNAs on the proliferation of H1755 cells expressing a control vector or CRKL^{silent mutant}. The shCRKL#2 and shCRKL#3 target the 3′UTR of endogenous CRKL mRNA.
(C) Effect of CRKL suppression on cleavage of PARP and Caspase-3 proteins in NSCLC cells. Immunoblots of PARP and Caspase-3 proteins in H1755 and HCC1833 cells expressing the indicated shRNAs.
(D) Effects of mutations on CRKL function in H1755 cells. Left, Schematic of CRKL mutants. Right, Effect of expressing wild-type or mutant CRKL on the proliferation of the H1755 cells expressing a CRKL-specific shRNA (shCRKL#3). Data represent mean + s.d. of six replicate measurements. * indicates p<0.0001 as compared to cells expressing a control vector and shCRKL#3.

(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.

(A) Immunoblot of CRKL in AALE cell lines overexpressing a control vector or CRKL, as compared to NSCLC cells that harbored CRKL amplifications. Relative intensity of bands is determined.
(B) Anchorage independent growth of AALE cell lines stably overexpressing a control vector, wild-type or mutant CRKL. Colony number indicates colonies greater than 0.2 mm in diameter 4 weeks after plating. Data represent mean + s.d. of six replicate determinations from two independent experiments. Wild-type CRKL and CRKL mutants that induce anchorage independent growth are marked in red.
(C) Overexpression of CRKL in AALE cells increased the phosphorylation of SRC. Immunoblot of phospho-Y419 SRC proteins in AALE cells overexpressing a control vector, CRKL or CRKL^W160L mutant.
(D) Immunoblot of phospho-T185/Y187 ERK1/2 and phospho-S473 AKT in AALE cell lines overexpressing wildtype or mutant CRKL. Wild-type CRKL and CRKL mutants that induced anchorage independent growth in (B) are marked in red.
(E) Effect of CRKL overexpression on CRKL-C3G interaction and phosphorylation of C3G in AALE cells. CRKL immune complexes in AALE cells expressing indicated constructs were isolated and immunoblotting for phosphorylated C3G and total C3G proteins was performed using specific antibodies.
(F) Overexpression of CRKL in AALE cells increased RAP1 activity. Pull-down assay for GTP-bound RAP1 proteins were performed followed by immunoblotting for RAP1 proteins. Immunoblot of RAP1 proteins in total lysates before pull-down assay was used as loading control. Relative intensity of bands is determined.
(G) Effect of CRKL suppression on RAP1 activity in NSCLC cells that harbored amplification of CRKL. Pull-down assays were performed to assess the RAP1-GTP levels in NSCLC cells 4 d after infection with a control shRNA targeting GFP or a CRKL-specific shRNA (shCRKL#1) followed by immunoblotting for RAP1. Immunoblot of RAP1 proteins in total lysates was shown. Relative intensity of bands is shown.
(H) Effect of expressing RAP1 on cell proliferation of H1755 cells after CRKL suppression. Each H1755 cell line expressing a control vector, or wildtype RAP1A or RAP1B or constitutively active mutants, RAP1A^63E or RAP1B^V12, was infected with control shRNAs or a CRKL-specific shRNA (shCRKL#3). Cell proliferation was measured 6 d after infection with shRNA. Data represent mean + s.d. of six replicate measurements. * indicates p<0.0001 as compared to cells expressing a control vector and shCRKL#3.
(I) Effect of suppression of SRC, C3G or RAP1 on CRKL-induced anchorage independent growth. Left, Immunoblots of SRC, C3G or RAP1 proteins in CRKL-overexpressing AALE cells expressing a control shRNA targeting GFP or each gene-specific shRNA. The shRNAs that suppressed the target protein the best were marked in red color. The antibody for RAP1 detects both RAP1A and RAP1B. Right, Anchorage independent growth of AALE cells expressing indicated constructs. Colony number indicates colonies greater than 0.2 mm in diameter 4 weeks after plating. Data represent mean + s.d. of six replicate determinations from two independent experiments. * indicates p<0.001 as compared to cells expressing CRKL and shGFP.
(J) Effect of suppression of SRC, C3G or RAP1 on phospho-ERK1/2 levels in CRKL-overexpressing AALE cells. Immunoblots of phospho-ERK1/2 in CRKL-overexpressing AALE cells expressing indicated shRNAs. The shRNAs marked in red were validated in (I). (K) Effect of dasatinib treatment on anchorage independent growth of CRKL-overexpressing AALE cells. Anchorage independent growth of CRKL-overexpressing AALE cells that expressed wildtype SRC or mutant SRC or a control vector in the presence of dasatinib at indicated concentration. Colony number indicates colonies greater than 0.2 mm in diameter 4 weeks after plating. Mean + s.d. are shown. * indicates p<0.001 compared to cells expressing a control vector.
(L) Effect of dasatinib treatment on phospho-SRC and phospho-ERK1/2 in CRKL-overexpressing AALE cells. AALE cells expressing indicated constructs were exposed to dasatinib at indicated concentration for 6 h followed by immunoblotting.

(A) Overexpression of CRKL increased RAS activity. The levels of GTP-bound RAS in AALE cells overexpressing a control vector or CRKL were measured by a pull-down assay followed by immunoblotting for RAS. Total RAS levels in total lysates were used as loading control. Positive and negative technical controls were obtained by incubating the total lysates with non-hydrolyzable analog of GTP (GTPγS) or GDP, respectively, before pull-down assays.
(B) Overexpression of CRKL increased in vitro BRAF kinase activity. The BRAF proteins in AALE cells expressing indicated constructs were isolated by immunoprecipitation. The kinase activity was assessed by incubating with substrate proteins (MEK1). Immunoblots of phospho-MEK1 and BRAF proteins in the isolated BRAF immune complexes after kinase activity assay are shown.
(C) Immunoblot of phospho-S338-RAF1 in AALE cell lines overexpressing wildtype or mutant CRKL.
(D) Interaction between CRKL and SOS1 in AALE cells overexpressing CRKL. CRKL immune complexes were isolated followed by immunoblotting for SOS1 or CRKL proteins in AALE cells expressing indicated constructs.
(E) CRKL-induced anchorage independent growth required SOS1-RAS-BRAF/RAF1 signaling. Left, Immunoblots of SOS1, KRAS, BRAF, RAF1 or ARAF proteins in CRKL-overexpressing AALE cell lines expressing a control shRNA targeting GFP or each gene-specific shRNA. ShRNAs that suppressed more than 50% of target protein levels were marked in red color. Right, Anchorage independent growth of AALE cells expressing indicated constructs. Colony number indicates colonies greater than 0.2 mm in diameter 4 weeks after plating. Data represent mean + s.d. of six replicate determinations from two independent experiments. * indicates p<0.0001 as compared to cells expressing CRKL and shGFP.

(A) Overexpression of CRKL promotes anchorage independent growth of AALE cells. Anchorage independent growth of AALE cell lines stably expressing the indicated constructs is shown. Colony number indicates colonies greater than 0.2 mm in diameter 4 weeks after plating. Data represent mean + s.d. of six replicate determinations from two independent experiments.
(B) Overexpression of CRKL cooperated with NF1 suppression in inducing tumor formation of AALE cells. AALE cell line expressing indicated constructs was implanted subcutaneously into immunodeficient mice (2×10⁶ cells per injection site). The frequency of tumor formation and the number of tumor formation/number of injections within 5 months after implantation are depicted.
(C) Pull-down assay for RAS-GTP levels. AALE cell lines expressing indicated constructs were subjected to pull-down assay for RAS-GTP levels followed by immunoblotting for RAS. Relative intensity of RAS-GTP levels was determined. Immunoblot of RAS proteins in the total lysates was used as loading control. Immunoblot of NF1 proteins is also shown.

(A) Effect of overexpressing CRKL on gefitinib sensitivity in HCC827 cells that harbored EGFR mutation. Cell proliferation was measured 72 h after treatment with gefitinib. Data represent mean ± s.d. of triplicate measurements relative to untreated cells.
(B) Effect of overexpressing CRKL on phosphorylation levels of EGFR, ERK, AKT and SRC in response to gefitinib. HCC827 cells expressing a control vector or CRKL were exposed to gefitinib at indicated concentrations for 6 h and cell lysates were collected for immunoblotting with indicated specific antibodies.
(C) Effect of overexpressing CRKL on its interaction with SOS1 proteins. HCC827 cells expressing a control vector or CRKL were exposed to 1 μM gefitinib for 6 h. After isolating CRKL immune complexes from cell extracts, immunoblotting for SOS1 and CRKL proteins was performed.
(D) Effect of SOS1 suppression on CRKL-induced gefitinib resistance. Cell proliferation of each HCC827 cell line expressing indicated constructs 3 d after treatment with gefitinib. Data represent mean ± s.d. of six replicate determinations relative to untreated cells for each cell line..
(E) Effect of SOS1 suppression on phospho-ERK levels in CRKL-expressing HCC827 cells in response to gefitinib. Cells expressing indicated constructs were exposed to 1 μM gefitinib for 6 h followed by immunoblotting with indicated specific antibodies.
(F) Effect of overexpressing CRKL on the interaction of p85 with CRKL and ERBB3 proteins. HCC827 cells expressing a control vector or CRKL were either untreated or treated with 1 μM gefitinib for 6 h. After isolating p85 immune complexes, immunoblotting for CRKL, phospho-Y1289 ERBB3, total ERBB3 and p85 proteins was performed.
(G) Effect of GDC-0941 on CRKL-induced gefitinib resistance. Cell proliferation of CRKL-expressing HCC827 cells 3 d after treatment with gefitinib alone or in combination with GDC-0941 at indicated concentrations. Data represent mean ± s.d. of triplicate measurements.
(H) Effect of GDC-0941 on phospho-AKT levels in CRKL-expressing HCC827 cells in response to gefitinib. Cells were exposed to 1 μM gefitinib alone or in combination with GDC-0941 at indicated concentrations for 6 h followed by immunoblotting with specific antibodies.
(I) Acquired amplification of CRKL in an EGFR inhibitor-resistant lung tumor. FISH analysis of a NSCLC obtained from a patient prior to EGFR inhibitor therapy (left) or after acquiring drug resistance (right) using a CRKL-specific probe (green) and chromosome 22 telomere-specific probe (red). Nuclei were stained with DAPI (blue).