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. Author manuscript; available in PMC: 2019 Jul 1.
Published in final edited form as: Mol Cancer Ther. 2018 Oct 23;18(1):17–27. doi: 10.1158/1535-7163.MCT-18-0328

Figure 5: Effect of FRNK transduction on FAK expression/activity, proliferation, apoptosis, and Rac1 expression in SCLC cell lines transduced with FAK shRNA.

Figure 5:

NCI-H446 cell lines were first stably transduced with FAK shRNA or no-target (NT) shRNA and then stably transduced with doxycycline-inducible FRNK-expression plasmid or pCLX empty vector as control. After transduction, they were submitted to puromycin and blasticidin-selection for two weeks. Finally, they were treated with doxycycline for 48h before the experiments.A. FAK and PARP p85 expression/activity evaluation by WB. Whole cell lysates from SCLC cell lines were resolved with SDS-PAGE and blots were incubated with antibodies against total FAK (125 kD), FRNK (41 kD), phospho-FAK (Tyr397) (125 kD), PARP p85 (85 kD), and β-Actin (45 kD) for normalization. Decreased FAK and phospho-FAK (Tyr397) expression was observed by WB in cell lines double-transduced with FAK shRNA and FRNK as compared to those transduced with NT shRNA and PCLX. Increased FRNK expression was observed in cell lines double-transduced with FAK shRNA and FRNK when treated with doxycycline. Increased PARP p85 expression, a marker of apoptosis, was observed in cells expressing FRNK. WB densitometric quantification is available in Supplementary Fig.S1. B. Cell proliferation evaluation by WST-1 assay. SCLC cell lines were cultured for four days. Inhibition of proliferation was observed in cell lines double-transduced with FAK shRNA and FRNK and expressing FRNK after treatment with doxycycline. Optical density (OD) in Y-axis reflects the proportion of metabolically active cells. Error bars represent mean +/− standard deviation (SD) (n=5). All the graphs represent one of five independent experiments with similar results. *** P ≤ 0.001. C. Effects of FRNK on Rac1 activity. NCI-H446 SCLC cell lines were double-transduced with FAK shRNA and a doxycycline-inducible FRNK vector or with no-target (NT) shRNA and pCLX empty vector as control. 1. FAK and FRNK expression evaluation by WB. Whole cell lysates from SCLC cell lines were resolved with SDS-PAGE and blots were incubated with antibodies against total FAK (125 kD), FRNK (41 kD), and β-Actin (45 kD) for normalization. Decreased FAK expression was observed by WB in cell lines double-transduced with FAK shRNA and FRNK as compared to those transduced with NT shRNA and PCLX. Increased FRNK expression was observed in cell lines double-transduced with FAK shRNA and FRNK when treated with doxycycline. WB densitometric quantification is available in Supplementary Fig.S1. 2. Rac1 activation evaluation by Rac pull down assay for activated GTPases. Whole cell lysates from SCLC cell lines were enriched in activated GTPases using a GST-PAK affinity assay (GTPases pull down assay). Enriched eluates were resolved with SDS-PAGE and blots were incubated with antibodies against Rac1 (21 kD) and β-Actin (45 kD) for normalization. In control cells double-transduced with NT shRNA and PCLX, WB revealed low activated Rac1 expression at baseline, while treating them with GTP significantly increased its expression. In cells double-transduced with FAK shRNA and FRNK, activated Rac1 expression was present in the absence of doxycycline, while doxycycline-induced FRNK expression significantly decreased its expression. WB densitometric quantification is available in Supplementary Fig.S1.