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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 4: Effect of FAK shRNA and FAK-related non kinase (FRNK) transduction on FAK expression/ activity, cell proliferation, cell cycle, and apoptosis in SCLC cell lines.

Figure 4:

A. Two SCLC cell lines were stably transduced with FAK shRNA or no-target (NT) shRNA as control, and submitted to puromycin selection for two weeks. 1. FAK expression and activity evaluation by WB. Whole cell lysates from these two cell lines were resolved with SDS-PAGE and blots were incubated with antibodies against total FAK (125 kD), phospho-FAK (Tyr397) (125 kD), and β-Actin (45 kD) for normalization. Significant decrease of FAK expression and phosphorylation (Tyr397) was observed by WB in SCLC cell lines transduced with FAK shRNA as compared to those transduced with NT shRNA. WB densitometric quantification is available in Supplementary Fig.S1. 2. Cell proliferation evaluation by WST-1 assay. SCLC cell lines were cultured for three days. No significant difference in cell proliferation was observed by WST-1 between cells transduced with FAK shRNA and those transduced with NT shRNA. Optical density (OD) in Y-axis reflects the proportion of metabolically active cells. Error bars represent mean +/− SD (n=5). All the graphs represent one of five independent experiments with similar results. *** P ≤ 0.001. 3. Cell cycle evaluation by flow cytometry. SCLC cell lines were stained with anti-BrdU antibody and PI, and the staining was quantified by FACS. No significant difference in cell cycle was observed between cells transduced with FAK shRNA and those transduced with NT shRNA transfection. Error bars represent mean +/− SD from three independent experiments. ***P ≤ 0.001. B. NCI-H446 cell lines were stably transduced with doxycycline-inducible FRNK-expression plasmid or empty vector (pCLX) as control, and submitted to blasticidin-selection for two weeks. Cells were treated with doxycycline for 48h before the experiments. 1. 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. Significant increase of FRNK expression was confirmed by WB in SCLC cell lines transduced with FRNK and treated with doxycycline as compared to those not treated with doxycycline or transduced with pCLX empty vector. Significant increase of PARP p85 expression, a marker of apoptosis, was also observed by WB in cells expressing FRNK, while total FAK and phospho-FAK (Tyr397) expression remained unchanged. 2. Cell proliferation evaluation by WST-1 assay. SCLC cell lines were cultured for five days. Inhibition of proliferation was observed by WST-1 in cell lines expressing FRNK as compared to those not expressing it. Optical density (OD) in Y-axis reflects the proportion of metabolically active cells. Error bars represent mean +/− SD (n=5). All the graphs represent one of five independent experiments with similar results. *** P ≤ 0.001 3. Cell cycle evaluation by flow cytometry. SCLC cell lines were stained with anti-BrdU antibody and PI, and the staining was quantified by FACS. DNA synthesis was decreased in cell lines expressing FRNK as compared to those not expressing it. Error bars represent +/− SD from five independent experiments. *** P ≤ 0.001.