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. 2008 Nov 19;100(22):1592–1605. doi: 10.1093/jnci/djn379

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© The Author 2008. Published by Oxford University Press.

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Effect of overexpressed in lung cancer 1 (OLC1) overexpression on NIH3T3 and MBE cells. A) Immunoblot of untransfected NIH3T3 cells (Parental), NIH3T3 cells transfected with empty vector (Vector), and NIH3T3 clones (OLC1-C1 through C3) transfected with OLC1 DNA, using a rabbit polyclonal anti-OLC1 antibody. Blots were probed with monoclonal mouse anti–β-actin antibody to control for protein loading and transfer. One representative of three independent experiments is shown. B) The tetrazolium salt 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide proliferation assay of NIH3T3 cell clones in (A). H-Ras^Val12–transformed NIH3T3 cells were used as a positive control. Means and 95% confidence intervals (CIs, error bars) are shown. Error bars represent 95% CIs for three independent experiments performed in triplicate. *OLC1-C1, C2, C3, or H-Ras^Val12–transformed NIH3T3 cells vs Vector, P < .001. C) Numbers of colonies in the soft agar assay from the three OLC1 NIH3T3 cell clones (C1–C3), the NIH3T3 parental line, and vector control cells. Colonies larger than 200 μm in diameter were counted. Means and 95% CIs (error bars) are shown from three independent experiments performed in triplicate. D, E) Tumors observed in nude mice inoculated NIH3T3 parental, vector, or OLC1-C1 (white arrows) cells. Tumor size was measured every 3 days; the error bars in (E) represent 95% CIs of the mean volume of eight tumors induced by OLC1-C1. F) Immunoblot analysis of OLC1 protein expression in MBE cell clones stably overexpressing OLC1 using the antibody in (A). Clone 4 (C4, also MBE-OLC1) was used in subsequent experiments because it had the highest level of OLC1 expression. G) In vitro growth of MBE-OLC1 and MBE-vector cells. Error bars represent 95% CIs from a representative experiment (counted from triplicate wells) of three independent ones. *MBE-OLC1 vs MBE-vector cells, P = .005; **P < .001. H) Colony formation assay of MBE-OLC1 and MBE-vector cells. Means and 95% CIs (error bars) are shown from three independent experiments performed in triplicate. *MBE-OLC1 vs MBE-vector cells, P < .001. I) Cell cycle analysis of MBE-OLC1 and MBE-vector cells. Cells (1 × 10⁶) were stained with propidium iodide, and their DNA content was determined using flow cytometry. One representative experiment of three independent experiments is shown.

Effect of overexpressed in lung cancer 1 (OLC1) overexpression on NIH3T3 and MBE cells. A) Immunoblot of untransfected NIH3T3 cells (Parental), NIH3T3 cells transfected with empty vector (Vector), and NIH3T3 clones (OLC1-C1 through C3) transfected with OLC1 DNA, using a rabbit polyclonal anti-OLC1 antibody. Blots were probed with monoclonal mouse anti–β-actin antibody to control for protein loading and transfer. One representative of three independent experiments is shown. B) The tetrazolium salt 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide proliferation assay of NIH3T3 cell clones in (A). H-Ras^Val12–transformed NIH3T3 cells were used as a positive control. Means and 95% confidence intervals (CIs, error bars) are shown. Error bars represent 95% CIs for three independent experiments performed in triplicate. *OLC1-C1, C2, C3, or H-Ras^Val12–transformed NIH3T3 cells vs Vector, P < .001. C) Numbers of colonies in the soft agar assay from the three OLC1 NIH3T3 cell clones (C1–C3), the NIH3T3 parental line, and vector control cells. Colonies larger than 200 μm in diameter were counted. Means and 95% CIs (error bars) are shown from three independent experiments performed in triplicate. D, E) Tumors observed in nude mice inoculated NIH3T3 parental, vector, or OLC1-C1 (white arrows) cells. Tumor size was measured every 3 days; the error bars in (E) represent 95% CIs of the mean volume of eight tumors induced by OLC1-C1. F) Immunoblot analysis of OLC1 protein expression in MBE cell clones stably overexpressing OLC1 using the antibody in (A). Clone 4 (C4, also MBE-OLC1) was used in subsequent experiments because it had the highest level of OLC1 expression. G) In vitro growth of MBE-OLC1 and MBE-vector cells. Error bars represent 95% CIs from a representative experiment (counted from triplicate wells) of three independent ones. *MBE-OLC1 vs MBE-vector cells, P = .005; **P < .001. H) Colony formation assay of MBE-OLC1 and MBE-vector cells. Means and 95% CIs (error bars) are shown from three independent experiments performed in triplicate. *MBE-OLC1 vs MBE-vector cells, P < .001. I) Cell cycle analysis of MBE-OLC1 and MBE-vector cells. Cells (1 × 10⁶) were stained with propidium iodide, and their DNA content was determined using flow cytometry. One representative experiment of three independent experiments is shown.