Evaluation of sialidase NEU3 effect on cell viability with or without gefitinib.

<p>MTT test was performed on HSAEC1, HCC4006 and H1734 cell lines transfected with either the empty vector (mock) or pcDNA3.1-HsNEU3 and then treated or not with 27 nM or 1 μM gefitinib for 36 h <i>post</i>-transfection. Data were normalized on control cells transfected with the empty vector (A). Cell viabilities of mock and NEU3 transfected cells are reported after treatment with either 27 nM (B) or 1 μM gefitinib (C). Data were normalized on control cells without drug. Values are presented as means ± standard error (SE).</p

Sensitivity to gefitinib.

<p>(A) Dose-response curves of human lung cell lines to gefitinib. Cell survival was determined by MTT assay in the absence or the presence of different gefitinib doses (0, 0.01, 0.1, 1 and 10 μM) for 72 h. Viabilities are expressed as a percentage of the untreated control ± standard error (SE). (B) Nonlinear Regression of experimental data for HSAEC1 and HCC4006 cells lines was obtained using a Four Parameter Logistic Curve f1 = min + (max-min)/(1 + (x/EC₅₀)^(-Hillslope)). (C) Cell death of HSAEC1, HCC4006 and H1734 cells lines was determined by flow cytometry through PI staining. The cells were treated with different gefitinib concentration ranging from 0.01 to 10 μM for 72 h and stained by PI dye. The zero concentration was defined as a control and cell death was expressed as a percentage of the control ± standard error (SE).</p

EGFR phosphorylation levels after EGF stimulation, sialidase NEU3 overexpression and gefitinib treatment.

<p>(A) Representative Western-blot analyses performed on a normal lung cell line (HSAEC1) and NSCLC cell lines (HCC4006 and H1734) transfected either with the empty vector (mock) or pcDNA3.1-HsNEU3. Cells were treated for 3 h with 1 μM gefitinib, followed by the addition of EGF (20 ng/mL) for 15 min. Protein extracts were separated on a 10% SDS-PAGE and probed with anti-EGFR, anti-P-EGFR antibodies. GAPDH was used as a loading control. The experiments were performed in triplicate. (B)–(C)–(D) Densitometric analysis was performed with Scion Image Software. Values are expressed by comparing the data obtained after EGF stimulation with those obtained in the absence of EGF (B); by comparing the data obtained after transfection with NEU3 with those obtained after transfection with the empty vector (mock) (C); by comparing the data obtained after gefitinib treatment with those obtained without gefitinib administration (D). Statistical analyses were performed using Student’s t-test. Values are presented as means ± standard error (SE). *p<0.05, **p<0.01, and ***p<0.001 (Student’s t-test).</p

ERK phosphorylation levels after EGF stimulation, sialidase NEU3 overexpression and gefitinib treatment.

<p>(A) Representative Western-blot analyses performed on normal lung cell line (HSAEC1) and NSCLC cell lines (HCC4006 and H1734) transfected with either the empty vector (mock) or pcDNA3.1-HsNEU3. Cells were treated for 3 h with 1 μM gefitinib, followed by the addition of EGF (20 ng/mL) for 15 min. Protein extracts were separated on a 10% SDS-PAGE and probed with anti-ERK1/2, anti-P-ERK1/2 antibodies. Vinculin was used as a loading control. The experiments were performed in triplicate. (B)–(C)–(D) Densitometric analysis was performed with Scion Image Software. Values are expressed by comparing the data obtained after EGF stimulation with those obtained without EGF (B); by comparing the data obtained after transfection with NEU3 with those obtained after transfection with the empty vector (mock) (C); by comparing the data obtained after gefitinib treatment with those obtained without gefitinib administration (D). Statistical analyses were performed using Student’s t-test. Values are presented as means ± standard error (SE). *p<0.05, **p<0.01, and ***p<0.001 (Student’s t-test).</p

IC₅₀ values for gefitinib.

<p>IC₅₀ values for gefitinib.</p

Akt phosphorylation levels after EGF stimulation, sialidase NEU3 overexpression and gefitinib treatment.

<p>(A) Representative Western-blot analyses performed on normal lung cell line (HSAEC1) and NSCLC cell lines (HCC4006 and H1734) transfected with either the empty vector (mock) or pcDNA3.1-HsNEU3. Cells were treated for 3 h with 1 μM gefitinib, followed by the addition of EGF (20 ng/mL) for 15 min. Protein extracts were separated on a 10% SDS-PAGE and probed with anti-Akt, anti-P-Akt antibodies. Vinculin was used as a loading control. The experiments were performed in triplicate. (B)–(C)–(D) Densitometric analysis was performed with Scion Image Software. Values are expressed by comparing the data obtained after EGF stimulation with those obtained without EGF (B); by comparing the data obtained after transfection with NEU3 with those obtained after transfection with the empty vector (mock) (C); by comparing the data obtained after gefitinib treatment with those obtained without gefitinib administration (D). Statistical analyses were performed using Student’s t-test. Values are presented as means ± standard error (SE). *p<0.05, **p<0.01, and ***p<0.001 (Student’s t-test).</p

Relative quantification of <i>NEU3</i> and <i>EGFR</i> mRNA levels by quantitative real-time PCR in lung cancer cell lines.

<p>The relative expression levels of sialidase <i>NEU3</i> (A) and <i>EGFR</i> (B) in lung cancer cell lines were calculated with the Livak method (2^[-ΔΔC(T)]) and were expressed as a fold change, using <i>Pol2</i> gene as internal reference control and the HSAEC1 normal lung cell line as calibrator. Values are presented as means ± standard error (SE).</p

NEU3 transfection evaluation and NEU3 membrane localization.

<p>(A) Relative quantification of NEU3 mRNA levels by qPCR on HSAEC1, HCC4006 and H1734 cell lines transfected with either the empty vector (mock) or pcDNA3.1-HsNEU3. (B) Representative Western-blot analyses performed on membrane fractions. Proteins were separated on a 10% SDS-PAGE and probed with anti-NEU3 antibody. Transferrin receptor was used as a membrane marker. The experiments were performed in quadruplicate. (C) Densitometric analysis was performed with Scion Image Software. Values are expressed by comparing the data obtained after transfection with pcDNA3.1-HsNEU3 with those obtained after transfection with the empty vector (mock). Values are presented as means ± standard error (SE). ***p<0.001 (Student’s t-test).</p

Confocal images of Neuro2A cells transfected with the wild-type or the mutant construct.

<p>To investigate intracellular distribution of CRH, cell were fixed in PFA and probed with mouse polyclonal anti-GM130 (red) for Golgi visualization and rabbit polyclonal anti-CRH (green) antibodies.</p

Ability to express CRH in Neuro2A cells transiently transfected with wild-type or mutant preproCRH construct.

<p>A) CRH levels of expression detected by realtime quantitative PCR in not transfected (NT) or transfected cells (wt or p.Pro30Arg) at three different times: 24 h, 48 h and 72 h. Each bar represents the mean ± S.E.M. (<i>n</i> = 3) of mRNA levels normalized to the basal CRH expression in Neuro2A cells (NT values) and to a housekeeping control gene (b-Actin). * t = −3.676 and p = 0.020 compared with wt at 24 h; ** t = 5.274 and p = 0.002 compared with wt at 24 h. B) Densitometric analysis of CRH immunoreactive proteins in subcellular fractions of the Neuro2A cells. Each bar represents the mean ± S.E.M. (<i>n</i> = 3) and protein content is expressed in arbitrary units. C) Levels of secreted CRH protein measured by ELISA. The ability of cells to secrete the CRH hormone was evaluated by measuring the protein level in cultured media of cells transfected either with the wild-type or the mutant construct at 24 h or 48 h after the transfection. Each bar represents the mean ± S.E.M. (<i>n</i> = 2) and protein content is expressed as % in respect to the mean value of wt 24 h (assumed equal to 100%).* t = −7.403 and p = 0.005 compared with wt at 24 h; ** t = 7.796 and p = 0.004 compared with wt at 24 h.</p