Figure 1.
Expression of GLP1R and cell proliferation assays.
RT-PCR analysis of the expression of the GLP1R in all the neuronal cell lines and in pancreas taken as the positive control. Amplicon length = 240 basepairs M 100 bp = Molecular weight marker 100 basepairs (A). Cell proliferation assay performed in SK-N-AS and SH-SY5Ycells after treatment with 0.3 µM exendin-4 (in 2% FBS) for 24 h. Cpm = counts per minute; C 2% = control cells in 2% FBS, EXE = exendin-4. +EXE vs. C 2%, p>0.05). C 10% = control cells in 10% FBS (standard medium, used as the positive control) (B).
Figure 2.
Effect of 24 h 0.3 µM exendin-4 treatment on cell adhesion as assessed by Bengal rose assay. Data are mean ± SE of three independent experiments. EXE = exendin-4. * = p<0.05 vs related control (FBS 2%); FBS 10% was the positive control.
Figure 3.
Cell adhesion assay on different ECM proteins and uPAR expression.
Representative experiment on the effect of 0.3 µM exendin-4 on the adhesion of SH-SY5Y and SK-N-AS cells on different ECM proteins. * = p<0.05 vs. related control (A); Bengal rose adhesion assay performed on SK-N-AS (B) and SH-SY5Y (C) cells plated on vitronectin and treated with 0.3 µM exendin-4 for 24 h. A representative experiment of three independent experiments is shown in each case. * = p<0.05 vs. control; expression of uPAR as assessed by real-time RT-PCR in SK-N-AS and SH-SY5Y cells treated with 0.3 µM exendin-4 for 24 h. Mean percentage ± SE of four independent experiments. EXE = exendin-4. * = p<0.05 vs. control cells (D).
Figure 4.
Effects of exendin-4 on SK-N-AS and SH-SY5Y in 2D or 3D matrigel cultures.
Representative 400X phase-contrast inverted microscope fields of: SH-SY5Y control (A) and exendin-4-treated (B) cells after 1 h plating on top of matrigel; SK-N-AS control (C) and exendin-4-treated (D) cells after 3 h plating on top of matrigel: the arrows show the adherent cells in contrast to detached, rounded and refractile cells. Representative 100X phase-contrast inverted microscope fields of SH-SY5Y control (E) and exendin-4-treated (F) cells, SK-N-AS control (G) and exendin-4-treated (H) cells, FNC control (I) and exendin-4-treated (L) cells after plating inside matrigel for 48 h; long thin neuritic protrusions are indicated by the arrows and are suggestive of a more differentiated phenotype. EXE = exendin-4.
Figure 5.
Expression of the neuronal markers MAP2, Tau and SYP after plating cells inside matrigel for 48 h, assessed by real-time RT-PCR. Results are expressed as mean fold increase ± SE vs. related control, taken as 1, of three independent experiments. * = p<0.05 vs. related control cells.
Figure 6.
Analysis of passive properties and ionic current densities in FNC and SK-N-AS cells.
Data were evaluated until 48 h in culture. Effect of Exendin-4 (EXE) on RMP (A), on membrane capacitance, Cm (B), and resting specific membrane conductance Gm/Cm (C). * p<0.05 EXE- treated cells vs. the related control; § and §§ p<0.05 and <0.01 SK-N-AS 48 h vs. 24 h; †<0.05 and †† <0.01 SK-N-AS+Exendin-4 48 h vs. 24 h. FNC at 48 h, SK-N-AS at 24 and 48 h data are from 26, 28 and 32 cells, respectively. Effect of EXE on ISAC/Cm (D), INa/Cm (E), ICa,T/Cm (F) and ICa,L/Cm (G). * and ** p<0.05 and P<0.01 EXE treated cells vs. the related control; §, §§ and §§§ p<0.05, <0.01 and <0.001 SK-N-AS 48 h vs. 24 h; †<0.05, †† <0.01 and ††† 0.001 SKNAS +EXE 48 h vs. 24 h. ISAC/Cm INa/Cm, ICa,T/Cm and ICa,L/Cm in FNC at 48 h, SK-N-AS at 24 and 48 h data in each experimental condition are from 14–18 cells.
Figure 7.
Analysis of voltage-dependent Na+ and Ca2+ channels in FNC and SK-N-AS cells.
Typical INa traces recorded in a SK-N-AS cell. The voltage threshold of INa was at −50 mV (A). Effect of Exendin-4 (EXE) on INa amplitude (B). In A, B numbers represent the voltages eliciting the maximal INa. C) Normalized I–V plots represent the data evaluated at the current peak in all the cells investigated; the Boltzmann fits (Eq. 1) are superimposed to the experimental data. D) Normalized data related to INa activation and inactivation and superimposed Boltzmann fit in control SK-N-AS and under exendin-4 treatment; the Boltzmann curves for activation are determined from panel C by the equation: and inactivation from eq. 2; Boltzmann parameters listed in Table 1. Data represent mean ± SE from 26–43 cells. Representative ICa,tot traces obtained in a control (E) and in exendin-4 treated FNC cell (F). The arrow in the −50 mV trace indicates the presence of a first component as a fast-activating current, ICa,T. High-voltage-activated and slowly inactivating current (ICa,L, HVA) as a second component starting from −40 mV. Ca2+ currents elicited by a voltage step at −20 mV without (Cont) and in the presence of nifedipine (Nif), Cd2+ and Ni2+(G). Representative ICa,T recorded at a holding potential of –50 mV without (H), and with exendin-4 (I). Normalized I–V plots determined at the current peaks in control and under exendin-4 treatment related to ICa,T (L) and ICa,L (M). Normalized activation and inactivation data for T- (N) and L-type Ca2+ current (O) in control and under exendin-4 treatment, with the related Boltzmann fit superimposed to the data. The related Boltzmann parameters are listed in Table 1. In each experimental condition, data are from 18 to 23 cells.
Table 1.
Boltzmann parameters of activation and inactivation curves for INa and T- and L-type Ca2+ currents.
Figure 8.
Effect of 0.3 µM exendin-4 (EXE) on FBS-induced migration of cells as assessed by Boyden chambers migration assay. Results are reported as mean percentage of migrated cells/field ± SE of four independent experiments, considering at least 10 random fields for each experimental point. * = p<0.05 vs. control (A); representative phase contrast inverted microscope pictures of migrated SH-SY5Y control (B) or exendin-4-treated (C) cells (50X magnification); SK-N-AS control (D) or exendin-4-treated (E) cells; FNC control (F) or exendin-4-treated (G) cells (400X magnification).
Figure 9.
Cell migration in response to different stimuli and CXCR4 expression.
Representative experiment showing the effect of exendin-4 on the migration of NB cells and FNC cells. NB cells were induced to migration also by hMSC-conditioned culture medium (CM) (A). Expression of CXCR4 detected by real-time RT PCR and reported as fold-increase vs. related control. EXE = Exendin-4 (B). * = p<0.05 vs. related control.
Table 2.
Mean percentage ± SE of migrated cells in response to SDF-1, IGF-1 or PDGF in the presence of exendin-4, compared to untreated cells, taken as 100%.
Figure 10.
Invasion ability evaluation on exendin-4 treated cells.
Representative experiment from three independent experiments on the invasive ability of SK-N-AS and SH-SY5Y cells after treatment with 0.3 µM exendin-4 (A); real-time RT-PCR analysis of the expression of MMP-9 and TIMP-1 in NB cells after treatment with 0.3 µM exendin-4 for 6 h. * = p<0.05 vs. related control (B). Size of the cell colonies grown in soft agar 7, 14 or 21 days after suspension, with or without (C = control) exendin-4. Data are reported as mean percentage vs. related control of three replicates. EXE = exendin-4; * = p<0.05 vs. related control; # = p<0.05 vs. C 7d; § = p<0.05 vs. C 14d (C).