Effects of leptin on the migration of untreated and sPLA₂-IIA-treated cells.

<p>Cell mobility was assayed using the 96-well Boyden chamber system. Serum-starved 1321N1 cells were untreated or treated with the indicated concentration of leptin for 30 min, in the absence (A) or presence of kinase inhibitors (B). Cells were allowed to migrate for 2 h to DMEM, FCS or leptin. *p<0.001 vs untreated cell towards DMEM, **p<0.001 vs untreated cells towards FCS, ^†p<0.001 vs leptin-treated cell towards DMEM, ^††p<0.001 vs leptin-treated cell towards FCS, ^†††p≤0.001 vs untreated cell towards leptin, n = 3. Serum-starved 1321N1 cells were treated as indicated for 30 min (loaded into top wells), and then allowed to migrate for 2 h to DMEM (C), or treated with medium or sPLA₂-IIA (sPLA₂) for 30 min (loaded into top wells), and then allowed to migrate for 2 h to DMEM or leptin (loaded into the bottom wells) (D). ^‡ p≤0.001, n = 3.</p

Effect of leptin+sPLA₂-IIA treatment on the activation of intracellular kinases in 1321N1 cells.

<p>1321N1 cells were stimulated with 0.1 μM of leptin, 0.2 μg/ml of sPLA₂-IIA (sPLA₂) or both, for the indicated time intervals. Src phosphorylation was analyzed by flow cytometry analysis (A), and ERK, Akt, P70S6K and rS6 phosphorylation were determined by immunoblotting, using phospho-specific antibodies (B).</p

Effect of kinase inhibitors on the intracellular signaling mediators activated in leptin-treated cells.

<p>1321N1 cells were incubated without or with 0.5 μM of leptin or 0.4 μM of EGF for 15 min in the presence or absence of the indicated inhibitors. Lysates were analysed by western blotting with the specified antibodies.</p

1321N1 astrocytoma cells express the leptin receptor (ObR) and leptin stimulates EGFR-independent cell proliferation.

<p>(A) 1321N1 cells immunofluorescence for ObR expression without and with primary anti-ObR antibody (H-300). Representative histograms. Unspecific staining, solid grey curves. OB-R expression in cells treated as mentioned in the plot, open black curves (B) Cells stimulated for 18 h with the indicated agonist: Expression of ObR was analyzed by flow cytometry. Serum-starved 1321N1 cells were stimulated for 24 h with increasing concentrations of leptin (C), or with the indicated agonist in the presence or absence of specific inhibitors (D). Cell proliferation was measured as explained in Materials and Methods. *p≤0.001 vs untreated control, ⁺p≤0.001 vs EGF, n = 3</p

Effect of leptin and sPLA₂-IIA on phosphatases activity in 1321N1 cells.

<p>1321N1 cells were stimulated with 0.1 μM of leptin, 0.2 μg/ml of sPLA₂-IIA (sPLA₂) or both, for the indicated times. Ser/Thr phosphatase (A) and Tyr phosphatase (B) activity was measured in the cell lysate. *p≤0.001 vs untreated control, #p≤0.001 vs sPLA₂ alone, n = 3.</p

Effect of leptin+sPLA₂-IIA treatment on EGFR transactivation in 1321N1 cells.

<p>1321N1 cells were stimulated with 0.1 μM of leptin, 0.2 μg/ml of sPLA₂-IIA (sPLA₂) or both, in the presence or absence of 2.5 μM of AG1478. (A) At the indicated times, EGFR phosphorylation was analyzed by flow cytometry analysis. (B) After 24 h cell proliferation was measured as explained in Materials and Methods. *p≤0.001 vs control without inhibitor, ^#p≤0.001 vs sPLA₂ treated without inhibitor and ^‡p≤0.001 vs leptin+sPLA₂ treated without inhibitor. (C) Phosphorylation of signaling proteins was determined by immunoblotting at the indicated times.</p

Effect of leptin in sPLA₂-IIA- and EGF-induced cell proliferation.

<p>Serum-starved 1321N1 cells were stimulated for 24 h with the indicated concentrations of leptin, sPLA₂-IIA (sPLA₂) and EGF. Cell proliferation was measured as explained in Materials and Methods. *p≤0.001 vs untreated control, ^#p≤0.001 vs sPLA₂ alone, ^†p≤0.001 vs leptin alone, n = 3</p

Actions of leptin and sPLA₂-IIA on mouse primary astrocytes.

<p>Astrocytes were stimulated with leptin, sPLA₂-IIA (sPLA₂) or both, as indicated. (A, C) After 24 h cell proliferation was measured as explained in Materials and Methods. *p≤0.001 vs untreated control cells, #p≤0.001 vs 0.2 μg/ml of sPLA₂ alone, ^†p≤0.001 vs 0.1 μM of leptin alone, n = 3. (B, D) After 15 min, phosphorylation of signaling proteins was determined by immunoblotting on the cell extracts. (E) At the indicated times, the activity of Ser/Thr- and Tyr-phosphatase was measured in the cell lysate. *p≤0.001 vs untreated control, #p≤0.001 vs sPLA₂ alone, n = 3.</p

Representative immunocytochemistry images of cardiac myofibroblasts examined by fluorescence microscopy.

<p>Vimentin staining (A), α-smooth muscle actin (α-SMA) staining (B) and vimentin and α-SMA staining merged (C). Magnification 40X. Scale bar: 50 µm.</p

Effect of erythrodiol and uvaol on the fibrotic effect of angiotensin II in cardiac myofibroblasts.

<p>Time course of angiotensin II (Ang II; 1 µM)-stimulated collagen I protein production (A). Effect of erythrodiol (ERY; 5 µM) or uvaol (UVA; 5 µM) on CTGF (B), collagen I (C) and galectin 3 (D) protein expression in angiotensin II-treated cardiac myofibroblasts for 12 hours. Representative immunoblots of 4 experiments. Values are mean ± SEM of four assays. *p<0.05 <i>vs</i> vehicle. †p<0.05 vs angiotensin II. Quantification of band intensities was measured by densitometry and normalized to respective α-tubulin.</p