Effect of delphinidin and VEGF on melanoma cell (Fig 2A) and HUVEC proliferation (Fig 2B).

<p>(A) B16-F10 melanoma cells were incubated for 24, 48 and 72 h in cell culture medium containing 1% human serum with different additions. NT: non treated cells. DMSO: in presence of the solvent DMSO. and in presence of 10 μg/ml delphinidin, 10 ng/ml VEGF or 10 μg/ml delphinidin + 10 ng/ml VEGF. Cell number is expressed in arbitrary units. In each group n = 5. (B) HUVECs were incubated for 24, 48 and 72 h in cell culture medium containing 1% human serum, in absence of any additions (Control) and in presence of 10 μg/ml delphinidin, 10 ng/ml VEGF or 10 μg/ml delphinidin + 10 ng/ml VEGF. Cell proliferation rate is expressed in % of control (n = 12). In comparison with control: ** (<i>P</i> < 0.01), **** (<i>P</i> < 0.0001). In comparison with the respective incubation times—[Delphinidin+VEGF] versus Delphinidin: ¤ (<i>P</i> < 0.05)—[Delphinidin+VEGF] versus VEGF: #### (<i>P</i> < 0.0001).</p

Effect of delphinidin on mouse tumor development.

<p>Mouse xenografts and treatments were conducted as described in Materials and Methods. (A) Experimental protocol and delphinidin effect on mouse weight. Mice were receiving delphinidin (●) or the vehicle of delphinidin as control (○). (B) Effect of delphinidin on tumor weight. On the 30^th day, mice were sacrificed and tumors were dissected. In comparison with control: * (<i>P</i> < 0.05).</p

Effect of delphinidin and VEGF on ERK1/2 (Fig 6A), p38 MAP kinase activation (Fig 6B), CREB (Fig 6C) and ATF1 (Fig 6D) transcription factor activation.

<p>HUVECs were treated for 30 minutes with or without 10 μg/ml delphinidin, in absence or presence of 10 ng/ml VEGF. Analysis of 30 μg protein was performed in each condition. (A) Densitometry represents the ratio of phosphorylated (a) over total (b) ERK1/2 and is expressed in % of control (n = 4). In comparison with control: * (<i>P</i> < 0.05), ** (<i>P</i> < 0.01), *** (<i>P</i> < 0.001). In comparison with VEGF: ## (<i>P</i> < 0.01). (B) Densitometry represents the ratio of phosphorylated (a) over total (b) p38 and is expressed in % of control (n = 4). In comparison with control: ** (<i>P</i> < 0.01). In comparison with VEGF: # (<i>P</i> < 0.05). (C) Densitometry represents the ratio of phosphorylated (a) over total (b) CREB and is expressed in % of control (n = 4). In comparison with control: * (<i>P</i><0.05). In comparison with VEGF: # (<i>P</i><0.05). (D) Densitometry represents the ratio of phosphorylated ATF1 (a) over total CREB (b) and is expressed in % of control (n = 4). In comparison with control: **** (<i>P</i> < 0.0001). In comparison with VEGF: ## (<i>P</i> < 0.01).</p

Effect of LY-294002 and delphinidin on cellular proliferation.

<p>HUVECs were incubated for 24, 48 and 72 h in cell culture medium containing 1% human serum with different additions. (A) Basal proliferation: no addition (Control), in presence of 10 μM LY-294002, 10 μg/ml delphinidin, and 10 μM LY-294002 + 10 μg/ml delphinidin. (B) VEGF-stimulated proliferation: no addition (Control), 10 ng/ml VEGF, 10 μM LY-294002 + 10 ng/ml VEGF, 10 μg/ml delphinidin + 10 ng/ml VEGF, 10 μM LY-294002 + 10 μg/ml delphinidin + 10 ng/ml VEGF. Cell proliferation rate is expressed in % of control (n = 3). In comparison with control for (A) and (B): * (<i>P</i><0.05); ** (<i>P</i><0.01); *** (<i>P</i><0.001); **** (<i>P</i><0.0001). In comparison with the respective incubation times–[LY-294002+VEGF] versus VEGF: § (<i>P</i> < 0.05)–[LY-294002+Delphinidin+VEGF] versus [LY-294002+VEGF]: † (<i>P</i> < 0.05).</p

Effect of delphinidin and VEGF on VEGFR2.

<p><b>VEGFR2 Phosphorylation</b>: (A) HUVECs and (B) B16-F10 were treated with delphinidin (10 μg/ml) for 3 h and then incubated without or with VEGF (10 ng/ml) for 30 min. HUVEC was cultured in M199/RPMI 1640 (50:50, v/v) containing 2 μmol/ml ultraglutamine I, 100 U/ ml penicillin, 100 μg/ ml streptomycin, 2.5 μg/ml fungizone and supplemented with 20% serum, i.e. 10% of human serum (HS) + 10% of heat-inactivated foetal calf serum (high serum culture medium) as described previously [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0145291#pone.0145291.ref016" target="_blank">16</a>]. B16-F10 was cultured in DMEM supplemented with 100 U/ ml penicillin, 100 μg/ ml streptomycin and 10% heat-inactivated foetal calf serum. After treatment cells were harvested, cell lysates were prepared and the VEGFR2 expression and phosphorylation were determined. (A) and (B) represent the ratio of phosphorylated over total VEGFR2. Data are means ± SEM of n = 5 experiments. *<i>P</i> < 0.05 versus DMSO, ** <i>P</i> < 0.01 versus VEGF, ** <i>P</i> < 0.01 versus VEGF. <b>VEGFR2 expression</b>. (C) HUVECs and (D) B16-F10 were treated with delphinidin (10 μg/ml) for 3 h and then incubated without or with VEGF (10 ng/ml) for 30 min for VEGFR2 phosphorylation or 24 h for VEGFR2 expression. (A) and (B) represent the ratio of phosphorylated over total VEGFR2. Data are means ± SEM of n = 5 experiments. * <i>P</i> < 0.05 versus DMSO, ** <i>P</i> < 0.01 versus VEGF, ** <i>P</i> < 0.01 versus VEGF. <b>Effect of delphinidin, VEGF and Ki8751 on HUVECs proliferation</b> (E). HUVECs were incubated for 24 and 48 h in cell culture medium containing 1% human serum with different experimental conditions: Control, 10 ng/ml VEGF, 10 nM Ki8751 + 10 nM VEGF, 10 μg/ml delphinidin + 10 nM VEGF, 10 nM Ki8751 + 10 μg/ml delphinidin + 10 nM VEGF. Cell proliferation rate is expressed in % of control (n = 5). In comparison with control: *<i>P</i> < 0.05, ** <i>P</i> < 0.01. in comparison with the respective incubation times -[Ki8751 + VEGF] versus VEGF: # <i>P</i> < 0.05, ## <i>P</i> < 0.01 –[delphinidin+ VEGF] versus VEGF: § <i>P</i> < 0.05, §§ <i>P</i> < 0.01 –[Ki8751+delphinidin+VEGF] versus [Ki8751+VEGF]: ££ <i>P</i> < 0.01.</p

Effect of U-0126 and delphinidin on cellular proliferation.

<p>HUVECs were incubated for 24, 48 and 72 h in cell culture medium containing 1% human serum with different additions. (A) Basal proliferation: no addition (Control), in presence of 10 μM U-0126, 10 μg/ml delphinidin, and 10 μM U-0126 + 10 μg/ml delphinidin. (B) VEGF-stimulated proliferation: no addition (Control), 10 ng/ml VEGF, 10 μM U-0126 + 10 ng/ml VEGF, 10 μg/ml delphinidin + 10 ng/ml VEGF, 10 μM U-0126 + 10 μg/ml delphinidin + 10 ng/ml VEGF. Cell proliferation rate is expressed in % of control (n = 9). In comparison with control for (A) and (B): * (<i>P</i> < 0.05); ** (<i>P</i> < 0.01); *** (<i>P</i> < 0.001); **** (<i>P</i> < 0.0001). In comparison with the respective incubation times—[U-0126+Delphinidin] versus Delphinidin: ¤ (<i>P</i> < 0.05); ¤¤¤ (<i>P</i> < 0.001)—[U-0126+VEGF] versus VEGF: §§ (<i>P</i> < 0.01); §§§ (<i>P</i> < 0.001)—[Delphinidin+VEGF] versus VEGF: ## (<i>P</i> < 0.01); ### (0.001); #### (<i>P</i> < 0.001)—[U-0126+Delphinidin+VEGF] versus [U-0126+VEGF]: †† (<i>P</i> < 0.01).</p

A-SAA mRNA expression in the skin and the serum of psoriatic and control patients.

<p>A-SAA mRNA expression in (A) the skin and (B) the serum of psoriatic patients is compared to healthy controls. Positive correlations of (C) serum A-SAA and CRP protein levels and (D) A-SAA mRNA expression in the skin and A-SAA protein concentrations in the serum of psoriatic patients. A-SAA mRNA levels from psoriatic skins are increased with (E) psoriasis severity, as evaluated by PASI, (F) disease duration, (G) cigarette smoking and (H) metabolic syndrome, respectively. A-SAA mRNA expression from 37 psoriatic skins was compared to 28 healthy skins and quantified by RT-qPCR. A-SAA protein concentrations in 17 psoriatic sera were determined by immunonephelemetry and compared to those of 11 healthy sera. Values are expressed as mean ± SEM. Statistical comparisons were performed using t test or Spearman rank correlation test (*p<0.05; **p<0.01; ***p<0.0001; ns, non-significant).</p

Skin and liver expression of A-SAA in a mouse model of psoriasiform dermatitis.

<p>C57BL/6 mice were treated daily during six days with imiquimod 5% cream (IMQ), with Vaseline (VAS) or were not treated (NT). (A) SAA1/2 and (B) SAA3 mRNA expression was determined by RT-qPCR. All data represent mean ± SEM relative expression to <i>GAPDH</i>. Statistical comparisons were performed using t test (*p<0.05; **p<0.01; ns, non-significant).</p

Expression of proinflammatory mediators by A-SAA-stimulated NHEK and in synergy with IL-17A.

<p>(A) NHEK were incubated with A-SAA (10 μg/ml) for 24 hours and the expression of TNF-α, S100A7, S100A8, hBD2, CCL20 and A-SAA was determined by RT-qPCR. (B) IL-17A (10 ng/ml) had a synergistic effect with rA-SAA. After A-SAA and IL-17A costimulation, mRNA expression of S100A7, hBD2 and A-SAA were further increased compared to A-SAA or IL-17A alone. Three independent experiments with duplicates were performed. Values are expressed as mean ± SEM fold change above unstimulated NHEK. Statistical comparisons were performed using t test (*p<0.05; **p<0.01; ***p<0.001).</p

Characteristics of psoriatic patients.

<p>Data are expressed as Mean ± SD or percentage (%). Abbreviations: PASI (Psoriasis Area and Severity Index), BMI (Body Mass Index).</p