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. 2021 May 28;12:3213. doi: 10.1038/s41467-021-23002-5

Fig. 2. Sand fly salivary yellow proteins are the neutrophil chemoattractants in sand fly saliva.

Fig. 2

Neutrophils from C57BL6 bone marrow (a–d, j) or from human periheral blood (e, f, k) were added to a transwell plate top chamber and P. duboscqi salivary gland homogenate (SGH) or recombinant proteins were added to the bottom chamber. After 3 h, cells in the bottom chamber were quantified. P. duboscqi SGH was treated with Proteinase K (Ptk) (a), heated for 30 min at 70 °C (b) or treated with 90% of ethanol (EtOH) (b). Increasing amounts of rPduM10 or rPduM35 (c); rPduM10 (0.5 μM), rPduM35 (0.5 μM) or rPduM10 + rPduM35 (0.25 μM of each protein) (d), increasing amounts of rLJM17 or rLJM11 (e), or rLJM17 (0.5 μM), rLJM11 (0.5 μM) or rLJM17 + rLJM11 (0.25 μM of each protein) (f). g–i Validation of yellow proteins as neutrophil chemoattractants by EZ-TAXIScan assay. N-Formylmethionyl-leucyl-phenylalanine (fMLP) was used as a positive control, and Hanks’ Balanced Salt Solution (HBSS) as baseline control. g, h Chemotaxis parameters were analyzed by Imaris. g Chemotaxis mean speed was computed as total path length divided by time. h Each track is plotted from the central point showing XY displacement. i Still images of neutrophils at two different time points. Red squares show migrated neutrophils. Neutrophils were pretreated with pertussis toxin (PTx; 2 μg/mL), wortmannin (1 μM) or BAPTA-AM (10 μM) and their migration towards a combination of rPduM10 + rPduM35 (j) or rLJM11 + rLJM17 (k) (0.25 μM each) was assayed. Cumulative results of at least three (a–f, j, k) or two (g–i) independent experiments are presented as means ± SEM; a n = 10, 7, 9, 6, 5; **P = 0.0043, ****P < 0.0001. b n = 3, 3, 3, 2, 3; ***P = 0.0003, ****P < 0.0001. c n = 3; *P = 0.0123, *P = 0.0104, *P = 0.0208, **P = 0.0081. d n = 3; **P = 0.0042, ***P = 0.0007, ****P < 0.0001. e n = 8, 5, 6, 5, 7, 7, 7; *P = 0.0446, **P = 0.0387, ****P < 0.0001. f n = 4; ***P = 0.0003, ****P < 0.0001. g n = 3, 4, 2; *P = 0.0162. j n = 10, 10, 6, 8, 8; **P = 0.0032, **P = 0.0093, **P = 0.0021 ***P = 0.0003. k n = 10, 18, 12, 5, 5; **P = 0.0018 ***P = 0.0001, ****P < 0.0001. P calculated by one-way ANOVA test, except (g) two-tailed unpaired t-test.