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. 2020 Oct 26;3:615. doi: 10.1038/s42003-020-01333-1

Fig. 1. Expression of ICOSL is required for OPN-mediated cell migration and is blocked by ICOS-Fc.

Fig. 1

a M14 and JR8 cell lines with high expression of ICOSL (ICOSLhigh), b PCF2 and A2058 cell lines with low ICOSL expression (ICOSLlow), and c A2058 ICOSLhigh-transfected cells were treated or not with ICOS-Fc (2 μg ml−1) and then plated onto the apical side of Matrigel-coated filters in 50 μl serum-free medium. The cells were allowed to migrate for 8 h to the lower chamber containing 10 μg ml−1 OPN or 20% FBS as chemotactic stimuli. The cells that migrated to the bottom of the filters were stained using crystal violet and all counted (quadruplicate filter) using an inverted microscope. Dotted line refers to untreated cells (NT). Data are expressed as the number of migrated cells per high-power field (***P < 0.001 refers baseline, ##P < 0.001 refers to OPN-induced migration (n = 5 technical replicate). d Relative quantification of ICOSL gene in silenced HUVEC using siRNA1 and siRNA2; cells treated with non-specific siRNA (SCR-siRNAscr) served as controls. Data are shown as gene expression relative to the expression of the endogenous control GAPDH (2−ΔCtmethod) and T test was used; **P < 0.01 (n = 3–4 technical replicate). e Assessment of migration of silenced HUVECs, via a Boyden chamber assay, in response to OPN and VEGF (***P < 0.001 refers to OPN-mediated migration in scr sample) (n = 7–8 technical replicate). f Schematic overview of the three different mutants of ICOSL generated. g ICOSL expression on transfected HeLa cells as assessed by flow cytometry. In the dot plots, gray, gray dotted, and black lines indicate the intracytoplasmic mutant (IC), the tailless mutant (TL), and the wild-type molecule (WT), respectively. The black filled histogram shows the negative control. h HeLa migration induced by OPN or FBS was assessed via a Boyden chamber assay. Cells migration was evaluated as described in a, b, and c (**P < 0.01 refers to baseline) (n = 5 technical replicate). All data are expressed as means ± standard error. For migration experiments one-way ANOVA with post-hoc Tukey multi-comparison test was used.