Fig. 6

Effects of PD-L1 knockout from human astrocyte cells (U87MG) on MCP-1 (CCL2) production. A Schematic representation of human PD-L1 gene structure consisting of 7 exons (EX1–EX7, NM_014143.3) and the 20 bp guide RNA (gRNA) that was aligned to its target site in exon 3. B and C Representative histograms of PD-L1 expression from WT U87MG cells versus PD-L1 exon-3 KO U87MG cells. WT U87MG and PD-L1 exon-3 KO U87MG cells were treated with medium, IL-1β, TNF-α, IFN-γ, or their combinations for 24 h as indicated. Each of these cytokines was used at 10 ng/mL. Cells were subjected to surface staining of PD-L1. D Representative plots of flow cytometry showing the effects of PD-L1 KO on the production of MCP-1. WT U87MG and PD-L1 exon-3 KO U87MG cells were treated with medium or a mixture of cytokines (IFN-γ, IL-1β, and TNF-α at 10 ng/mL each) for 24 h. Cells were subjected to intracellular staining of MCP-1 to determine MCP-1 production. E Pooled data showing the effects of the effects of PD-L1 KO on the production of MCP-1 in response to stimulation with the mixture of cytokines. All experiments were repeated at least four times. ns, not significant; **p < 0.01