Fig. 3: Knockdown p110δ attenuated TNFα and IL-6 secretions by disturbing their trafficking out of Golgi complex in TiPs-stimulated macrophages.

A TiPs-induced secretions of TNFα and IL-6 from p110δ-knockdown macrophages were detected by ELISA assay. B mRNA productions of TNFα and IL-6 were examined by qPCR assay in p110δ-knockdown RAW264.7 upon 8 h TiPs stimulation. C Protein levels of TNFα and IL-6 were analysed by WB assay after 8 h TiPs stimulation with Brefeldin A (BFA), an inhibitor to hinder the transit from Endoplasmic Reticulum (ER) to Golgi complex. D, E After treated with TiPs for 2 h, p110δ-knockdown RAW264.7 were imaged with confocal microscopy to detect TNFα distribution. D Macrophages were co-treated with TiPs and 10 μM TAPI-1 to detect the surface distribution without permeabilization. It reflected the secretion of TNFα. The white bar, 10 μm. E Permeabilization was performed to detect the intracellular distribution of TNFα, which reflecting the cellular residual TNFα. The white bar, 10 μm. F After treated with TiPs for 2 h, p110δ-knockdown RAW264.7 were imaged with confocal microscopy to detect cellular IL-6 distribution. The white bar, 10 μm. G Surface TNFα and intracellular TNFα were analysed respectively by FCM assay after 2 h TiPs stimulation with or without 10 μM IC87114. For surface TNFα detection, permeabilization was not applied and 10 μM TAPI-1 (TACE inhibitor) was employed to block the cleavage of TNFα. The surface TNFα detection could reflect the secretion of TNFα. For intracellular detection, permeabilization was conducted without TAPI-1 treatment. And the intracellular detection could reflect the cellular residual TNFα. H, I Activation of MAPK signaling pathway (H) and NF-κB signaling pathway (I) were examined after 1 h TiPs stimulation in p110δ-knocdown or p110δ-overexpressed macrophages. All data were concluded from at least three independent assays. Statistic data were displayed as mean ± SEM and were conducted unpaired t test analysis or one-way ANOVA analysis to determine significant difference. * p < 0.05, ** p < 0.01, *** p < 0.001 compared with the negative group.