Fig. 7: SPRY1 activates inflammation via NF-κB-dependent mechanisms in ADAP-deficient macrophages.

A–C WT and ADAP knockdown RAW 264.7 cells were transfected with non-targeting control siRNA (si-NC) or siRNA targeting SPRY1 (si-SPRY1). Twenty-four hours post-transfection, cells were either left untreated or stimulated with LPS (1 μg/ml, 6 h). RT-qPCR results showing the mRNA expression of TNF-α (A), IL-1β (B), and IL-6 (C) are presented. n = 3. Data are expressed as mean ± SEM. P values were from a two-way ANOVA with multiple comparison. ***P < 0.001. ns nonsignificant. D WT and ADAP knockdown RAW 264.7 cells were transfected with either SPRY1-specific siRNA or non-targeting siRNA as a control. Forty-eight hours post transfection, cells were either left unstimulated or stimulated with 1 μg/ml LPS for 15 min. Whole-cell lysates were collected and subjected to Western blot analysis using anti-phospho-p65 and anti-p65 antibodies. E–G WT and ADAP knockdown RAW 264.7 cells were reconstituted via lentiviral transduction with either an empty vector or a SPRY1 overexpression (SPRY1-OE) vector. Cells were left unstimulated or stimulated with LPS (1 μg/ml) for 6 h. Total RNA was extracted and subjected to RT-qPCR analysis for mRNA expression of TNF (E), IL-1β (F), and IL-6 (G). Data were normalized to Gapdh expression. Statistical significance was assessed by an unpaired two-tailed Student t-test. *P < 0.05, **P < 0.01, ***P < 0.001. H WT and ADAP knockdown RAW 264.7 cells were reconstituted via lentiviral transduction with either an empty vector or a SPRY1 overexpression (SPRY1-OE) vector. Cells were left unstimulated or stimulated with LPS (1 μg/ml) for 15 min. Cell lysates were subjected to Western blot analysis using the indicated antibodies.