Fig 1.
Relative mRNA expression of proinflammatory cytokines in SNX25 knockdown RAW 264.7 cells after LPS stimulation.
(A–C) RAW 264.7 cells were stimulated with LPS (1μg/ml) for 8 h and mRNA was extracted immediately. RT-qPCR analyzed the relative expression of IL-1β, TNF-α, and IL-6. β-actin was used as an endogenous control. All data are expressed as means ± SD. Circles indicate the individual experimental values. * P < 0.05 *** P < 0.001, n = 6, Welch’s t-test. (D–F) RAW 264.7 cells were stimulated with LPS (1μg/ml) for various times and mRNA was extracted immediately. RT-qPCR analyzed the relative expression of IL-1β, TNF-α, and IL-6. β-actin was used as an endogenous control. All data are expressed as means ± SD. n = 3.
Fig 2.
Relative protein expression of proinflammatory cytokines in SNX25 knockdown RAW 264.7 cells after LPS stimulation.
(A–C) RAW 264.7 cells were stimulated with LPS (1μg/ml) for 12 h. The expression of IL-1β, TNF-α, and IL-6 was analyzed by Western blotting. GAPDH was used as a loading control. Data are expressed as means ± SD. Circles indicate the individual experimental values. * P < 0.05, n = 5.
Fig 3.
Activation of MAPK signaling pathways in SNX25 knockdown RAW 264.7 cells.
(A–C) RAW 264.7 cells were stimulated with LPS (1μg/ml) for 20 min. The expression of p-ERK, p-p38 and p-JNK was analyzed by Western blotting. ERK, p38, and JNK were used as loading controls. Data are expressed as means ± SD. Circles indicate the individual experimental values. * P < 0.05, n = 4.
Fig 4.
Activation of NF-κB signaling pathways in SNX25 knockdown RAW 264.7 cells.
(A) RAW 264.7 cells were stimulated with LPS (1μg/ml) for 10 min. p-p65 expression was analyzed by Western blotting. p65 was used as a loading control. Circles indicate the individual experimental values. Data are expressed as means ± SD. n = 7. (B, C) RAW 264.7 cells were stimulated with LPS (1μg/ml) for 30 min. The expression of p65 in whole cells, cytoplasm and nucleus was analyzed by Western blotting. GAPDH, α-Tubulin and LaminB1 were used as a loading control. Data are expressed as means ± SD. Circles indicate the individual experimental values. * P < 0.01; ns, not significant, n = 3 (whole cells), n = 7 (nucleus and cytoplasm).
Fig 5.
Activation of IκBα and IKKβ in SNX25 knockdown RAW 264.7 cells.
(A and B) RAW 264.7 cells were stimulated with LPS (1μg/ml). Expression of IκBα and p-IκBα was analyzed by Western blotting. GAPDH was used as a loading control. Data are expressed as means ± SD. Circles indicate the individual experimental values. * P < 0.05 ** P < 0.01, n = 4 (p-IκBα) n = 6 (IκBα). (C) RAW 264.7 cells were stimulated with LPS (1μg/ml). Expression of p-IKKβ was analyzed by Western blotting. GAPDH was used as a loading control. Data are expressed as means ± SD. Circles indicate the individual experimental values. *** P < 0.001, n = 4. (D) MG132-induced accumulation of polyubiquitinated IκBα was analyzed by Western blotting. GAPDH was used as a loading control.
Fig 6.
Model illustrating that SNX25 regulates proinflammatory cytokine expression by affecting IκBα ubiquitination.
SNX25 inhibits ubiquitination of IκBα and the dissociation of IκBα/NF-κB (p65 and p50) complex and prevents nuclear translocation of NF-κB and subsequent cytokine expression.