Fig 2: Knockdown of miR-363-3p blocks the NF-?B activation by targeting S1PR1. (a) Relative mRNA levels of S1pr1 in lung tissues of mice in different groups. (b) Relative protein levels of S1PR1 in lung tissues of mice in different groups. (c) Relative protein levels of NF-?B p65 (cytoplasm), NF-?B p65 (nucleus), p-IKK Ser180/Ser181, IKK, p-I?B-a Ser32/36, and I?B-a in lung tissues of mice in different groups. (d) Representative images of dual immunofluorescence staining with S1PR1 and F4/80 in lung tissues of mice in different groups. (e) A luciferase reporter assay was used to measure the relative luciferase activities of S1PR1-3’-UTR wt and mut in RAW264.7 cells. Results were expressed as means ± SD, *P<0.05.

Fig 3: Low SLC7A2 upregulates CXCL1 expression by transactivating P65 expression.A Western Blotting the SLC7A2, phosphorylated P65 at ser536 (p-P65Ser536), P65, P50, p-IKBa, IKBa, ß-actin was used as control. B qRT-PCR was used to detect that relative mRNA expression of CXCL1 in the indicated cells with treatment with NF-k?B inhibitor, JSH-23 (10 µm, 24 h). The data are shown as the mean ± SD from the at least three independent experiments; *P < 0.05, **P < 0.01. C ELISA was used to detect that relative expression of CXCL1 in the indicated cells with treatment with NF-?B inhibitor, JSH-23 (10 µm, 24 h). The data are shown as the mean ± SD from the at least three independent experiments; *P < 0.05, **P < 0.01. D The expression of phosphorylated P65 was analyzed in the nuclear and cytoplasmic fractions of Huh7-shSLC7A2 cells and MHCC97H-SLC7A2 cells. The laminB1 and ß-actin were as control respectively. E Luciferase reporter assay was showed in the indicated co-transfected with pCMV-SLC7A2 and the CXCL1 promoter-luciferase construct. The data are shown as the mean ± SD from the at least three independent experiments; **P < 0.01. F Truncated and mutated CXCL1 promoter constructs were co-transfected with PCMV-SLC7A2, and the relative luciferase activity was confirmed. The data are shown as the mean ± SD from the at least three independent experiments; *P < 0.05. G ChIP assay confirmed the direct binding of P65 to the CXCL1 promoter in HCC cells. The data are shown as the mean ± SD from the at least three independent experiments; *P < 0.05. H ChIP assay confirmed the direct binding of P65 to the CXCL1 promoter in human HCC tissues. The data are shown as the mean ± SD from the at least three independent experiments; *P < 0.05. I Immunofluorescence of p-P65Ser536 of indicated HCC cells. J Huh7-shSLC7A2 cells treated with inhibitors of PI3K, JNK, ERK, p38, STAT3, and then western blotting was used to detect the expression of SLC7A2 and p-P65Ser536 in the nuclear as well as the total and phosphorylate expression of AKT, ERK, JNK, p38, STAT3.

Fig 4: Effects of miR-200c-3p on NF-?B activation in LPS-treated BV2 cells. A The expression of NF-?B p65 was examined by immunofluorescence staining at 400 × and 800 × magnification in LPS-treated BV2 cells with miR-200c-3p mimics transfection. Representative western blot for B nuclear NF-?B p65 and C p-NF-?B p65 in LPS-treated BV2 cells with miR-200c-3p mimics transfection. Data were represented as mean ± SD (n = 3) and analyzed by one-way analysis of variance (ANOVA) followed by Tukey’s multiple comparison test. #p < 0.05 vs. Con group; *p < 0.05 vs. LPS or the indicated group

Fig 5: Blockade of EVs release by GW4869 inhibited intestinal inflammatory responses via inhibiting the activation of the STING pathway.A–D The expressions of p-STING, STING, p-IRF3, IRF3, p-p65, and p65 in colon tissues from Wild type (WT), STING−/−, WT murine colitis, WT murine colitis treated with GW4869, and STING−/− murine colitis models were determined by western blot. WT murine colitis was abbreviated as WT + DSS in the illustrations. WT murine colitis treated with GW4869 was abbreviated as WT + DSS (GW4869 IP). STING−/− murine colitis was abbreviated as WT + DSS. The activation of the STING pathway was observed in the WT murine colitis model and was inhibited in WT murine colitis treated with GW4869. E, F The proportion of TUNEL-positive cells was quantified to estimate intestinal apoptotic level in WT, WT murine colitis, WT murine colitis treated with GW4869, and STING−/− murine colitis models. The level of apoptosis was higher in the WT murine colitis model and lower in WT murine colitis treated with GW4869, and STING−/− murine colitis models. Representative images were shown. Scale bar = 50 µm. G The expression of inflammatory cytokines including IL-6, TNF-α, and IFN-β in the homogenates of murine colon tissues was measured by ELISA (n = 5–9/group). All results were representative of at least three independent experiments. Data were displayed as mean values ± SD. *P < 0.05, **P < 0.01, ***P < 0.001.