Fig 1: IL-1β treatment increased IL-1R1 and p-IRAK-1 expression in RBECs (n=4). (A) Immunoreactive bands of IL-1R1 (80 kDa) and β-actin (42 kDa). (B) The bar graph shows increased IL-1R1 expression in the IL-1β group compared with that in the control group (**P<0.01). (C) Immunofluorescence images showing the expression of CD31+ RBECs (a and d, green), IL-1R1 (b and e, red), and the co-localization of IL-1R1 and RBECs (c and f). Enhanced IL-1R1 immunofluorescence was evident in the IL‑1β group compared with the control group. Scale bars (a-f): 10 μm. (D) Immunoreactive bands of p-RIAK-1 (77 kDa), t-IRAK-1 (77 kDa) and β-actin (42 kDa). (E) The bar graph shows increased p-IRAK-1 expression in the IL-1β group compared with the control group (**P<0.01). The protein expression of p‑IRAK‑1 was significantly suppressed with IL‑1Ra treatment (*P<0.05). RBECs, rat brain capillary endothelial cells; IL-1β, interleukin-1β; IL‑1R, interleukin‑1 receptor; IL‑1Ra, interleukin‑1 receptor antagonist; HC, high concentration of carbon dioxide; ns, non‑significant.
Fig 2: Fluoxetine treatment reduces MMP‐1 expression in CSE treated SAEC via inhibition of TLR‐4 signaling. A, SAEC cultures were treated with 5% CSE ±10‐µmol/L fluoxetine for 24 h. Media was harvested, and MMP‐1 levels were assessed by ELISA. CSE treated cultured secreted significantly more MMP‐1 as compared to controls (5.2 ± 1.3 vs. 2.1 ±0.3 ng/mL, respectively; *** p ≤ 0.001, n = 12). Treatment with 10‐µmol/L fluoxetine reduced CSE stimulated MMP‐1 secretion to control levels (2.6 ± 0.3 ng/mL). B, Real‐time qPCR was performed on SAEC cultures treated with 5% CSE ±10‐µmol/L fluoxetine for 24 h. CSE treatment upregulated MMP‐1 mRNA expression as compared to controls (1.82 ± 0.11 vs. 0.92 ± 0.06 RQ, respectively; ** p ≤ 0.01, n = 6). This increase was significantly inhibited by treatment with fluoxetine (Fluo) (1.82 ± 0.11 vs. 1.11 ± 0.20 RQ, respectively; # p ≤ 0.05, n = 6). C, Treatment with CSE significantly increased TLR‐4 expression (0.23 ± 0.11 vs. 2.11 ± 0.42 RQ, respectively; *** p ≤ 0.001, n = 6). This increase was inhibited by addition of 10‐µmol/L fluoxetine to CSE stimulated cultures (2.11 ± 0.42 RQ vs. 0.51 ± 0.09 RQ, respectively; ### p ≤ 0.001, n = 6). D, Western blot demonstrating fluoxetine mediated inhibition of the CSE induced increase in IRAK phosphorylation
Fig 3: Expression of MYD88 S257D and L265P promote NF-kB activation.A Phosflow analysis for IRAK1(Threonine 209) in OCI-LY1 and OCI-LY7 transduced with different MYD88 mutants. Cells were transduced with an empty vector (EV) or an expression vector for MYD88 (WT, S257D, S257A or L265P) and sorted for GFP expression. Untransduced, GFP negative, cells were used as negative control. IRAK1(Thr209) phosphorylation levels are shown relative to cells expressing WT MYD88. The mean ± SD of four independent experiments is shown. **P < 0.01 using 1-way ANOVA with Dunnett’s multiple comparisons test. B NF-kB activity measured by NF-kB-dependent luciferase reporter assays in OCI-LY1 and OCI-LY7 transduced with different MYD88 mutants. Cells were stably transduced with different MYD88 mutants and subsequently transfected with plasmid DNAs encoding for NF-κB promoter-driven firefly luciferase and a Renilla luciferase as control. Cells were allowed to recover for 48 h after transfection. Firefly luciferase activity was first normalized to the activity of Renilla luciferase. Then, luciferase activity was normalized to cells expressing WT MYD88. The mean ± SD of four independent experiments performed is shown. *P < 0.05; **P < 0.01 using 1-way ANOVA with Dunnett’s multiple comparisons test. C, E RT-qPCR analysis of CD80 (C) or HCK (E) expression in OCI-LY1 and OCI-LY7 transduced with different MYD88 mutants. Cells were allowed to recover for 72 h before RNA isolation. RPLP0 was used as an input control and data are normalized to the EV control expression levels. The mean ± SD of four independent experiments performed in triplicate is shown. **P < 0.01 using 1-way ANOVA with Dunnett’s multiple comparisons test. D Flow cytometric analysis showing membranous CD80 staining in OCI-LY1 and OCI-LY7 transduced with different MYD88 mutants. One representative experiment of three independent experiments is shown. F Immunoblot analysis for HCK in OCI-LY1 and OCI-LY7 transduced with different MYD88 mutants. β-actin was used as loading control.
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