Fig 1: L-THE inhibites the expression of IL-23 and chemokines in vitro. (A) Q-PCR analysis of IL-23p19 and TNF-a in mRNA in BMDCs were treated with 4 µg/ml IMQ for indicated time points after pretreatment with L-THE for 12 h. (B) ELISA analysis of IL-23 protein in BMDCs were treated with 4 µg/ml IMQ for indicated time points after pretreatment with L-THE for 12 h. (C) Q-PCR analysis of CXCL1-3, and CCL2 mRNA in mouse primary keratinocytes were treated with 100 ng/ml IL-17A for indicated time points after pretreatment with L-THE for 12 h. (D) ELISA analysis of CXCL1 protein in mouse primary keratinocytes were treated with 100 ng/ml IL-17A for indicated time points after pretreatment with L-THE for 12 h. (E) Q-PCR analysis of IL-17RA mRNA in mouse primary keratinocytes were treated with 100 ng/ml IL-17A for indicated time points after pretreatment with L-THE for 12 h. Data is representative of three independent experiments. p values are determined by two-way ANOVA. *, p < 0.05; **, p < 0.01; ***, p < 0.001.
Fig 2: Ischemia-reperfusion (IR) induces production of chemokines and infiltration of immune cells.A Hierarchical clustering analysis of the differentially expressed mRNAs in IR group compared to Sham group (green, low; red, high). Three significantly upregulated mRNAs: CXCL1, CXCL2, and CCL2 (fold-change > 10) were highlighted in red box. B KEGG pathway enrichment analysis for upregulated mRNAs. C–E Time course of chemokine CXCL1 (C), CXCL2 (D), and CCL2 (E) expression. Data represent mean ± SEM. n = 6–8. **p < 0.01 versus Sham group. F Immunofluorescence staining of Gr-1 and F4/80 in the kidney 6 h and 24 h after renal IR. Gr-1 was used as a marker of neutrophils, and F4/80 was used as a marker of macrophages. Scale bar, 100 µm. G Quantification of Gr-1-positive neutrophils and F4/80-positive macrophages in the kidneys. Data represent mean ± SEM. n = 6. **p < 0.01.
Fig 3: IRAR regulates chemokine expression.A Co -expression network of IRAR and differentially expressed mRNAs. The solid line indicates positive regulation and the dotted line indicates negative regulation. B qRT-PCR analysis of IRAR in mTECs transfected with IRAR overexpressing lentivirus (LncRNA-OE) or Negative lentivirus (LncRNA-NC). C–E qRT-PCR analysis of CXCL1 (C), CXCL2 (D), CCL2 (E) in mTECs transfected with IRAR overexpressing lentivirus or Negative lentivirus. F Western blot analysis of CXCL1, CXCL2 and CCL2 in mTECs transfected with IRAR overexpressing lentivirus or Negative lentivirus.. Data are means from three independent experiments. G–J qRT-PCR analysis of IRAR (G), CXCL1 (H), CXCL2 (I) and CCL2 (J) in mTECs. mTECs were transfected with GapmeR IRAR (GapmeR-Hypoxia) or Negative control (Control-Hypoxia) for 48 h, then treated with hypoxia. K RNA fluorescence in situ hybridization (FISH) assays indicated co-expression between IRAR and CXCL1, CXCL2, CCL2 in the kidney at 24 h after ischemia reperfusion. Scale bar, 100 µm. L RNA immunoprecipitation (RIP) experiments were performed using antibodies against CXCL1, CXCL2 and CCL2. RIP enrichment was determined relative to the input controls. M RNA pulldown assays were performed in tubular epithelia cells to examine the association of IRAR with CXCL1, CXCL2 and CCL2. Data represent mean ± SEM. n = 4. **p < 0.01.
Fig 4: Inhibition of IRAR reduces ischemia-reperfusion (IR)-induced production of chemokines and infiltration of immune cells.A–C ELISA of CXCL1 (A), CXCL2 (B), CCL2 (C) in the kidney 24 h after renal IR (n = 6–8). D Immunofluorescence staining of Gr-1 and F4/80 in the kidney 6 h after renal IR. Gr-1 was used as a marker of neutrophils and F4/80 was used as a marker of macrophages. Scale bar, 100 µm (n = 6–8). E Quantification of Gr-1-positive neutrophils and F4/80-positive macrophages in the kidneys. Data represent mean ± SEM. n = 4. *p < 0.05, **p < 0.01.
Fig 5: Compound 21 significantly ameliorates LPS-induced pro-inflammatory response (cytokine production) in macrophages in a dose-dependent manner. RAW 264.7 macrophage cells were cultured with LPS (100 ng/mL) and co-treated with different concentrations of C21, as indicated, or vehicle for 24 h. An amount of 500 ng of total RNA was reverse transcribed into complementary DNA, and then gene expression was detected using SYBR Green-based RT-qPCR. PPIA was used as an endogenous control. Protein expression was evaluated via ELISA. Cell culture supernatants were collected following a similar experimental design and centrifuged at 2000× g × 10 min for complete removal of cells’ debris, and then the secreted cytokines/chemokines were assessed. The mRNA expression level of pro-inflammatory cytokines and chemokines IL-1ß (A), TNF-a (B), and CXCL1 (C). The protein expression level of pro-inflammatory cytokine/chemokines IL-1ß (D), TNF-a (E), and CXCL1 (F). Values are the mean ± SD of 3–4 independent experiments analyzed by one-way ANOVA. ns p > 0.05, * p < 0.05, ** p < 0.01.
Supplier Page from Abcam for Mouse CXCL1 ELISA Kit (GRO alpha)