Fig 1: Analysis of cytokine expression levels. (A) Western blotting confirmed NF-?B, MMP2, and MMP9 expression levels. ImageJ software (NIH) was used for image processing to verify band intensities as a result of semi-quantitative western blots. NF-?B expression level in CaSR overexpression group was significantly higher than that in empty vector and negative control groups. NF-?B, MMP2, and MMP9 expression levels were significantly lower in CaSR knockdown group than in empty vector and negative control groups (p < 0.001). (B) PTHrP expression levels in CM from modified A549 cells co-cultured with macrophages were detected by ELISA. PTHrP expression level was significantly higher in CaSR overexpression group than in empty vector and negative control groups. PTHrP expression level was significantly lower in CaSR knockdown group than in empty vector and negative control groups (p < 0.001). (C–E) IL-6, IL-8, and IL-11 expression in CM from modified A549 cells co-cultured with macrophages were detected by ELISA. IL-6, IL-8, and IL-11 expression levels were significantly higher in CaSR overexpression group than in empty vector and negative control groups. Expression levels of these three factors were significantly lower in CaSR knockdown group than in empty vector and negative control groups (p < 0.001). **p < 0.01, ***p < 0.001.
Fig 2: KRT8 is required for the IL-11-mediated metastatic phenotype in ccRCC(A) IL-11 mRNA levels were determined by qRT-PCR, and IL-11, p-STAT3, and STAT3 protein levels were determined by western blotting. (B) IL-11 overexpression restored Caki-1-SH-mediated migration and invasion ability. In contrast, IL-11 knockdown significantly reduced 786-O-KRT8 cell migration and invasion. (C) Representative images of the mice over time after the tail vein injections with each type of renal cancer cell. The statistical analysis is shown in (D). (E) Representative H&E-stained images of the lung metastatic loci in each group in (C). The statistical analysis is shown in (F).
Fig 3: TMED3 promotes cell migration by increasing IL-11 expression.(A) Differentially expressed genes in HepG2-siTMED3 cells compared with control cells. The top 10 up- and down-regulated genes are listed on the right. (B,C) Relative IL-11 expression in cells with stable TMED3 knockdown or overexpression and paired controls. IL-11 expression was analyzed by western blot and normalized to ß-actin. (D,E) The IL-11 concentration in culture medium from cells with stable TMED3 knockdown or overexpression and paired controls was determined by ELISA. n = 3, mean ± SD, the Student’s t test, *p < 0.05 versus GFP control, **p < 0.01 versus GFP control. (F,G) Relative P-STAT3 and STAT3 expression levels in cells with stable TMED3 knockdown or overexpression and paired controls were analyzed by western blot and normalized to ß-actin. (B–G) n = 3.
Fig 4: KRT8 activates IL-11/STAT3 signaling in ccRCC(A) Genes that are differentially expressed in Caki-1-SH cells compared with Caki-1-NC cells. (B) Relative IL-11 mRNA and protein expression levels in KRT8-knockdown cells, KRT8-overexpression cells and paired controls. IL-11 expression was analyzed by western blotting and normalized to ß-actin. (C) IL-11 concentrations in the culture media from stable KRT8-knockdown or KRT-overexpression cells and paired controls were determined by ELISA. (D) Relative p-STAT3 and STAT3 expression levels in stable KRT8-knockdown cells, KRT8-overexpression cells and paired controls were analyzed using western blotting and were normalized to ß-actin. (E) IL-11 mRNA expression levels in paired ccRCC tissue samples (n=109). (F) The correlation between KRT8 mRNA levels and IL-11 mRNA levels was measured in the same set of ccRCC tissues that was assessed in (E).
Fig 5: Setmelanotide increases IL-6 and IL-11 mRNA and protein secretion via MC4R. Setmelanotide (10 µM, black bars) significantly increased mRNA expression of IL-6 and IL-11 in MC4R+ astrocytes under control and inflammatory conditions (B,E) and not in mock cells (A,D). Likewise, setmelanotide increased IL-6 and IL-11 protein production and secretion as measured in supernatant under basal and inflammatory conditions (C,F). The effects of setmelanotide were completely blocked by MC4R antagonist SHU9119 (10 µM, gray bars, B,C,E,F). Student's t-test (mock) and One-way ANOVA (MC4R+), n = 2–3 independent experiments with 3 technical replicates per experiment for mRNA levels. For ELISA, supernatant was collected from 3 independent stimulations. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.
Supplier Page from Abcam for Human IL-11 ELISA Kit