FIGURE 2.

IL6‐AS1 promotes the expression of inflammatory cytokines. (A and B) qRT‐PCR analysis of relative gene expression (including IL‐6, IL‐8, NFκB1, IKκB, RELa, CCL2, and CCR7) in HBF cells (two‐way ANOVA, n = 3 biological replicates). (C and D) qRT‐PCR analysis of relative gene expression as indicated in HFL1 cells (two‐way ANOVA, n = 3 biological replicates). (E and F) Multiplex immunoassay analysis of protein expression of nine cytokines and chemokines (including IL‐6, IL‐8, VEGF, IL‐1α, MIF, CXCL1, and CXCL2) regulated by IL6‐AS1 (two‐way ANOVA, n = 3 biological replicates). (G) qRT‐PCR analysis of interleukin (IL) 6 expression after cotransfection with IL6‐AS1 small interfering RNA (siRNA) (siIL6‐AS1‐1) and an IL6‐AS1 overexpression vector in HBF and HFL1 cells (one‐way ANOVA, n = 3 biological replicates). (H) ELISA analysis of IL‐6 expression after cotransfection with IL6‐AS1 siRNA (siIL6‐AS1‐1) and an IL6‐AS1 overexpression vector in HBF and HFL1 cells (one‐way ANOVA, n = 3 biological replicates). (I and J) qRT‐PCR analysis of IL‐6 expression in knockdown (I) and overexpressing (J) IL6‐AS1 HBF and HFL1 cells in response to stimulation with lipopolysaccharide. Cells were transfected with IL6‐AS1 siRNA (siIL6‐AS1‐1) or control for 48 h and then exposed to LPS (1 μg/ml) for 6 h (one‐way ANOVA, n = 4 biological replicates). (K and L) ELISA analysis of IL‐6 expression in knockdown (K) and overexpressing (L) IL6‐AS1 HBF and HFL1 cells in response to stimulation with LPS (one‐way ANOVA, n = 4 biological replicates). Error bars represent means ± SD. *p < 0.05, **p < 0.01, and ***p < 0.001