Figure 3.

Proviral Integration site for Moloney murine leukemia virus 1 (Pim-1)/nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and IL-6/JAK2/STAT3 form a positive feedback loop. A: lung fibroblasts derived from patients with idiopathic pulmonary fibrosis (IPF) were cultured for 24 h in the presence of an IL-6 neutralizing antibody (IL-6 NAb, 10 μg/mL), a JAK2 inhibitor (AZD 1480, 10 µM), or a STAT3 inhibitor (LLL12, 1 µM) before RNA isolation and qPCR analysis. RNA transcript levels are quantified relative to the same representative nontargeting (NT) sample, using GAPDH as a housekeeping gene. n = 3 biologically independent and experimentally independent experiments (**P < 0.01 vs. the control treated groups). B: lung fibroblasts derived from patients with IPF were transfected with nontargeting (NT) siRNA or siRNA targeting PIM1 for 72 h before collecting whole cell protein for Western blot analysis. Representative images are shown and band density for three independent experiments was quantified (*P < 0.05, **P < 0.01 vs. the NT control group). C: lung fibroblasts derived from patients with IPF were transfected with nontargeting (NT) siRNA or siRNA targeting PIM1 and cultured for 72 h before collecting RNA for qPCR analysis. RNA transcript levels are quantified relative to the same representative NT sample, using GAPDH as a housekeeping gene. n = 3 independent experiments (***P < 0.001 vs. the indicated group). D: positive feedback model. Pim-1 phosphorylates p65/RelA, supporting NF-κB transcriptional activity that stimulates IL-6 synthesis. IL-6 signals through its receptor system and JAK2/STAT3 to transcribe PIM1. Tools used throughout our studies are highlighted in yellow. Schematic was prepared in part using Motifolio Scientific Illustration Toolkits.