Fig 1: Upstream regulators of LIF expression. (a–e) LIF upregulated the expression of itself in NRK49F cells through ERK-EGR1 axis. NRK49F cells were treated with LIF for 24 h. (a) Real-time PCR showed that LIF upregulated itself in NRK49F cells. Relative Lif mRNA levels were shown as fold induction over controls after normalization with gapdh, respectively. *P < 0.01 versus controls (n = 3). (b and c) Western blot analyses (b) and quantitative data (c) showed that LIF induced the expression of itself and EGR1 in a dose-dependent manner. #P < 0.05, *P < 0.01 versus controls (n = 4). (d and e) Western blot analyses (d) and quantitative data (e) showed that knocking down EGR1 diminished LIF-induced LIF production. #P < 0.05, *P < 0.01 versus NC+LIF (n = 4). (f) Upstream regulators of Lif expression. NRK49F cells were treated with OSM (20 ng/ml) and IL11 (10 ng/ml). (g) Upstream regulators of Lif expression. NRK49F cells were treated with TGF-ß1 (8 ng/ml), TNF-a (8 ng/ml) and Ang II (10 nM/ml) for 24 h. Real-time PCR measured the mRNA level of Lif. Relative Lif mRNA levels were showed as fold induction over controls after normalization with gapdh, respectively. *P < 0.01 versus controls (n = 4). (h) The mRNA level of Lif were induced by TGF-ß1 induced LIF expression in a dose-dependent manner in NRK49F cells. *P < 0.01 versus controls (n = 4). (i and j) Western blotting Representative Western blot (i) and quantitative data (j) showed that the protein level of LIF were induced by TGF-ß1 in a dose-dependent manner in NRK49F cells. #P < 0.05, *P < 0.01 versus controls (n = 4). (k) NRK49F cells were treated with TGF-ß1 (8 ng/ml) for 24 h, and the LIF concentration in the culture medium was determined by ELISA. *P < 0.01 versus controls (n = 4). Data were expressed as means ± SD. Student's t-test in (f), (g) and (k) or one-way ANOVAs (Least-Significant Difference test or Dunnett's T3 test) in (a), (c), (e), (h) and (j) was used for determining the P values while comparing between each pair of groups, respectively.