Fig. 8.

MX, an inhibitor of USP11, reduces LPA1 levels and lessens LPS-induced lung inflammation. a. MLE12 cells were treated with DMSO or MX (5 μM) for 1 h prior to LPA treatment (5 μM, 30 min). Cell lysates were subjected to immunoprecipitation with an ubiquitin antibody, followed by LPA1 immunoblotting. Input lysates were analyzed by immunoblotting with LPA1 and β-actin antibodies. b. MLE12 cells were treated with MX (5 μM) for 0–8 h. Cell lysates were analyzed by immunoblotting with LPA1 and β-actin antibodies. c. MLE12 cells were transfected with LPA1-V5 or LPA2-V5 plasmid, and then cells were treated with MX (0–10 μM) for 6 h. Cell lysates were analyzed by immunoblotting with antibodies to V5 and β-actin antibodies. d. MLE12 cells were treated with MX (5 μM, 6 h) prior to LPA treatment (5 μM, 10 min). Cell lysates were analyzed by p-Erk1&2, Erk1&2, p-p38 MAPK, p38 MAPK, p-I-κB, and β-actin antibodies. Representative immunoblots were from at least three independent times. e. HBEpCs were treated with MX (5 μM, 6 h) prior to LPA treatment (1 μM, 3 h), and then cells were collected and IL-8 mRNA levels were examined by RT realtime PCR. f. HBEpCs were treated with MX (5 μM, 6 h) prior to LPS treatment (10 μg/ml, 3 h), and then cells were collected and IL-8 mRNA levels were examined by RT realtime PCR. g. MLE12 cells were treated with MX (5 μM, 6 h) prior to LPS treatment (10 μg/ml, 6 h), and then cells were collected and KC mRNA levels were examined by RT realtime PCR. h–k. C57/BL6 mice (6–8/group) were i.t. administrated with MX (0.25 mg/kg body weight) for 1 h, and then followed by i.t. LPS (2 mg/kg body weight) for 24 h. BAL were collected and BAL protein levels were measured by protein assay (h), IL-6 (i) and KC (j) levels were examined by Elisa. Lung tissues were fixed and stained with H&E (k).