Error bars indicate mean SD (**, P 0

Error bars indicate mean SD (**, P 0.01; ***, P 0.001; NS, not significant). RhoA regulates YAP activity through F-actin cytoskeleton polymerization/ depolymerization independent of Hippo pathway kinases We next investigated how RhoA regulates YAP activity. (VP) were used to explore the effect of CD44-RhoA-YAP signaling blockade on mechanics-induced fibroblast activation and CS-induced pulmonary fibrosis. Results: Matrix stiffness could induce nuclear translocation of the Yes-associated protein (YAP) through CD44 in fibroblasts. This effect required RhoA activity and F-actin cytoskeleton polymerization but was independent of Hippo pathway kinases, Mst 1 and Lats 1, forming CD44-RhoA-YAP signaling pathway. Pharmacological upstream blocking by CD44 antibody or downstream blockade of YAP by DHI or VP could attenuate fibroblast migration, invasion, proliferation, and collagen deposition. Furthermore, CD44-RhoA-YAP signaling blockade could alleviate CS-induced fibrosis and improve pulmonary function identified that the signaling transduction was dependent on RhoA activation and F-actin cytoskeleton polymerization, but not on Hippo pathway kinases Mst 1 and Lats 1. Furthermore, we utilized anti-CD44 antibody or gradient-dose dihydrotanshinone I (DHI, a lipophilic component of traditional Chinese medicine Salvia Miltiorrhiza Bunge) 12 as well as verteporfin (VP, a small molecule YAP inhibitor) 13 and verified that the CD44-RhoA-YAP signaling blockade could affect fibroblast function and postpone fibrosis Carbazochrome progression in experimental silicosis. This thesis may shed light on a new molecular mechanism and a specific molecular target to postpone CS-induced fibrosis. Materials and Methods Animals and treatments Male C57BL/6 mice (6-8 weeks) were purchased from SLAC Laboratory Animal Co. Ltd. (Shanghai, China). All mice were raised in a pathogen-free facility, provided with a standard mice feedstuff and water ad libitum. The animals were acclimatized for a week before starting the experiments. All animal experiments were approved by the Animal Care and Use Committee at China Medical University. The silicosis model mice were described previously 14-16. Study 1: Male C57BL/6 mice received one-time intratracheal instillation of 50 L CS suspension (10 mice per group). Mice were randomized to receive weekly intraperitoneal injection of rat anti-mouse CD44 antibody (IM7) or isotype control rat IgG2b (300 g in 500 L saline). As shown in Figure Carbazochrome S1A, treatment in this study began at day 7 after CS instillation and all mice were sacrificed at indicated time points under anesthesia. Study 2: Male C57BL/6 mice received one time either intratracheal instillation of Carbazochrome 50 L CS suspension or sterile saline (10 mice per group). Mice receiving CS suspension were randomized to receive I: DMSO vehicle control, II-IV: daily intragastrical administration of DHI at doses of 150, 75, or 37.5 mg/kg dissolved in DMSO, respectively, and V: intraperitoneal injection of verteporfin at the dose of 100 mg/kg dissolved in DMSO every other day. Treatments in this study began at Terlipressin Acetate day 7 after CS instillation and all mice were sacrificed at indicated times under anesthesia (Figure S1B-C). Lung tissues were obtained for further analyses. Pulmonary function assay Body weight as well as minute volume, tidal volume, and breathing frequency of the male C57BL/6 mice, which received sterile saline, CS, CS+anti-CD44, CS+VP, and CS+DHI150 mg/kg were recorded prior to administering different treatments. These parameters were recorded again at week 1, 2, 3, 4, 5, 6, 7, and 8. We used minute volume, tidal volume, and breathing frequency relative to body weight to describe the mice pulmonary function. Cytotoxicity assay of DHI The cytotoxicity of DHI was assessed by the MTT assay. NIH-3T3 fibroblasts cell line, purchased from the National Infrastructure of Cell Line Resource (Beijing, China) , were cultured in 96-well plates at an initial density of 5104 cells/well in a serum-free Dulbecco’s Modified Eagle Medium (DMEM) overnight. Subsequently, NIH-3T3 fibroblasts were treated with DHI at different concentrations for 24 hours. Then DMEM was removed and replaced with 20 L MTT (0.5 mg/mL) for 4 hours followed by 150 L DMSO for 10 minutes. The absorbance of the dissolved formazan crystals was measured at 570 nm by a 96-well multimode plate reader (Figure S2). 2D cell culture and treatments The 2D Col-gel was acquired from Bioruo (Beijing, China). NIH-3T3 fibroblasts were cultured at 37C with 5% CO2 and grown in DMEM supplemented with 10% FBS, 100 U/mL penicillin, 100 g/mL streptomycin, and 10 mM HEPES. Following overnight serum deprivation (DMEM without FBS), NIH-3T3 cells were seeded on coverslips or plastic dishes coated with either.