Supplementary Figure 3

Supplementary Materials and Methods Pulmonary fibrosis model in rat A total of 60 SPF male Wistar rats (6-week-old) weighing 190–210 g and have never mated were used in the study. All animals were obtained from Shanghai Super-B&K Animal Laboratory Co., Ltd. (Shanghai, China), with Certification No. SCXK 2013-0016. All animals were housed under specific pathogen-free conditions in accordance with the provision and general recommendation of the Chinese Experimental Animals Administration Legislation. The 6-week-old SPF male Wistar rats were randomly divided into 9 treatment groups with 3 rats. According to the improved method[1,2], modeling method for a dministering BLM in a single dose after one week of adaptive feeding. In the experiments, equal volume of saline was administered as a control group. Rats from the PF model group underwent intranasal instillation of a single dose of 3.5 μg/g and 7 μg/g BLM in saline (100 μl, 150 μl and 200 μl). All groups were sacrificed at the 28th day. The lung tissue of rats were embedded in sections, then treated with H&E staining. Quantitative RT-PCR analysis For RNA isolation, tumors were homogenized and suspended, and RNA was extracted using the RNAspin Mini Kit (GE Healthcare, Waukesha, WI, USA) according to the manufacturer's instructions. For cDNA synthesis, 1μg of total RNA was reverse-transcribed using oligo-dT primers and the Superscript Amplification System (Life Technologies, Carlsbad, CA, USA). Quantitative RT-PCR was carried out using SYBR Green PCR Master Mix (Life Technologies). The PCR amplification program consisted of an initial polymerase activation at 94°C for 5 min, followed by 35 cycles at 94°C for 30 s, 59.5°C for 40 s and 72°C for 30 s. Amplification of GAPDGH, a relatively invariant internal reference RNA, was performed in parallel, and cDNA amounts were standardized to equivalent GAPDGH mRNA levels. Supplementary Fig. 1 Scheme and process of BLM-induced Pulmonary fibrosis animal experiment. Supplementary Fig. 2 Effect of different doses of BLM-induced alteration in lung histology of rats. Photomicrograph of sections of lungs of normal (BLM 0 mg/kg); low-dose BLM ( BLM 3.5 mg/kg) groups and high-dose BLM (BLM 7.0 mg/kg) groups. Rats treated with nasal BLM instillation of three dose at 100μL, 150μL, and 200μL. Photographs were taken with a high-resolution microscope, scale bar, 50μm. The data presented were obtained from three independent experiments. Supplementary Fig. 3 Effect of of XJXBCQ on Smad7 expression in MRC-5 cells. (A and B) Western blot assay was conducted to detect the levels of Smad7 in MRC-5 cells. MRC-5 cells were treated with TGF-β1 for 48 h firstly, Then cells were treated with different reagents as previous methods. Western blot with an antibody to GAPDH was used to ensure equal loading of proteins in each lane. Bolts were photographed and quantified for each sample. The data presented were obtained from three independent experiments. Each value is presented as the mean ± SD of three independent experiments. Supplementary Fig. 4 The effect of XJXBCQ on smad2 and smad7 mRNA levels in MRC-5 cells. Real-time quantitative PCR was performed to detect smad2 and smad7 mRNA in vitro. The data represented here were calculated from the means ± SD from triplicate experiments. Supplementary Fig. 5 Effect of of XJXBCQ and up-smad2 on MRC-5 cells proliferation. Cells were treated with various concentrations of XJXBCQ and analyzed by CCK-8 analyses as previously. Reference 1. Kandhare AD, Bodhankar SL, Mohan V, Thakurdesai PA . Effect of glycosides based standardized fenugreek seed extract in bleomycin-induced pulmonary fibrosis in rats: Decisive role of Bax, Nrf2, NF-κB, Muc5ac, TNF-α and IL-1β. Chem Biol Interact. 2015;237:151-165. 2. Hu J, Liu XL. Establishment and Pathological Evolution of Pulmonary Fibrosis Model in Mice Induced by bleomycin nasal instillation. Chinese Journal of Experimental Animals. 2009;17(5):352-354.

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