Ab16502

TSCs, cocultured with macrophage, were digested and collected for cell slit. Palovarotene or LDN193189 and induction treatment were given according to the experimental group for 30 min. After washing the cells with PBS, 4% paraformaldehyde (Biosharp, # BL539A) was added, and the cells were fixed after 20 min at room temperature. PBS washes were given twice again, and 0.1% Tx-100 (Beyotime, # P0096) was added. After 15 min at room temperature, the liquid turned transparent, and 5% BSA/PBS was added, and the cells were incubated at 37°C for 1 h. The TPPP3 (Biorbyt, #5-F10) or CCR7 (Abcam, # ab32527) or p65 (Abcam, # ab16502) or Smad5 (Affinity, #AF5119) or Id1 (Proteintech, #18475-1--AP) antibody working fluid was added, and the cells were incubated at 4°C overnight. After overnight incubation, the samples were washed 3 times in PBS and were incubated at 37°C for 1 h with anti-rab-CY3 (ABCAM, #AB6939) working solution. The samples were washed 3 times in PBS and were incubated at room temperature for 15 min with Hoechst (ABCAM, #AB228551) and were washed 3 times in PBS. Lastly, the samples were seeded and photographed under a fluorescence microscope.

TSD was extracted by the research group, and the total saponin content was 61.7% [17 ]. Other reagents were colchicine (purity rate >99%) (Meilunbio Co., Ltd., J1225AS), colchicine tablets (Xishuangbanna Pharmaceutical Co., Ltd.), MSU (Shanghai Yuanye Biotechnology Co., Ltd., S24J9I64369), rat IL-1β ELISA kits, rat TNF-α ELISA kits (Wuhan Boster Bios Co., Lot : EK0393 and EK0526, respectively), TLR4 antibody, MyD88 antibody, NF-κB antibody, β-actin antibody (Abcam, ab13556, ab2064, ab16502, and ab8227, respectively), Lamin B antibody (Affinity, AF5161), PCNA antibody (Abbkine, A01040), GAPDH antibody (Servicebio, GB12002), HRP-labeled goat antirabbit IgG, HRP-labeled goat antimouse IgG (Abbkine A21020 and A21010), and ECL chemiluminescence kit (Thermo 32109).

Lung tissue sections were fixed in, dewaxed in dewaxing solution, and dehydrated in an ethanol series. After natural cooling, the sections were placed in PBS (PH7.4) to repair antigen and block endogenous peroxidase. The tissue was uniformly covered with 3%BSA in the tissue chemical circle and closed at room temperature for 30 min. Add PBS to the sections in a certain proportion of Phosphoinositide 3-kinase (PI3K) (Abcam, ab191606, 1:200), Akt (Abcam, ab179463, 1:200), nuclear factor kappa-B (NF-κB) p65 (Abcam, ab16502, 1:500), p-NF-κB p65 (Affinity Biosciences, AF2006, 1:250), IKB-α (Proteintech, 10268-1-AP, 1:250) antibodies, and the sections were placed flat in a wet box at 4 °C for overnight incubation. Corresponding secondary antibodies were added for incubation under ambient temperature for 50 min. After hematoxylin counterstain and ethanol dehydration, the sections were sealed by neutral gum with a cover glass and interpreted under a white light microscope. Intensities were quantified with Image Pro Plus 6.0 software. Three separate fields from each section and three tissue samples from each group were assessed.