2.8. Cell experiments
PASMCs were plated into 6-well or 96-well flat bottom culture plates. Upon appropriate confluence, PASMCs were starved with low serum (0.1% FBS) for 24 h. Then, plasma-derived exosomes were suspended in exosome-free FBS (Exo-FBS™ Exosome-depleted FBS, System Biosciences). The volume of plasma was used to quantify the exosomes. PASMCs were treated with the above suspended exosomes for 24h. Thereafter, Western blotting, IF, proliferation and scratch wound healing assay were performed.
PASMCs were treated with SCH772984 (1, 5, 25, 50, 100, 500 nM) under normal culture conditions for 1 h, and then continually were exposed to normoxia (21% O2) for 24 h. Then, the cell viability was measured by CCK8 assay. Based on toxicological results, PASMCs were treated with SCH772984 (1, 5, 25, 50, 100 nM) under normal culture conditions for 1 h, and then were exposed to normoxia (21% O2) for 24 h. According to the cell viability assay, the optimal dose of SCH772984 on PASMCs was determined.
The KLF-4 small interference RNA (siRNA) and riboFECT TM CP transfection reagent were purchased from Ribobio (Guangzhou, China). All protocols were performed according to the manufacturers’ instructions as previously described(Zhu et al., 2017).