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).