4 6 diami dino 2 phenylindole dapi p0131

Reagents including TubA (HY-13271A, purity≥98%) were purchased from MedChemExpress (NJ, USA), TBHP solution (416665) and 3-methyladenine (3-MA, M9281) were obtained from Sigma-Aldrich Chemical Company (WI, USA), 4,6-Diami-dino-2-phenylindole (DAPI, P0131) was purchased from Beyotime (Shanghai, China). Primary antibodies were included as the following: HDAC6 (12834–1), GAPDH (10494-1), Bcl-2 (26593-1), HO-1 (10701-1), SOD1 (10269-1), Beclin 1 (11306-1), Aggrecan (13880-1), MMP13 (18165-1) and α-tubulin (66031-1) from Proteintech (IL, USA); Bax (ab182733), Collagen II (ab185430), ADAMTS5 (ab41037), Lamp2 (ab13524) and p62 (ab211324) from Abcam (MA, USA); ATG5 (12994) and acetyl-α-tubulin (5335) from CST (MA, USA); cleaved caspase 3 from (AF7022) from Affinity Biosciences (OH, USA); LC3B (NB600-1384) from NOVUS Biologicals (CO, USA); secondary antibodies against mouse, rat and rabbit were purchased from Proteintech; Alexa Fluor FITC (488) or Cy5 (647) donkey anti-rabbit/mouse/rat secondary antibodies were purchased from Abcam.

After the Transwell upper layer of BeWo cells formed a tight single-cell layer, forskolin (final concentration of 50 μM, IF0200, Solarbio, Beijing, China) was added to the upper layer to promote trophoblast syncytialization.
To verify the trophoblastic syncytialization, forskolin-treated BeWo cells (after 72 h) were subjected to immunofluorescence staining for the E-Cadherin protein. In brief, BeWo cells were fixed by adding paraformaldehyde. The fixed cells were then incubated with E-Cadherin Rabbit mAb primary antibody (3195T, Cell Signaling Technology, Inc., Boston, MA, USA) at 4 °C overnight. Alexa Fluor 594 rabbit secondary antibody (A11012, Invitrogen, Carlsbad, CA, USA) was then added and incubated for 1 h. Finally, the cell nuclei were stained using 4′,6-diamidino-2-phenylindole (DAPI, P0131, Beyotime Biotechnology, Shanghai, China). The fusion of cells and the red fluorescence intensity of E-Cadherin were observed under laser confocal microscopy (Olympus, FV2000, Tokyo, Japan).