Bs 0754r

As previously reported, the immunofluorescence (IF) experiment procedure was carried out (Liu et al., 2018a ). The primary antibodies used in the IF experiment were as follows: mouse anti-BrdU monoclonal antibody (1:200, G3G4, DSHB, IA), rabbit anti-PCNA antibody (1: 100, bs-0754R, Bioss, Beijing, China). The secondary antibodies used in the IF experiment were as follows: goat anti-mouse IgG (H + L)-TRITC (1:100, BS11502, Bioworld Technology, Inc., Minneapolis, MO), goat anti-rabbit antibody (1:50, HA1016, HuaBio, Hangzhou, China). After that, the IX70 fluorescence microscope (Olympus, Tokyo, Japan) was used to observe the fluorescence image.

The total proteins were extracted from cells by RIPA lysis buffer (Beyotime) with phenylmethanesulfonyl fluoride (PMSF; Beyotime) and then separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE; Beyotime) transferring to polyvinylidene difluoride membranes (PVDF; Millipore, MA, USA). Primary antibodies against type I collagen (COL1) (1:1000, Bioss, Beijing, China), runt-related transcription factor-2 (RUNX2) (1:1000, 12556, CST, MA, USA), ALP (1:1000, ab67228, Abcam), NRF2 (1:1000, ab62352, Abcam), NAD(P)H quinone oxidoreductase 1 (NQO1) (1:10,000, ab80588, Abcam), CD206 (1:1000, ab125028, Abcam), iNOS (1:1000, 18985-1-AP, Proteintech, Wuhan, China), IDO (1:1000, ab211017, Abcam), IL-1β (1:1000, A1112, Abclonal), AhR (1:1000, ab190797, Abcam), PCNA (1:500, bs-0754R, Bioss), GAPDH (1:2000, Bioss) were used. The secondary antibody goat anti-rabbit IgG H&L (HRP) (1:5000, ab205718, Abcam) was used. The images were detected by using ChemiDocTM MP Imaging System (Bio-Rad, USA).

The coverslips were placed in the bottom of 24-well plates. After adjusting to 2×10⁵ cells.mL^-1, 1 mL single Skov3 cell suspension was added to each well of the plates and
then incubated for 24 hours at 5% CO2 and 37 °C. Subsequently, the cells were treated with different concentrations of ATZ for 48 hours, which was prepared for proliferating
cell nuclear antigen (PCNA) IHC staining. For IHC staining, standard IHC procedures were carried out as previously described ( 21 (link) ).
Rabbit anti-human PCNA (1:300, Bioss, bs-0754R) was used as the primary antibody, and biotinylated goat anti-rabbit IgG (1:500, Bioss, bs-0295G-Bio) was used as the secondary antibody.
The slices were evaluated by Image-Pro Plus 6.0, and positive cells were identified as cells with brown staining. The results were repeated at least three times independently.