Bl739a

The fixation of HPMECs was performed using 4% paraformaldehyde followed by permeabilization using 1% Triton X-100 (Biosharp, China, BS084), followed by blocking for 10 min with quick blocking buffer (Beyotime, China, P0260) at room temperature. The paraffinized lung tissue sections were dewaxed, and antigen repair was performed, followed by blocking. Then, cells and lung tissues were incubated with Nrf2 (Proteintech, China, #16396-1-AP, 1:300) and p-p65 (CST, USA, #3033, 1:1600) primary antibodies for 10–16 h at 4 ^∘C. After washing the excess primary antibodies the next day three times with pre-cooled PBS for 10 min, the tissue samples were incubated with the respective fluorescent CoraLite594-conjugated Goat Anti-Rabbit IgG (H+L) (Proteintech, China, #SA00013-4, 1:300) secondary antibodies. The mixture was then stained with an antifade mounting medium for fluorescence (with DAPI) (Biosharp, China, BL739A). The cells and tissues were then observed and photographed under laser confocal microscopy.

Decalcification of the specimens was performed with 10% disodium ethylenediaminetetraacetic acid (EDTA-Na₂, Solarbio Beijing, China) at 4 °C for 30 d. Then, decalcified tissue embedded into paraffin wax was sliced into 5 μm sections. The sections were baked for 1 h at 60 °C and antigen repair was performed with trypsin antigen retrieval solution (BL333A, Biosharp, China). After blocking with goat serum for 2 h, the sections were incubated with primary antibodies overnight at 4 °C, then washed three times for 5 min. Finally, they were sealed with antifade mounting medium with DAPI (BL739A, Biosharp, China). In order to detect the bone regeneration, phenotype switching of macrophages, and angiogenesis in vivo, the tissue sections were incubated with primary antibodies anti-ALP (1:500, ab65834, Abcam), anti-Runx2 (1:500, ab76956, Abcam), anti-CD68 (1:250, ab955, Abcam)/anti-iNOS (1:200, ab15323, Abcam), anti-CD68/anti-CD206(1:200, ab64693, Abcam) and anti-CD31 (1:300, ab28364, Abcam). The fluorescent slides were finally covered by the cover glass using mounting medium containing DAPI. The fluorescent images were obtained with the fluorescence microscope, the fluorescence intensity of ALP and the number of Runx2⁺ cells, CD206⁺/CD68⁺ cells, iNOS⁺/CD68⁺ cells and CD31⁺ cells in the defects were counted and measured by Image J software.

SKOV3 and A2780 cells grown on glass coverslips and were fixed in 4% formaldehyde for 10 min and then permeabilized for 15 min in 1% Triton X-100. After blocked with 5% bovine serum albumin for 1 h at room temperature, cells were incubated in primary antibodies including γH2AX (1:100) and RAD51 (1:500) overnight at 4 °C and then in Alexa 488-conjuated secondary antibody (1:500, Thermo Fisher Scientific, A-11070) for 1 h at room temperature. Glass coverslips were mounted in anti-fade mounting medium containing DAPI (Biosharp, BL739A) and analyzed under laser scanning confocal microscopy (FV1200, Olympus Corp, Tokyo, Japan).