Sirt1

Cells after treatment were collected and dissolved in RIPA buffer (Beyotime, Nantong, China) and protein concentration was measured using the BCA protein assay kit. A total of 50 μg protein in each sample was isolated by 15% SDS-PAGE and then imprinted on PVDF membrane (Beyotime, Nantong, China). The membrane was blocked and incubated with primary antibodies overnight at 4 °C. The blots were incubated with HRP-conjugated secondary antibody at room temperature for 1 h on the next day. The ECL assay kit (Beyotime, Nantong, China) was used to reveal the protein bands. The density of each band was normalized to the expression of GADPH (Cat. No: ab8245, Abcam, Cambridge, MA, USA) or VDAC1 (Cat. No. ab15895, Abcam). The other primary antibodies used include Cytochrome c (Cat. No: ab13575, Abcam), SIRT1 (Cat. No. ab110304, Abcam), SIRT3 (Cat. No. ab217319, Abcam), LC3B (Cat. No. ab192890, Abcam), PINK1 (Cat. No. ab23707, Abcam), Parkin (Cat. No: ab77924, Abcam), Nrf2 (Cat. No. ab137550, Abcam), Nrf2 (Acetyl-Lys599) (Cat. No. HW147, Signalway Antibody, Nanjing, China). All experiments were repeated three times, and the gray values were quantified using Image J software (version 1.8.0 for Windows, National Institutes of Health, NY, USA).

Keloid tissues and its surrounding normal skin tissues were homogenized, lysed in RIPA for 30 min, and centrifuged at 12,000 r/min for 5 min. The supernatant was collected and the protein concentration was determined with BCA method. Similarly, proteins were also extracted from cells. After SDS-PAGE electrophoresis, the proteins were transferred to PVDF membranes. The membrane was blocked with 5% skimmed milk for more than 2 h, and washed with TBST for 5 min/time for 3 times. Then, the diluted primary antibodies against RUNX3 (1:2000; Abcam, USA), EZH2 (1:2000; Abcam, USA), AC-EZH2 (1:2000; Abcam, USA), SIRT1 (1:2000; Abcam, USA), CyclinD1 (1:2000; Abcam, USA), CDK2 (1:2000; Abcam, USA), CDK4 (1:2000; Abcam, USA), and β-actin (1:2000; Abcam, USA) were added and incubated overnight at 4° C. After incubation with goat anti-rabbit/goat anti-mouse secondary antibody (1:5000; Abcam, USA) for 70 min at room temperature, the membrane was subjected to ECL luminescence color development.

Placental section slides were prepared by the Northern Ireland Biobank (38 (link)) using fresh tissue before being subjected to immunofluorescence staining for FKBPL (Cat. no.: 10060-1-AP; Proteintech, UK), SIRT-1 (Cat. no.: ab110304; Abcam, UK), PlGF (Cat. no.: ab180734, Abcam, UK) and VEGF-R1 (Cat. no.: AF321, R&D systems, USA). Tissue slides were imaged using a Leica DMi8 fluorescence inverted microscope using the same magnification (20x) and exposure. Analysis was performed using Image J software (NIH, US) by selecting six random fields of view per section and measuring the intensity, with the assessor blind to patient group. Protein expression was quantified as previously described (39 (link)).

Hydrogen peroxide (H₂O₂, #MKBK8393V) and paraformaldehyde solution (#SZBC2290V) were purchased from Sigma-Aldrich. 5-Bromo-4-chloro-3-indolyl-β-D-galactosidase (X-gal, 11680293001) was purchased from Roche. Antibodies against the following proteins were used: DRG2 (Proteintech group, 14743-1-AP), acetyl-p53 (ac-p53, Cell Signaling Technology (CST), #2525), p53 (CST, #2524), p21^Waf1/Cip1 (Santa Cruz Biotechnology (SCBT), sc-397), cyclin D1 (SCBT, sc-717), p16^INK4α (BD Pharmingen, 551163), SIRT1 (Merck Millipore, 07-131), SIRT1 (Abcam, ab110304), acetyl-NF-κB p65 (ac-NF-κB p65, CST, #3045), NF-κB p65 (SCBT, sc-8008), phosho-Histone H2A.X (p-H2A.X, CST, 9718 s), and β-actin (SCBT, sc-1616). Secondary antibodies for immunoblotting were from SCBT (goat anti-mouse IgG-HRP, sc-2005; mouse anti-rabbit IgG-HRP, sc-2357; donkey anti-goat IgG-HRP, sc-2020), and those for immunofluorescent staining were from Invitrogen (goat anti-rabbit Alexa Fluor 568, A-11011; goat anti-mouse Alexa Fluor 488, A-11001; goat anti-mouse Alexa Fluor 568, A-11004). DMF (6,4′-dihydroxy-7-methoxyflavanone) was obtained from the Standardized Material Bank for New Botanical Drugs (no. NNMBP012), Wonkwang University (Republic of Korea). DMF (>98%) was isolated from the heartwood of Dalbergia odorifera [25 (link)] and was dissolved in DMSO (0.05% in final culture concentration).