Proliferating cell nuclear antigen (pcna)

Paraffin embedded sections were deparaffinised with Histoclear and rehydrated through graded ethanols. Heat induced antigen retrieval was performed in a microwave oven using Tris-EDTA buffer (10 mM Tris, 1 mM EDTA, 0.05 % Tween 20, pH 9.0) for 20 min and the slides were allowed to cool down prior to blocking with 3 % H₂O₂. After blocking with 5 % (v/v) goat serum in phosphate buffered saline (PBS), sections were incubated overnight at 4 °C with the primary antibodies. After a PBS wash, the sections were incubated with secondary antibody at 37 °C for 30 min. Visualization of a positive reaction was by means of a peroxidase substrate solution containing 0.02 % (wt/Vol) H₂O₂ and 0.1 % (wt/Vol) 3,3′-diaminobenzidine tetrahydrochloride (ZSBIO, Beijing, China) in PBS to produce a brown color, then the sections were counterstained with hematoxylin. A negative control, with the primary antibody replaced by mouse IgG antibody, was always included. The primary antibodies used were anti-proliferating cell nuclear antigen antibody (PCNA) (Merck, Millipore, Darmstadt, Germany) diluted 1:400 to identify cell proliferation. PCNA positive cells were counted in 10 randomly selected × 200 high-power fields under a microscope (OLYMPUS FSX100). The PCNA index was calculated according to the following formula: number of PCNA positive cells/total cell count × 100 % [15 (link)].

After homogenization, all skin tissues were lysed for 30 min with ice-cold radioimmunoprecipitation assay lysis buffer (CoWin Biosciences, Taizhou, China). Protein lysate (15 μg; concentration determined by a BCA assay) was subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE; CoWin Biosciences, Taizhou, China) on a 6% gel for collagen I and collagen III; 10% gel for Aurora B, CK14, and CK15; 12% gel for PCNA, pH3, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) at 80 V for 1.5 h, and then transferred to polyvinylidene fluoride membranes (Merck, Darmstadt, Germany). The membranes were blocked with 5% bovine serum albumin in PBS at room temperature for 1.5 h, followed by incubation with primary antibodies against collagen I (1:1000; Cell Signaling Technology, Danvers, MA, United States), collagen III (1:1000; Cell Signaling Technology, Danvers, MA, United States), PCNA (1:1000; Abcam), Aurora B (1:1000; Abcam), pH3 (1:200; Abcam), CK14 (1:1000; Proteintech), CK15 (1:2000; Proteintech), and GAPDH (1:20,000; Proteintech) at 4°C overnight. Quantitative analysis was performed on the immunoreactive bands using ImageJ software (v.1.53k; NIH, Bethesda, MD, United States).

The proteins were resolved by SDS–PAGE using homemade or precast gels (Bio-Rad) and transferred to a nitrocellulose membrane. Antibodies against the following proteins were used: Ser345 phospho-Chk1 (#2348; Cell Signaling Technology), Chk1 (#sc-8408; Santa Cruz), PCNA (#P8825; Sigma-Aldrich), Ser33 phospho-RPA32 (#ab221887; Abcam), RPA32 (#NA18; Calbiochem), DNA-PKcs (#ab1532; Abcam), PARP1 (#sc-8007; Santa Cruz), UBA1 (#4891s; Cell Signaling or #sc-53555; Santa Cruz), TOPBP1 (#A300-111A; Bethyl Laboratories), ATR (#A300-137A; Bethyl Laboratories), Thr1989 phospho-ATR (#GTX128145; GeneTex), and MBP (#E80329; New England Biolabs).

The antibodies used in this work were against: H3 (Abcam, ab1791, dilution 1/2000), H2B (Abcam, ab1790, dilution 1/2000), phosphorylated CHK1 (Cell Signaling, 2341 S, dilution 1/250), PCNA (Sigma, P8825, dilution 1/2500), RPA34^{62 (link)} (dilution 1/500), MCM3^{61 (link)} (dilution 1/2000), CDC45^{63 (link)} (dilution 1/1000), ELYS^{31 (link),64 (link)} (dilution 1/500), MCM4^{63 (link)} (dilution 1/1000), anti-Chk1 (dilution 1/500), anti-ORC5 (dilution 1/1000), anti-CDC6^{63 (link)} (dilution 1/500), OCT4 (Abcam, ab19857, dilution 1/500), actin (Sigma, A4700, dilution 1/500), HRP-linked ECL anti-mouse IgG (GE Healthcare, NA931V, dilution 1/4000), HRP-linked ECL anti-rabbit IgG (GE Healthcare, NA934V, dilution 1/4000) (For details see Supplementary Table 7).