A0262

Western blot was performed using published protocols [16 (link)] with antibodies directed against OC-STAMP (2051. PB1; FabGennix Inc.), collagen type I (Col I) (ab34710, Abcam), α-SMA (ab32575, Abcam), E-cadherin (ab76055, Abcam), N-cadherin (ARG23870,Arigo), p-Smad2/3 (8828 s, Cell Signaling Technology, MA, USA), Smad2/3 (5678, Cell Signaling Technology), Phospho ataxia telangiectasia and Rad3-related protein (p-ATR, DF7512, Affinity), Phospho ataxia telangiectasia mutated (p-ATM, AF8410, Affinity), p-p53-S15 (AP0083, Abclonal), p21 (ab109520, Abcam), p16 (A0262, Abclonal), p-PERK (DF7576, Affinity), p-IRE1α (ab48187, Abcam), Phospho-nuclear factor-kappaB (p-NF-κB, ARG51516, Arigo), transforming growth factor-β1 (TGF-β1, ARG56429, Arigo), TGF-β receptor I (TGFβR1, A16396, Abclonal), and TGF-β receptor II (TGFβR2, ARG59501, Arigo) at a concentration of 1 : 1000.

The protein extracts from breast cancer tissues and cells were electro-separated and transferred to PVDF membrane which was then incubated with primary antibodies of mouse anti-KDM5A (1:1000, ab78322, Abcam, UK), rabbit anti-p16 (1:1000, A0262, ABclonal), mouse anti-Fbxo22 (1:1000, sc-100736, Santa Cruz), rabbit anti-E-cadherin (1:1000, #3195, Cell Signaling), rabbit anti-N cadherin (1:1000, #13,116, Cell Signaling), rabbit anti-Vimentin (1:1000, #5741, Cell Signaling), rabbit anti-H3K4me3 (A2357, 1:1000, ABclonal), and rabbit anti-GAPDH (1:1000, #2118, Cell Signaling, internal reference) overnight at 4 °C. HRP-labeled goat anti-mouse (1:10,000, BA1050, Boster, Wuhan, China) or goat anti-rabbit IgG (1:10,000, BA1054, Boster) secondary antibodies were incubated with the membrane for 1 h at ambient temperature. The films were exposed in an Amersham Imager 600 (UK). Gray-scale analysis was then performed using ImageJ.

NPCs were lysed with RIPA lysis buffer (Fudebio, Hangzhou, China) containing PMSF (Fudebio, Hangzhou, China) in an ice bath. After boiling with loading buffer (Fudebio), proteins from the samples were electrophoresed using SDS–PAGE gels at 80 V for 2 h and transferred to 0.22 μm polyvinylidene difluoride (PVDF) membranes (Millipore, MA, USA) at 280 mA for 120 min. The membranes were blocked with 5% skim milk powder in TBST for 2 h at room temperature and incubated with antibodies specific for β-actin (AF5001, Beyotime), MCM7 (A11325, Abclonal, Wuhan, China), p21 (27296-1-AP, Proteintech, Wuhan, China), p16 (A0262, Abclonal), aggrecan (13880-1-AP, Proteintech), collagen II (28459-1-AP, Proteintech), MMP-13 (18165-1-AP, Proteintech), and MMP-3 (17873-1-AP, Proteintech) overnight at 4 °C. The next day, the PVDF membranes were washed with TBST and incubated with an HRP-conjugated secondary antibody (Fudebio). After washing with TBST, bands were detected using a ChemiDoc Touch Imaging System (Bio–Rad, Hercules, CA, USA) with a chemiluminescence kit (Fudebio).

Paraffin-embedded sections (5 μm) were stained with H and E and immunohistochemical (IHC) staining. The slides were incubated overnight at 4°C with mouse anti-HPV16 E7 (1:50, orb10573, Biorbyt), rabbit anti-P16 (1:100, A0262, Abclonal), rabbit anti-RB1 (1:100, 10048-2-Ig, Proteintech), rabbit anti-Ki67 (1:100, ab16667, Abcam), rabbit anti-CDK2 (1:400, ab6538, Abcam), rabbit anti-E2F1 (1:200,12171-1-Ap, Proteintech), rabbit anti-TP53 (1:150, A16989, Abclonal), rabbit anti-14-3-3 Sigma (1:50, A1026, Abclonal) and rabbit anti-P21 (1:150, A11454, Abclonal) primary antibodies. Diaminobenzidine (DAB) was used for antibody detection. Images were photographed from three randomly selected fields using cellSens Dimension (version 1.8.1, Olympus). Staining was assigned a score using a semiquantitative six category grading system: 0, no staining; 1,1%to 10% staining; 2, 11% to 25% staining; 3, 26% to 50% staining; 4, 51% to 75% staining; and 5, >75% staining. Staining intensity was assigned a score using a semiquantitative four-category grading system: 0, no staining; 1, weak staining; 2, moderate staining; and 3, strong staining. Every core was assessed individually and the mean of three readings was calculated for every slide. The staining score was determined separately by two experts under the same conditions.

Rb1 (purity greater than 98%) was obtained from Victory (Sichuan, China). Antibodies against p21^Cip1 (ab188224), p16^INK4a (ab51243), ICAM-1 (ab179707), VCAM-1 (ab134047), PAI-1 (ab222754) and GAPDH (ab181603) were purchased from Abcam (MA, USA). Antibodies against collagen I (14695), collagen III (22734) and Axl (13196) were purchased from Proteintech Group (MA, USA). Antibodies against Gas6 (A8545) and p16^INK4a (A0262) were purchased from ABclonal Technology (Hubei, China). Modified Krebs-Henseleit (K-H) buffer, phenylephrine (a potent vasoconstrictor) and acetylcholine (Ach, an endothelial-dependent NO donor) were purchased from Sigma-Aldrich (MO, USA). All other reagents used were of analytical grade.