Pifithrin α

Inhibition of p53 was conducted by adding the famous p53 inhibitor pifithrin-α (PFT-α; Selleck, Houston, TX, USA) (20 μM) to the osteogenic induction medium described above and renewing the medium every 3 days. The lentiviral vector carrying specific p53-targeting shRNA was obtained from GenePharma Co., Ltd. (Shanghai, China; Gene ID: 24842). BMSCs were infected with the lentiviral vector at a multiplicity of infection of 100 according to the protocols described above. The sequence of p53 shRNA was 5′-GAG AAT ATT TCA CCC TTA A-3′ (Zheng et al., 2016 (link)).

Cisplatin was purchased from Sigma (St. Louis, MO, USA). Pifithrin-α was purchased from Selleck (Houston, TX, USA). Primary antibodies used in this study were from the following sources: rabbit polyclonal anti-cleaved caspase-3, mouse polyclonal anti-p53, and rabbit polyclonal anti-phospho-p53 (Ser15), GAPDH. The β-actin antibodies were from Cell Signaling Technology (Danvers, MA, USA). Rabbit polyclonal anti-fibronectin was from Abcam (Cambridge, MA, USA). Rabbit polyclonal anti-Collagen I and rabbit polyclonal anti-CTGF were from Novus Biologicals (Littleton, CO, USA). Mouse polyclonal anti-p21 was from Invitrogen (Carlsbad, CA, USA). Secondary antibodies were from Jackson ImmunoResearch (West Grove, PA, USA).

In vitro 3D HSE were generated as described previously^{7 (link)}. Briefly, a suspension of collagen type I (Biochrom, Berlin, GER) containing 1 × 10⁵ fibroblasts per ml was poured into cell-culture inserts (3 μm pore size; BD Bioscience, Bedford, MA, USA) and allowed to gel for 2 h at 37 °C. After equilibration with KGM-2 for 2 h, 1.5 × 10⁶ KC, in a total volume of 2 ml KGM-2, were placed on the collagen gel. After overnight incubation the medium was removed from both the inserts and external wells, and serum-free KC-defined medium (SKDM) was added only to the external wells. SKDM consists of KGM-2 (without bovine pituitary extract and epinephrine) supplemented with 1.3 mM calcium chloride, 50 μg/ml ascorbic acid, and 0.1% bovine serum albumin (all supplements from Sigma-Aldrich). SKDM was changed every second day for 7 days.
For some experiments HSE were cultured in SKDM containing 10 µM of the DNA methylation inhibitor 5-Aza-2′-Deoxycytidine (Sigma Aldrich), 0.1 nM of proteasome inhibitor MG132 (Merck, Calbiochem, Darmstadt, Germany), 20 nM of the autophagy inhibitor bafilomycin A1 (InvivoGen, San Diego, CA, USA) or 80 mM of the p53 inhibitor pifithrin-α (Selleckchem, Munich, Germany).

CAPE and p-nitro CAPE (CAPE-pNO₂) were synthesized as previously described⁴⁷ . The chemical structures are shown in Fig. 1A and B. The human colon cancer cell line HT-29 and HCT-116 were purchased from the Cell Bank at the Chinese Academy of Sciences (Beijing, China). Dulbecco’s modified Eagle medium-high glucose (DMEM-H) and foetal bovine serum (FBS) were purchased from Gibco/Invitrogen (Carlsbad, CA, USA). Dimethyl sulfoxide (DMSO), trypsin, streptomycin, penicillin, 3-[4,5-dimethyl-2-thiazolyl]-2,5- diphenyl-2-tetrazolium bromide (MTT), propidium iodide (PI) and Hoechst 33342 were obtained from Sigma Aldrich (St. Louis, MO, USA). P53 inhibitor (Pifithrin-α) was purchased form Selleck (USA). An Annexin V-FITC Apoptosis Detection Kit was purchased from Keygen Biotech Co Ltd. (Nanjing, China). RNase A was acquired from Promega (Madison, WI). Bcl-2, Bax, cleaved-caspase-3, pro-caspase-3, P38, P21^Cip1, P27^Kip, P53, c-Myc, CDK2, VEGF and β-actin antibodies were purchased from Proteintech Group Inc. (Wuhan, China). For treatment, CAPE and CAPE-pNO₂ were dissolved in DMSO and diluted to a final concentration with culture medium (DMSO concentration < 0.1%). All chemical reagents were used before the date of expiration.

Thirty-six-week-old C57BL/6J mice (Shanghai SLAC Laboratory Animal Co., Ltd., Shanghai, China) were randomly assigned to 5 groups (n = 6): Normal group, normal mice without any treatment; Vehicle group, depilated mice treated with normal saline; TSG group, depilated mice treated with 200 μM TSG (National Institute for the Control of Pharmaceutical and Biological Products, China); p53 inhibitor group, which received 200 mM Pifithrin-α (Selleck Chemicals, USA); Minoxidil group, which was treated with 2% Minoxidil (Sigma, USA). All medicines were performed topically on the upper back once per day for 2 weeks. Afterwards, mice were sacrificed and dorsal skins were fixed in 4% paraformaldehyde (PFA) (Sigma) or frozen in liquid nitrogen for further study. All animal experiment protocols were approved by the Animal Experiment and Care Committee of Shanghai Jiao Tong University School of Medicine.
Five groups of mice were all treated with the same depilated model, which was induced as previously described [19 (link)]. Briefly, a melted wax/rosin mixture (1 : 1) under general anesthesia was used on the dorsal skin which could peel off all hair shafts and immediately induce all follicles to turn into homogeneous growth phase [19 (link)].

Primary astrocyte cultures were replaced with the glucose-free DMEM and transferred into an anaerobic chamber flushed with 5% CO₂ and 95% N₂ (v/v) at 37 °C for 6 h. The astrocytes were incubated again in DMEM containing 10% FBS and returned to the normoxic incubator (95% air and 5% CO₂) at 37 °C for 24 h. For IL-33 treatment, cultured astrocytes (5 × 10⁵) were treated with 50 ng/mL IL-33 throughout the whole period of OGD/R (30 h), pretreatment with or without Ly294002 (1 μM; Selleck) for phosphorylation AKT-S473 analysis or P53 inhibitor Pifithrin-α (10 μM; Selleck) for cell apoptosis analysis. Cells without exposure to OGD/R were defined as control group.

Mouse bone marrow Lin⁻c-Kit⁺ cells were obtained by a combination of negative selection for Lin⁻ cells and positive selection for c-Kit⁺ cells using MicroBeads (Miltenyi Biotec Inc., Auburn, CA, USA) following the protocols provided by the manufacturer. They were seeded into wells of 24-well plates at 3 × 10⁵ cells per well and incubated with StemSpan™ SFEM (StemCell Technologies, Vancouver, Canada) supplemented with l-glutamine, penicillin-streptomycin (Invitrogen), and three cytokines: 20 ng/ml thrombopoietin, 125 ng/ml stem cell factor, and 50 ng/ml Flt-3 ligand (TSF; R&D Systems, Minneapolis, MN, USA). Some wells were supplemented with TSF alone or TSF with 50 ng/ml IL-33 following 200 cGy for 72 h and then collected for total cell counts and CFCs analysis. Some wells were cultured with TSF alone, TSF with 50 ng/ml IL-33, or TSF, IL-33, and 1 μM Ly294002 (Selleck), following 200 cGy for 30 min or 72 h and then collected for phosphorylation AKT-S473 analysis or CFCs assay, respectively. Other wells were cultured with TSF alone, TSF with 50 ng/ml IL-33, or TSF, IL-33, and P53 inhibitor Pifithrin-α (10 μM; Selleck), following 200 cGy for 72 h, and then collected for cell apoptosis analysis.