Skin samples used for immunohistochemistry (IHC) were obtained by punch biopsy under local lidocaine anesthesia from depigmented lesions of vitiligo patients (n=6; 2 male and 4 females) aged 16–37 years. All vitiligo patients had been diagnosed by dermatologists and reflectance confocal microscopy, according to published criteria (15,16): Hypopigmentation lesion of the skin with no associated inflammation; wood lamp examination accentuating the vitiligo lesion; biopsy showing complete absence of melanocytes with loss of epidermal pigmentation. Matched normal adult human skin specimens were obtained from 6 healthy adults (2 male and 4 female) aged 18–41 years undergoing plastic surgery.

Blisters (8 mm in diameter) were produced with a vacuum of 40 kPa for 60–90 min in healthy individuals and vitiligo patients (from depigmented areas). Blister roofs were collected and used for the isolation and culture of keratinocytes and protein analysis.

The present study was approved by the Institutional Review Board of the Third People's Hospital of Hangzhou (Hangzhou, China). Informed consent was obtained from each participant.

Skin tissues were fixed in 4% paraformaldehyde at room temperature for 20 min. IHC analysis was performed on 4-µm thick sections from paraffin-embedded clinical samples. Tissue slides were preheated in a 60°C oven for 2 h. The sections were immersed in xylene (I) at room temperature for 30 min, followed by xylene (II) to deparaffinize at room temperature for 10 min. Slides were subsequently immersed into 100% ethanol for 10 min, 95% ethanol for 5 min, 90% ethanol for 2 min, 80% ethanol for 2 min, 70% ethanol for 2 min and double distilled water for 2 min, all at room temperature. Slides were rinsed in PBS-T (0.01M PBS, pH 7.4; 0.02% KH₂PO₄, 0.29% N₂HPO₄, 0.02% KCl, 0.8% NaCl, 0.05% bovine serum albumin (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA), 0.05% Tween-20 and 0.0015% Triton X-100) and subsequently quenched with 3% peroxide-methanol at room temperature for 10 min for endogenous peroxidase ablation. Tissues were blocked in goat serum (1:100) at room temperature for 20 min prior to incubation with rabbit anti-human SUMO1 antibody (cat. no. ab32058; 1:1,000; Abcam, Cambridge, UK) for 2 h at 37°C. Slides were rinsed in PBS-T (3×5 min) and biotin-labeled secondary antibody (cat. no. ab6720; Abcam), 1:500, was added for 30 min at 37°C. After rinsing again in PBS-T (3×5 min), slides were visualized by 3,3-diaminobenzidine tetrahydrochloride at room temperature without light for 10 min (Thermo Fisher Scientific, Inc.). Sections were counterstained using hematoxylin (Thermo Fisher Scientific, Inc.) for 1 min at room temperature. Sliders were dehydrated with sequential ethanol washes of 1 min each starting with 75%, followed by 80% and finishing with a 100% ethanol wash. Images were captured using a BX51 microscope (Olympus Corporation, Tokyo, Japan).

The roofs of the blisters were washed twice with calcium-free Hanks' solution (Gibco; Thermo Fisher Scientific, Inc.), incubated with 0.25% trypsin solution (Gibco; Thermo Fisher Scientific, Inc.) at 37°C for 10 min, followed by incubation with 0.02% EDTA solution (Gibco; Thermo Fisher Scientific, Inc.) at 37°C for 10 min. Cells were separated from the epidermal sheet and observed under a stereo-microscope. The cell suspension was centrifuged (1,000 × g for 10 min at room temperature), resuspended with Medium 154 (Gibco; Thermo Fisher Scientific, Inc.) supplemented with Human Keratinocyte Growth Supplement (1:500; Gibco; Thermo Fisher Scientific, Inc.) with 100 U/ml penicillin and 0.1 mg/ml streptomycin. Cells were incubated in a CO₂-regulated incubator in humidified 95% air/5% CO₂ atmosphere at 37°C for approximately 3 weeks.

HaCat cells (American Type Culture Collection, Manassas, VA, USA) were cultured in DMEM supplemented with 10% fetal bovine serum (Gibco; Thermo Fisher Scientific, Inc.), 100 U/ml penicillin and 0.1 mg/ml streptomycin, in a CO₂-regulated incubator in humidified 95% air/5% CO₂ atmosphere at 37°C. The lentiviral shRNA vectors included empty (cat. no. SHC001), non-targeting control (cat. no. SHC002) and SUMO1 (5′-CGACCAATGCAAGTGTTCATA-3′; cat. no. PCA00244). The empty control consisted of any empty lentiviral vector without any shRNA, while the non-targeting control consisted of a lentiviral vector containing shRNA that does not target any known mammalian genes. Vectors were from the Sigma MISSON shRNA library (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany). HaCaT cells were plated at a density of 5×10⁶ in T75 flasks 20 h prior to infection. Each culture was infected at a MOI of 100 using HiPerFect transfection reagent (Qiagen GmbH, Hilden, Germany). Transduced cells were selected with puromycin and subsequent experiments were performed 72 h after transduction.

Epidermis tissue samples, and patient-derived and HaCaT keratinocytes, were lysed in denaturing radioimmunoprecipitation assay (RIPA) lysis buffer (50 mM Tris, pH 8.0; 150 mM NaCl; 1% NP-40; 1% sodium deoxycholate; 0.1% SDS) supplemented with 20 mM N-ethylmaleimide (Sigma-Aldrich; Merck KGaA) and protease and phosphatase inhibitors (Roche Diagnostics GmbH, Mannheim, Germany). The protein concentration was determined with a bicinchoninic acid (BCA) protein assay. SDS-PAGE (10%) was performed using the mini-gel system (Bio-Rad Laboratories, Inc., Hercules, CA, USA). Proteins were loaded at 20 µg/lane and transferred onto nitrocellulose membranes. The membrane was blocked with 5% non-fat milk in TBS-Tween-20 (TBST) buffer (50 mM Tris-HCl, pH 7.6; 150 mM NaCl; 0.1% Tween-20) for 1 h at room temperature, followed by overnight incubation at 4°C with specific primary antibodies: SUMO1 (cat. no. 4930S; CST Biological Reagents Co., Ltd., Shanghai, China), SENP1 (cat. no. ab108981, Abcam), Ubc9 (cat. no. 4876S; CST Biological Reagents Co., Ltd.), SAE1 (cat. no. 13585S; CST Biological Reagents Co., Ltd.), cyclin-dependent kinase (CDK)2 (cat. no. ab32147; Abcam), CDK6 (cat. no. ab124821; Abcam), proliferating cell nuclear antigen (PCNA; cat. no. ab29; Abcam), retinoblastoma protein (Rb; cat. no. 9309S; CST Biological Reagents Co., Ltd.), phosphorylated Rb (pRb; cat. no. 9308S; CST Biological Reagents Co., Ltd.) and β-actin (cat. no. sc-47778; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) at a 1:1,000 dilution. The membranes were washed with TBST buffer and incubated with IRDye 800CW goat anti-rabbit secondary antibody or IRDye 680 goat anti-mouse secondary antibody (LI-COR Biosciences, Lincoln, NE, USA) at a 1:10,000 dilution for 1 h at room temperature in the dark. The proteins were visualized using an Odyssey Infrared Imaging System (LI-COR Biosciences).

Cell suspension (100 µl/well) of SUMO1 and non-targeting control shRNA-transduced HaCaT cells were seeded in 96-well plates at 5,000 cells/well. HaCaT cells were routinely cultured in the laboratory of the present study. This cell line is a spontaneously transformed aneuploid immortal keratinocyte cell line from adult human skin and is commonly used to investigate the function of keratinocytes. The plates were preincubated in an incubator at 37°C under 5% CO₂. Cell Counting Kit-8 (CCK-8) solution (10 µl/well; Dojindo Molecular Technologies, Inc., Kumamoto, Japan) was added to each well. The plate was incubated for 1–4 h at 37°C. The absorbance at a wavelength of 450 nm was measured using a microplate reader.

Keratinocytes obtained from patients with vitiligo and healthy volunteers were lysed with RIPA buffer supplemented with protease and phosphatase inhibitors (Roche Diagnostics GmbH) at 4°C for 30 min. Protein concentration was determined using the BCA protein assay following centrifugation at 12,000 × g and 4°C for 10 min. 1 mg Proteins (1 mg) were incubated with 2 µg SUMO1 antibody (cat. no. 4930S; CST Biological Reagents Co., Ltd.) at 1:100 dilution for 16 h at 4°C with gentle inversion mixing. Subsequently, 50 µl protein A/G sepharose beads (Abmart, Inc., Berkeley Heights, NJ, USA) were added and incubated for 3 h at 4°C. The beads were collected and washed four times with RIPA buffer. The immunoprecipitated proteins were eluted by 1X SDS sample buffer (Beyotime Institute of Biotechnology, Shanghai, China) and SUMO-1 conjugated Rb was detected by western blotting with Rb primary antibody (cat. no. 9309S; CST Biological Reagents Co., Ltd.) as described above.

For cell cycle analysis, HaCaT keratinocytes were seeded at a density of 1×10⁶ cell/well in 6-well plates and fixed with prechilled 70% ethanol overnight at 4°C following transduction. Prior to analysis, cells were centrifuged (1,000 × g for 10 min at room temperature) and resuspended in 1 ml DNA staining solution (PBS containing 50 mg/ml propidium iodide and 200 mg/ml RNaseA) and 10 µl permeabilization solution at room temperature for 30 min in the dark. Both DNA staining and permeabilization solution were included in the Cell Cycle Staining kit (Hangzhou MultiSciences Biotech Co., Ltd., Hangzhou, China). Cells were incubated at 37°C for 30 min and immediately assayed on a flow cytometer (FACS Calibur; BD Biosciences, Franklin Lakes, NJ, USA) with ModFit LT (version 4.1; Verity Software House, Inc., Topsham, ME, USA).

All data are presented as the mean ± standard deviation of three independent experiments. Data were analyzed using unpaired Student's t-tests. SPSS version 17.0 (SPSS, Inc., Chicago, IL, USA) was used for all analyses. P<0.05 was considered to indicate a statistically significant difference.

To analyze the SUMOylation in the keratinocytes of patients with vitiligo, the current study collected punch biopsies from vitiligo lesions of patients and skin specimens from healthy adults. SUMO1-conjugated proteins in epidermis tissues were investigated via IHC and western blotting. The results revealed decreased levels of SUMO1-conjugated proteins in the depigmented lesion tissues of patients with vitiligo compared with healthy control samples (Fig. 1A-C). These results indicate that SUMO1 conjugation may be dysregulated in the depigmented lesions of patients with vitiligo. To confirm this at the cellular level, primary keratinocytes were cultured from vitiligo lesions (KVs) and adult healthy skin (KNs) and western blot analysis was performed. The results demonstrated that SUMO1-conjugated proteins were markedly decreased in KVs compared with KNs (Fig. 1D). In addition, the protein expression of Ubc9, which is a SUMO-activating and -conjugating enzyme, was also markedly decreased in KVs compared with KNs, whereas no marked alteration in the expression of SAE1 was detected between the two groups. By contrast, the protein levels of SENP1, one of the SUMO-specific proteases involved in deSUMOylation, was increased in KVs compared with KNs (Fig. 1E). In summary, these results indicated that vitiligo lesions may exhibit dysregulated SUMOylation and deSUMOylation.

Western blot analysis demonstrated that following transduction of HaCaT cells with SUMO1 shRNA knockdown vectors, the expression of SUMO1 was partially knocked-down compared with non-transduced, empty vector and non-targeting shRNA control groups (Fig. 2A). The cell proliferation rate was determined by a CCK-8 assay 72 h after transduction. The cell viability of SUMO1-knockdown HaCaT cells was demonstrated to be reduced compared with control cells transduced with non-targeting shRNA vectors at 48, 72 and 96 h (Fig. 2B). Furthermore, flow cytometry analysis demonstrated that the cell cycle was arrested in G1 phase in SUMO1-knockdown HaCaT cells compared with control HaCaT cells transduced with non-targeting shRNA (Fig. 2C and D). In addition, western blotting revealed decreased levels of CDK2, PCNA, CDK6 and pRb in SUMO1 knockdown HaCaT cells compared with control HaCaT cells transduced with non-targeting shRNA (Fig. 2E). PCNA is an important indicator of cell proliferation status. CDK2 and CDK6 belong to the CDK family, which drive the cell cycle through binding to their activating cyclins (17). Rb is a tumor suppressor, and its phosphorylation is essential for G0/G1 to S transition (18). These results indicate that dysregulation of cell cycle progression may be present in SUMO1 knockdown HaCaT human keratinocytes.

To investigate the SUMOylation status of proteins associated with the cell cycle in keratinocytes from lesions of patients with vitiligo, cell lysates were subjected to immunoprecipitation with an anti-SUMO1 antibody and Rb antibody. It was demonstrated that SUMOylated Rb was markedly decreased in KVs compared with KNs, whereas the expression of Rb in cell lysates from both groups was equal (Fig. 3). These results indicate that the deSUMOylation of Rb in keratinocytes may serve an important role in vitiligo.