Cell culture

Human melanoma cell lines A375 and A2058 were maintained in Dulbecco’s Modified Eagle’s medium (DMEM, Servicebio, Wuhan, China) and A875, SK-MEL-1, and SK-MEL-2 were cultured in Minimum Essential Medium (MEM, Solarbio, Shanghai, China). All mediums were supplemented with 10% fetal bovine serum (FBS, Tianhang Biotech, Zhejiang, China) and cultured at a 37 °C incubator with 5% CO2. Melanoma cells were purchased from Procell (Wuhan, China) or iCell Bioscience (Shanghai, China). Primary human skin melanocytes cells were purchased from iCell Bioscience (Shanghai, China) and cultured with special medium (iCell Bioscience).

Lentiviral vector construction and infection

The specific short hairpin RNAs (shRNAs) targeting KPNB1 were inserted into the pLVX-shRNA1 lentivirus (LV) vector (Fenghui Biotech, Changsha, China) to silence KPNB1 expression. The sequences of shRNA were 5’-cccTTCTCCGAACGTGTCACGTttcaagagaACGTGACACGTTCGGAGAAttttt-5’ (sh-NC); 5’-cccCAGAGCAGCTACAAGATAAATttcaagagaATTTATCTTGTAGCTGCTCTGttttt-3’ (sh1- KPNB1); 5’-cccGCTCAAACCACTAGTTATACAttcaagagaTGTATAACTAGTGGTTTGAGCttttt-3’ (sh2-KPNB1). To upregulate KPNB1 expression, a pLVX-IRES-puro lentivirus vector (Fenghui Biotech) containing a coding sequence of KPNB1 was constructed. The shRNA lentivirus was infected cells at a multiplicity of infection of 150 and the viral titer for the lentivirus vector was 108 TU/ml. After 48 or 72 h infection, cell lines with stable low or high expression of KPNB1 were obtained by the inclusion of antibiotics in the culture medium.

Cells treatment and transfections

For follow-up experiments, cells stably overexpressing or silencing KPNB1 were treated with cisplatin (DDP; 0, 4, 8, 16, 32, or 64 μM; Meilunbio, Dalian, China) for 48 h, treated with cycloheximide (CHX; 12.5 µg/mL; Meilunbio) for 0, 2, 4 or 8 h, or treated with MG132 (10 μM; MedChemExpress, Shanghai, China) for 6 h. Small interfering RNA (siRNA) of G3BP1 was purchased from JST Scientific (Wuhan, China). The sequences were: 5’-GCCACACCAAGAUUCGCCATT-3’ (si-NC); 5’-UGGCGAAUCUUGGUGUGGCTT-3’ (si-G3BP1). The si-NC or si-G3BP1 was transfected into melanoma cells with KPNB1 knockdown/overexpression for 48 h and the transfections were mediated by Lipofectamine 3000 (Invitrogen, Carlsbad, CA, USA).

Real-time quantitative PCR (RT-qPCR)

Quantitative RT-PCR was performed to detect the mRNA level of genes. Total RNA was extracted from cells using TriPure reagent, chloroform, and isopropanol following the manufacturer’s instructions. For mRNA detection, RNA was reverse transcribed by using BeyoRT II M-MLV reverse transcriptase (Beyotime, Shanghai, China), and quantitative RT-PCRs were conducted using gene-specific primers, SYBR Green (Solarbio), and 2 × Taq PCR Master Mix. Quantitative normalization was performed using the expression of β-actin. Relative mRNA expression was calculated using the 2−ΔΔCT method. Primer sequences were provided in Supplementary Table 1.

Western blot

Total protein was obtained from cells and tissues by using RIPA buffer (Beyotime) and phenylmethanesulfonyl fluoride (Beyotime). Nuclear protein was extracted from cells by using a Nuclear Protein Extraction kit (Beyotime) according to the manufacturer’s protocol. Cell lysates (15–30 μg protein) were loaded per lane and separated on SDS-PAGE gels (8%, 10%, or 15% separation gel). Next, the proteins were blotted onto the PVDF membrane. After being treated with 5% bovine serum albumin for 1 h, the membrane was incubated with primary antibodies (Shown in Supplementary Table 2) overnight at 4 °C followed by the incubation of the secondary antibody for 40 min. The secondary antibodies used were as follows: goat anti-rabbit IgG (No. SA00001-2; Proteintech; 1:10000) and goat anti-mouse IgG (No. SA00001-1; Proteintech; 1:10000). After washing with TBST, immunoreactions were visualized by using an ECL reagent solution (7 Sea biotech, Shanghai, China). Quantitative normalization was performed using the expression of β-actin. Histone H3 served as an internal control for nuclear protein detection.

Cell counting kit-8 (CCK-8) assay

The 5 × 103 melanoma cells were seeded into a 96-well plate. At 0, 24, 48, 72, and 96 h or treated with 0, 4, 8, 16, 32, and 64 μM DDP for 72 h, 10 μL CCK-8 (KeyGEN, Jiangsu, China) was added to each well and incubated for 2 h in a 37 °C incubator with 5% CO2. The absorbance at 450 nm was measured by a microplate reader (Biotek, Winooski, VT, USA).

Plate colony formation assay

Cells were seeded in Petri dishes (600 cells per petri dish) and then placed in a 37 °C incubator with 5% CO2 for 2 weeks. After washing with PBS, cells were fixed with 4% paraformaldehyde for 20 min at room temperature and stained with Wright-Giemsa stain solution (KeyGEN) for 5 min. After washing with water, cell count was performed under a microscope (Olympus, Tokyo, Japan).

Flow cytometry assay

Cells were cultured in a 37 °C incubator with 5% CO2 until adhere to the wall. For cell cycle detection, cells were harvested and fixed with ethanol (70%) for 2 h at 4 °C. For cytometry analysis, cells were treated with propidium iodide (PI; 25 μL) and RNase A (10 μL) for 30 min at room temperature in a dark room. Finally, cell cycle distribution was analyzed by a flow cytometer (ACEA Biosciences, San Diego, CA, USA). For apoptosis detection, cells were labeled with Annexin V-FITC (5 μL) and PI (5 μL) using an Apoptosis Detection kit (KeyGEN) according to the manufacturer’s instructions. After incubation for 15 min in the dark, apoptosis was measured by using a flow cytometer (ACEA Biosciences).

Wound healing

After the cells were cultured to the confluent state, 1 μg/mL mitomycin C was added into the medium for 1 h. Next, the monolayer of cells was scratched with a 200 μL pipette tip, and images of the scratch at time 0 h were captured. After the cells were cultured in a serum-free medium for 24 h in a 37 °C incubator with 5% CO2, images of the scratch at the time 24 h were obtained.

Transwell assay

The Transwell chamber was placed into a 24-well plate and coated with Matrigel. Next, cells (3 × 104 per well) were added to the upper chamber. The culture medium (800 μL) supplemented with 10% FBS was added to the lower chamber. After 24 h of incubation, cells were fixed with 4% paraformaldehyde for 25 min and stained with 0.4% crystal violet staining solution for 5 min. Images were obtained using a microscope (Olympus) and the number of migrated cells was counted.

In vivo tumor growth and pulmonary metastasis assay

All the mouse experiments were approved by the Ethics Committee of Shengjing Hospital of China Medical University and followed the National Institutes of Health Guide for the care and use of laboratory animals.

BALB/c nude mice aged 6 weeks were purchased from HFK Biotechnology Co., Ltd. (Beijing, China). For tumor growth, healthy nude mice were randomly divided into 5 groups (n = 8) named sh-NC, sh1-KPNB1, sh2-KPNB1, OV-NC, and OV-KPNB1. Cells (2 × 106 cells) stably overexpressing (SK-MEL-2 cells) or silencing (A375 cells) KPNB1 were subcutaneously into nude mice. Tumor volume was detected every 3 days, 21 days later, tumor tissues were taken out and weighed. The tumor tissues were fixed with 4% paraformaldehyde or stored in liquid nitrogen.

For pulmonary metastasis detection, animal grouping is the same as above. Cells (2 × 106 cells) infected with recombinant pLKO.1-EGFP-puro lentivirus (Fenghui Biotech) with KPNB1 overexpression (SK-MEL-2 cells) or silencing (A375 cells) were injected into nude mice through the tail vein. Seven weeks post-injection, tumor cells in the lung were reacted by a small animal living imager (Multimodal pro Light source 400 W; Bruker, Bremen, Germany). Next, nude mice were euthanized and tumor tissue was removed. The lung tissues were fixed with 4% paraformaldehyde for subsequent experiments. Based on previous studies, SK-MEL-2 and A375 cells were used for in vivo metastasis experiments [21, 22].

Immunohistochemistry (IHC)

Tumor tissues fixed with 4% paraformaldehyde were embedded in paraffin and sectioned (5 μm). Slides were then deparaffinized and rehydrated with graded alcohols. After blocking with normal goat serum for 15 min at room temperature, the slides were incubated with primary antibody against PCNA (No. A0264; Abclonal, Wuhan, China; 1:50) overnight at 4 °C. After washing with PBS, the slides were treated with paired secondary antibody (No. #31460; Thermo Fisher Scientific, Waltham, MA, USA; 1:500) for 1 h at 37 °C. The slides were then incubated with DAB color development reagent (Solarbio) and counterstained with hematoxylin. After dehydration and sealing, the slides were observed and pictured using a microscope (400×, Olympus).

Hematoxylin and eosin (H&E) staining

Lung tissues fixed with 4% paraformaldehyde were sectioned and stained with H&E to observe the lung metastasis of melanoma cells. Images were captured and pulmonary nodules were counted by using a microscope (40×, Olympus). The number of pulmonary nodules in the same area was quantitatively analyzed.

Immunofluorescence (IF) staining

Melanoma cells were fixed with paraformaldehyde (4%) for 15 min. After washing with PBS three times, cells were treated with 0.1% Triton X-100 for 30 min. Then, cells were incubated with normal goat serum (Solarbio) for 15 min at room temperature, followed by the incubation of primary antibody (G3BP1; No. 13057-2-AP; Proteintech; 1:50 or/and KPNB1; No. MAB8209-SP; Novus; 1:50) at 4 °C overnight. Cells were then incubated with goat anti-rabbit IgG labeled with Cy3 (No. A0516; Beyotime; 1:200) or Goat anti-mouse IgG labeled with FITC (NO. SA00003-1; Proteintech; 1:100) for 60 min in the dark at room temperature. After cells were washed in PBS and mounted with DAPI (No. D106471-5mg; Aladdin, Shanghai, China), a fluorescence microscope was used to obtain the images (400×, Olympus). Pearson correlation coefficient was analyzed based on previous studies [23].

Co-immunoprecipitation (co-IP) assay and ubiquitination assay

Proteins were isolated from melanoma cells and melanoma cells with KPNB1 knockdown or overexpression using Western and IP Lysis Solution (Beyotime). Next, the proteins were treated with IP-indicated antibody (1 μg) or paired mouse/rabbit-IgG (1 μg, Beyotime and Proteintech) at 4 °C overnight. Extracted proteins served as input controls. Protein A agarose beads were used to capture antigen-antibody complexes. After washing with PBS, the complexes were resuspended in loading buffer and boiled for 5 min to free the antigen and antibody. After, centrifugation, the supernatants were collected for western blot analysis. Primary antibodies used for co-IP were shown in Supplementary Table 3.

Statistical analysis

Statistical analysis was conducted by using GraphPad Prism 8.0 software. One-way ANOVA combined with Tukey’s post hoc test and unpaired t-test was used to analyze significance among groups. It is acceptable for small sample sizes when the effect size > 0.8 [24,25,26]. Effect sizes were calculated by Post Hoc Power Analysis to confirm the power of the analysis [27, 28]. All data were shown as the mean ± standard deviation (SD). P < 0.05 indicated statistical significance.