Monolayer and three-dimensional cell culture
Human dermal papilla cells (DPCs) were obtained from PromoCell (Heidelberg, Germany). DPCs were cultured with follicle dermal papilla cell growth medium (DPCGM; PromoCell, Heidelberg, Germany). Incubator gas tension was maintained at 21% O2 and 5% CO2 at 37°C. DPCs at passages 3 to 5 were used in the in vitro studies. For three-dimensional cell culture, DPCs (1 × 104 cells/well) were suspended in the culture medium and seeded onto nonadhesive 96-well plates (PrimeSurface Plate 96U, Sumitomo Bakelite Co., Japan). The formed spheroids were collected after brief washing with PBS and then lysed for RNA extraction.
Chemicals and short interfering RNAs (siRNAs)
The HIF-1a activator sildenafil was purchased from Sigma‒Aldrich (St. Louis, MO, USA). The sequences targeting HIF-1a and the control were synthesized by Integrated DNA Technologies Japan (Tokyo, Japan). The design of si-HIF-1a was as follows: 5’-GGGAUUAACUCAGUUUGAACUAACT-3’ and 3’-UACCCUAAUUGAGUCAAACUUGAUUGA-5’.
Informatics analysis
The scalp gene expression in patients with androgenetic alopecia (AGA) was assessed with the NCBI Gene Expression Omnibus (GEO) dataset (www.ncbi.nlm.nih.gov/geo, GSE90594). HIF1A mRNA expression levels (A_24_P56388 at probe) were evaluated between the healthy controls (n = 14) and AGA patients (n = 14). For gene set enrichment analysis (GSEA; www.broadinstitute.org/gsea), the mean value of A_24_P56388 (corresponding to HIF1A) was used as the criterion for categorizing the low- and high-HIF1A expression groups. A false discovery rate (FDR) less than 0.25 was considered statistically significant.
Western blotting
For protein extraction, 2D cultured cells were washed with PBS and sequentially lysed with RIPA lysis buffer (EzRIPA Lysis kit; ATTO, Tokyo, Japan). The cell lysates were incubated at 4°C for 15 min and centrifuged for 10 min at 14,000 × g. The supernatant was collected and mixed with 2× sodium dodecyl sulfate (SDS) sample buffer. The proteins were separated on SDS‒PAGE gels (Bio-Rad, Hercules, CA, USA) and transferred onto Immobilon-P membranes (Merck KGaA, Darmstadt, Germany). The membranes were blocked with 3% BSA in Tris-buffered saline containing 0.1% Tween-20 (TBST) solution for 1 h at room temperature and incubated with primary antibodies (HIF-1a, 1:1000 dilution, Abcam; GAPDH, 1:2000 dilution, Cell Signaling Technology) at 4°C overnight. The membranes were washed three times with TBST solution, incubated with a horseradish peroxidase-conjugated secondary antibody (1:2000 dilution, Cell Signaling Technology) for 1 h and visualized using chemiluminescent reagents (ECL Prime; GE Healthcare, Buckinghamshire, UK) and an Amersham imager 600 RGB (Cytiva, Tokyo, Japan). The relative HIF-1a expression levels were calculated using GAPDH as a reference protein.
Real-time quantitative reverse transcription polymerase chain reaction
Total RNA from spheroids or cultured cells was extracted using the RNeasy Mini Kit (Qiagen, Hilden, Germany), and cDNA synthesis was performed in a ReverTra Ace qPCR RT kit (TOYOBO, Japan). Quantitative real-time PCR (qRT‒PCR) on 96-well optical plates was performed using QuantStudio 3 (Thermo Fisher Scientific) with TB Green Premix EX Taq II (Takara Bio, Shiga, Japan). The sequences for qRT‒PCR primers were designed as follows: VCAN (5’-GGCACAAATTCCAAGGGCAG-3’, 3’- TCATGGCCCACACGATTAACA-5’), LEF1 (5’- CCCGATGACGGAAAGCAT-3’, 3’-TCGAGTAGGAGGGTCCCTTGT-5’), HIF1A (5’- TGCAGAATGCTCAGAGAAAGCGAA-3’, 3’- GCTGCATGATCGTCTGGCTGCT-5’), and GAPDH (5’- GCACCGTCAAGGCTGAGAAC-3’, 3’- TGGTGAAGACGCCAGTGGA-5’). The mRNA values of targeted genes were normalized to the GAPDH expression level.
Gene chip analysis
Total RNA was extracted from DPCs in 2D and spheroid culture at 3 d using the RNeasy Mini Kit (Qiagen) according to the manufacturer’s instructions. Gene expression profiling was performed using GeneChip Human Genome U133 Plus 2.0 arrays (Thermo Fisher Scientific). KEGG analysis was performed using the R package clusterProfiler. The upregulated genes of DPCs in spheroid culture compared to 2D culture were selected with the threshold of Log2FC > 1. The significantly upregulated genes in DPC spheroids were used for Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis with the Database for Annotation, Visualization, and Integrated Discovery (http://david.abcc.ncifcrf.gov/). The top 10 enriched pathways were identified with upregulated and differentially expressed genes (DEGs).
Statistical analysis
All generated data were expressed as the means and standard deviation or standard error of the mean. Two-tailed, paired Student’s t test was conducted to compare differences between the groups using GraphPad Prism 8 software. Differences were considered statistically significant when P < 0.05.