Human samples

Temporal and occipital human scalp skin was obtained from healthy donors (38–69 years old) undergoing routine face-lift surgery after informed consent and ethical approval (University of Muenster, no. 2015-602-f-S). No sample size calculation was performed. Number of three different donors was used due to the small availability of the tissue used in the study. This number of three was used in many previous studies, given statistical significance.

Tissue specimens

Scalp skin samples were either cut into small pieces (4 mm), embedded into OCT, and frozen in liquid nitrogen, or processed for HF microdissection25.

HF organ culture

Human scalp samples were obtained 1 day after face-lifting procedure (i.e., after overnight transport from collaborating surgeons) and used at the same day for microdissecting human anagen VI scalp HFs. The HF microdissection technique employed for setting up the classical Philpott assay25,26,62 used in the current study, removes all perifollicular tissue with the sole exception of the HF’s dermal sheath, and thus does not contain any other skin appendage structures (e.g., eccrine gland elements)25. Microdissected human scalp HFs were cultured at 37 °C with 5% CO 2 in a minimal media of William’s E media (WEM, Gibco, Life Technologies) supplemented with 2 mM of L-glutamine (Gibco), 10 ng/ml hydrocortisone (Sigma-Aldrich), 10 μg/ml insulin (Sigma-Aldrich), and 1% penicillin/streptomycin mix (Gibco)25,26,62. After microdissection, the HFs were first incubated in WEM for 24 h for re-equilibration. HFs after quality control (fully pigmented and presence in anagen VI phase) were randomly allocated to the different experimental groups.

Chemical stimulation of human microdissected HFs

After 24 h, WEM medium was replaced and HFs were treated with vehicle (0.1% DMSO), Sandalore® (50 and 500 µM; see Fig. 3 and Supplementary Note 1, Symrise), Phenirat® (in a ratio 1:1 to the agonist, Symrise), or Sandalore®+Phenirat® for 6 days for (immuno-)histology or 6 h for qRT-PCR.

For the IGF-1 neutralizing antibody experiments, IGF-1 neutralizing antibody (1 µg/ml, ab9572, Abcam) was added 30 min before adding Sandalore® to the corresponding groups. Culture medium was replaced every second day and after 6 days. HFs were then embedded in cryomatrix (Fisher Scientific), and snap frozen in liquid nitrogen for (immuno-)histology.

SiRNA transfection-knockdown OR2AT4 in organ-cultured HFs

Human anagen VI HFs were transfected using a commercial siRNA reagent system (Sc-45064, Santa Cruz) following the manufacturer’s instructions32,34,35. Briefly, stock solutions (10 µM) of siRNA OR2AT4 (gift from Prof. Hanns Hatt20) and siRNA control (scrambled oligo) were prepared using RNAse-free water. HF transfection was performed 24 h after microdissection for 6 h using either 100 mM OR2AT4 siRNA or control scramble siRNA. After 24 h of incubation with fresh WEM medium, HFs were collected per group in RNA later and stored at 4 °C for further RNA extraction and qRT-PCR analysis or immediately frozen in liquid nitrogen and stored at −80 °C for microarray analysis. Finally, fresh WEM medium was replaced every second day and after 5 days of culture, HFs were snap frozen in OCT for further quantitative (immuno-)histomorphometry analysis.

Histology

For histochemical visualization of melanin, Masson–Fontana staining was performed on frozen sections. Melanin was stained as brown dots26.

Immunofluorescence

OCT-embedded samples were sectioned (6 µm thickness for HF and 7 µm thickness for skin) with a Leica cryostat. For primary OR2AT420 (custom designed rabbit polyclonal antibody generated against the C-terminus sequence of OR2AT4 (Eurogentec, Liège, Belgium)), or cleaved-caspase-3 (#9661, clone Asp175, Cell Signaling) antibodies staining, tissue cryosections were fixed in 4% paraformaldehyde, pre-incubated with 10% of goat serum (for OR2AT4) or 5% goat serum +0.3% Tritton X-100 (for cleaved-caspase 3) and incubated with the corresponding primary antibody at 4 °C overnight (1/100 for OR2AT4 and 1/400 for cleaved-caspase 3). Secondary antibody incubation was performed at RT for 45 min. Counterstaning with DAPI (1 µg/ml) was performed to visualize nuclei.

Dermicidin protein was detected using tissue sections fixed in 4% paraformaldehyde, pre-incubated with 10% of goat serum, and incubated with a mouse anti-human Dermcidin antibody (Novus Biologicals, G-81, 1:200). Secondary antibody (Goat anti-mouse Alexa fluor 488) incubation was performed at room temperature for 45 min. Counterstaning with DAPI (1 µg/ml) was performed to visualize nuclei.

For TGFβ2 (Sc-90, Santa Cruz) and IGF-1 (Sc-1422, clone G-17, Santa Cruz31,32), tissue cryosections were fixed in acetone and endogenous peroxidase activity was blocked with 3% of H 2 O 2 (Merck Milipore). This step was followed by an avidin-biotin blocking step (SP2001, Vectorlabs) and a preincubation with TNB buffer (Tris HCl+NaCl+Casein). The corresponding primary antibody was incubated at 4 °C overnight (1/1000 for TGFβ2 and 1/250 for IGF-1). Secondary antibody incubation was performed at RT for 45 min before using the Tyramide signal amplification kit (NEL700001KT, Perkin Elmer). Counterstaning with DAPI was performed to visualize nuclei.

To stain apoptotic and proliferating cells, we used the apoptag kit (Merck Milipore) following the manufacturer’s protocol followed by Ki-67 staining25,26,33,63. Primary antibody was incubated overnight (Ki-67, M7240 Clone: MIB-1, DAKO, 1/20) after the TdT-enzyme step. The secondary antibody was incubated for 45 min at RT after the fluorescent-labeled anti-Digoxigenin step of the apoptag kit. Counterstaning with DAPI was performed to visualize nuclei. Negative controls were performed by omitting the primary antibody. Images were taken using a Keyence fluorescence microscope BZ9100 (Osaka, Japan) maintaining a constant set exposure time throughout imaging for further analysis.

Quantitative reverse transcriptase-PCR

Total RNA was isolated from whole microdissected HFs using RNeasy Mini Kit (Quiagen) following the manufacturer’s instructions described in the manufacturer’s protocol. RNA purity and concentrations were determined using the Nanodrop ND-1000 assay (Fisher Scientific). Reverse transcription of the RNA into cDNA was performed using the TetrocDNA Synthesis Kit (Bioline), according to the manufacturer’s instructions. RNA concentrations were adjusted between 50 to 500 nM for each sample set to allow further quantification comparison between samples and experiments after qRT-PCR. Controls were performed using the housekeeping gene GAPDH. Real-time quantitative polymerase chain reaction (qRT-PCR) was run in triplicate using TaqMan Fast Advanced Master Mix Product Insert and gene Expression Assay transcripts (Id: Hs01060665_g1 for ACTB, Hs02758991_g1 for GAPDH, and Hs02339277_s1 for OR2AT4, Applied Biosystem) on the qTower2.2 thermocycler. Real-time quantification plots and Ct values were collected and stored by the qPCRsoft2.1 software. The amount of the transcripts was normalized to those of the housekeeping gene using the ΔΔCT method using EXCEL.

Whole-genome microarray analysis

RNA isolation, sample processing, and microarray analyses (Agilent Technologies), as well as statistical evaluation, were performed by Arrows Biomedical GmbH (Muenster, Germany). Expressional alteration was considered to be significant only when ≥1.8-fold and equidirectional changes were observed in at least three of four patients (independent experiments). An additional analysis has been performed using 5-fold and equidirectional changes in the four different donors (independent experiments) in order to identify the top up and downregulated genes.

Human phospho-kinase array

In order to gauge which signaling pathways are regulated by the specific stimulation of OR2AT4, we performed a phospho-kinase array9. Total protein was isolated from whole microdissected HFs using a specific buffer from the Human Phospho-Kinase Array (ARY003B, R&D System), following the manufacturer’s protocol. Briefly, protein extracts were diluted and incubated overnight with the Human Phospho-Kinase Array. The array was washed to remove unbound proteins, followed by incubation with a cocktail of biotinylated detection antibodies. Streptavidin-HRP and chemiluminescent detection reagents were applied, and a signal was produced at each capture spot corresponding to the amount of phosphorylated protein bound.

Western blot analysis

Total protein was extracted from nine anagen and catagen microdissected human scalp HFs. Protein concentrations were determined using a Bradford assay (B6916, Sigma-Aldrich). Thirty micrograms of protein were subjected to 4–15% Mini-PROTEAN® TGX™ Precast gel (#4561083, Bio-Rad) and transferred to a nitrocellulose membrane (88018, Thermo Fisher Scientific), followed by incubation with the corresponding primary antibodies (PA5-71599 for OR2AT4, 1/1000, Thermo Fisher Scientific; and A3853 for Actin, 1/1000, Sigma-Aldrich) overnight at 4 °C. After incubation with peroxidase-conjugated secondary antibodies (WesternBreeze™ Chemiluminescent Kit, WB7106 and WB7104, Thermo Fisher Scientific), the bands were visualized using Chemocam imager 6.0 (Intas, Germany). Protein expression levels were normalized to corresponding actin levels. The uncropped blots are presented in the Supplementary Fig. 8.

Hair cycle score (HCS) and staging

HFs were microscopically evaluated for the hair cycle staging analysis using Masson–Fontana histochemistry and Ki-67/TUNEL immunostainings25,26. The HCS was also measured25,31, which consists of assigning an arbitrary unit for each stage of the hair cycle (Anagen VI = 100; Early catagen = 200; Mid-catagen = 300; and Late catagen = 400). After having classified each HF according to its hair cycle stage, following the previously defined objective classification criteria for organ-cultured human HFs26, for each experimental condition, the mean HCS was calculated. The closer the mean is to 100, the higher is the number of anagen VI HFs in a given group. The HCS provides a global read-out parameter that looks at all HFs in a given experimental group and synthesizes them into a single number, which reflects how close the majority of HFs is to either anagen VI or catagen and also permits statistical analysis that it is not possible with hair cycle staging. Therefore, hair cycle staging and the HCS are independent read-out parameters that complement each other.

Quantitative (immuno-)histomorphometry

Staining intensity was evaluated in well-defined reference areas by quantitative (immuno-)histomorphometry 31,32, using NIH ImageJ software (NIH, Bethesda, MD, USA).

Statistical analyses

All data are expressed as mean ± SEM (and variance is different between the groups) and were analyzed by one-way ANOVA or Kruskall–Wallis test and Dunn’s multiple comparisons test as post hoc test when more than two groups were compared or Student’s t-test or Mann–Whitney test when only two groups were compared (GraphPad Prism 6, GraphPad Software, San Diego, CA, USA) after performing d’Agostino and Pearson omnibus normality test. P < 0.05 was regarded as significant.