The Therapeutic Aryl Hydrocarbon Receptor-Modulating Agent Tapinarof Regulates SEMA3A Expression in Human Keratinocytes through NRF2

Semaphorin 3A (SEMA3A), a nerve repellent factor, is secreted by keratinocytes. Nerve density in the epidermis is associated with pruritis in atopic dermatitis (AD). Reduced epidermal SEMA3A levels reportedly cause nerve fiber elongation, resulting in intractable pruritis (Tominaga et al., 2014). SEMA3A treatments attenuate pruritis and skin inflammation (Negi et al., 2012Negi O. Tominaga M. Tengara S. Kamo A. Taneda K. Suga Y. et al.Topically applied SEMAphorin 3A ointment inhibits scratching behavior and improves skin inflammation in NC/Nga mice with atopic dermatitis.J Dermatol Sci. 2012; 66 (Epub Jan 21, 2012. PMID: 22381253): 37-43https://doi.org/10.1016/j.jdermsci.2012.01.007Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar; Yamaguchi et al., 2009). Furthermore, SEMA3A modulates functions of activated T cells, dendritic cells, and macrophages (Kiseleva et al., 2022). Therefore, SEMA3A upregulation in keratinocytes may aid the treatment of AD. To date, calcium-induced keratinocyte differentiation (Kamata et al., 2020Kamata Y. Tominaga M. Umehara Y. Honda K. Kamo A. Moniaga C.S. et al.Calcium-inducible MAPK/AP-1 signaling drives SEMAphorin 3A expression in normal human epidermal keratinocytes.J Invest Dermatol. 2020; 140 (Epub Jan 13, 2020. PMID: 31945349): 1346https://doi.org/10.1016/j.jid.2020.01.001Abstract Full Text Full Text PDF PubMed Scopus (6) Google Scholar) and baicalein (Yoshioka et al., 2021Yoshioka Y. Kamata Y. Tominaga M. Umehara Y. Yoshida I. Matsuoka N. et al.Extract of Scutellaria baicalensis induces SEMAphorin 3A production in human epidermal keratinocytes.PLoS One. 2021; 16 (PMID: 33905439; PMCID: PMC8078742)e0250663https://doi.org/10.1371/journal.pone.0250663Crossref Scopus (0) Google Scholar), have been identified as SEMA3A upregulators in human keratinocytes. Given that aryl hydrocarbon receptor (AHR), a ligand-activated transcription factor, mediates keratinocyte differentiation (Furue et al., 2018Furue M. Hashimoto-Hachiya A. Tsuji G. Antioxidative phytochemicals accelerate epidermal terminal differentiation via the AHR-OVOL1 pathway: Implications for atopic dermatitis.Acta Derm Venereol. 2018; 98 (PMID: 29972223): 918-923https://doi.org/10.2340/00015555-3003Crossref PubMed Scopus (30) Google Scholar) and that baicalein activates AHR (Li et al., 2021), we hypothesized that AHR may regulate SEMA3A expression in human keratinocytes. To test this, we analyzed SEMA3A expression in cultured normal human epidermal keratinocytes (NHEKs) treated with tapinarof, a therapeutic AHR-modulating agent. Tapinarof has been developed as a topical agent for psoriasis and AD (Furue et al., 2019Furue M. Hashimoto-Hachiya A. Tsuji G. Aryl hydrocarbon receptor in atopic dermatitis and psoriasis.Int J Mol Sci. 2019; 20 (PMID: 31683543; PMCID: PMC6862295): 5424https://doi.org/10.3390/ijms20215424Crossref Scopus (93) Google Scholar; Bissonnette et al., 2020) and improved pruritis and skin inflammation in a clinical study of AD (Paller et al., 2021Paller A.S. Stein Gold L. Soung J. Tallman A.M. Rubenstein D.S. Gooderham M. Efficacy and patient-reported outcomes from a phase 2b, randomized clinical trial of tapinarof cream for the treatment of adolescents and adults with atopic dermatitis.J Am Acad Dermatol. 2021; 84 (Epub Jun 2, 2020. PMID: 32502588): 632-638https://doi.org/10.1016/j.jaad.2020.05.135Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). We have reported that tapinarof treatment (up to 0.5 μM) induces AHR activation without cytotoxicity in NHEKs (Tsuji et al., 2022Tsuji G. Hashimoto-Hachiya A. Matsuda-Taniguchi T. Takai-Yumine A. Takemura M. Yan X. et al.Natural compounds tapinarof and Galactomyces ferment filtrate downregulate IL-33 expression via the AHR/IL-37 axis in human keratinocytes.Front Immunol. 2022; 13 (PMID: 35663970; PMCID: PMC9161696)745997https://doi.org/10.3389/fimmu.2022.745997Crossref Scopus (4) Google Scholar). We treated NHEKs with tapinarof (0.5 μM) for 24, 48, and 72 h and evaluated SEMA3A mRNA levels. Since SEMA3A expression is modified by calcium-induced keratinocyte differentiation (Kamata et al., 2020Kamata Y. Tominaga M. Umehara Y. Honda K. Kamo A. Moniaga C.S. et al.Calcium-inducible MAPK/AP-1 signaling drives SEMAphorin 3A expression in normal human epidermal keratinocytes.J Invest Dermatol. 2020; 140 (Epub Jan 13, 2020. PMID: 31945349): 1346https://doi.org/10.1016/j.jid.2020.01.001Abstract Full Text Full Text PDF PubMed Scopus (6) Google Scholar), we administered tapinarof under low (0.15 mM) (Figure 1A) or high (1.4 mM) (Figure 1B) calcium concentrations. Tapinarof treatment increased SEMA3A mRNA levels at 72 h under both calcium concentrations compared with vehicle. Since SEMA3A expression is high in the basal layer of the epidermis and gradually decreases with keratinocyte differentiation (Kamata et al., 2020Kamata Y. Tominaga M. Umehara Y. Honda K. Kamo A. Moniaga C.S. et al.Calcium-inducible MAPK/AP-1 signaling drives SEMAphorin 3A expression in normal human epidermal keratinocytes.J Invest Dermatol. 2020; 140 (Epub Jan 13, 2020. PMID: 31945349): 1346https://doi.org/10.1016/j.jid.2020.01.001Abstract Full Text Full Text PDF PubMed Scopus (6) Google Scholar), we performed subsequent experiments under low calcium concentrations. Tapinarof treatment (0.1, 0.2, and 0.5 μM) for 72 h increased SEMA3A mRNA levels in a dose-dependent manner (Figure 1C). Furthermore, we analyzed SEMA3A protein levels in NHEKs treated with tapinarof (0.5 μM) for 72 h using western blotting (Figure 1D) and immunofluorescent staining (Supplementary Figure S1A and S1B). We observed increased SEMA3A protein levels in the tapinarof-treated NHEKs. In addition, tapinarof treatment reduced mRNA levels of nerve growth factor (NGF) but not artemin (ARTN), axon guidance molecules (Figure 1E and 1F). We observed increased SEMA3A production in the culture supernatant (Figure 1G). Moreover, we confirmed increased SEMA3A production in the culture supernatant of three-dimensionally cultured NHEKs treated with tapinarof (0.5 μM) added to the culture medium for 72 h (Figure 1H). Since tapinarof is a dual activator of AHR and nuclear factor E2-related factor 2 (NRF2) (Smith et al., 2017Smith S.H. Jayawickreme C. Rickard D.J. Nicodeme E. Bui T. Simmons C. et al.Tapinarof is a natural AhR agonist that resolves skin inflammation in mice and humans.J Invest Dermatol. 2017; 137 (Epub Jun 6, 2017. PMID: 28595996): 2110-2119https://doi.org/10.1016/j.jid.2017.05.004Abstract Full Text Full Text PDF PubMed Scopus (188) Google Scholar) (Supplementary Figure S2A–I), we examined whether AHR and NRF2 are involved in the tapinarof-induced SEMA3A upregulation. We evaluated SEMA3A mRNA and protein levels in NHEKs transfected with siRNA against AHR or NRF2 and treated with tapinarof (0.5 μM) for 72 h. We confirmed that the siRNA transfection successfully knockdowned AHR and NRF2 (Supplementary Figure S1C–S1F). Knockdown of AHR or NRF2 canceled the increases in SEMA3A mRNA and protein levels induced by tapinarof treatment (Figure 1I, 1J, Supplementary Figure S1G, and S1H). Tapinarof-induced SEMA3A production in culture supernatant was also reduced by knockdown of AHR and NRF2 (Figure 1K). We reported that AHR is required for NRF2 activation since AHR knockdown by siRNA transfection inhibited nuclear translocation of NRF2 induced by ketoconazole, an AHR ligand (Tsuji et al., 2012Tsuji G. Takahara M. Uchi H. Matsuda T. Chiba T. Takeuchi S. et al.Identification of ketoconazole as an AhR-Nrf2 activator in cultured human keratinocytes: the basis of its anti-inflammatory effect.J Invest Dermatol. 2012; 132 (Epub Jul 14, 2011. PMID: 21753779): 59-68https://doi.org/10.1038/jid.2011.194Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar). To examine whether AHR is involved in NRF2 activation by tapinarof treatment, we treated AHR-knockdown NHEKs with tapinarof (0.5 μM) for 18 h and evaluated NRF2 localization using immunofluorescence staining. Tapinarof treatment induced the nuclear translocation of NRF2 in scramble siRNA-transfected NHEKs, which was partially inhibited in AHR-knockdown NHEKs (Supplementary Figure S3A, and S3B). Western blotting analysis of nuclear NRF2 expression supported this result (Supplementary Figure S3C, and S3D). These findings indicate that tapinarof treatment upregulates SEMA3A via the AHR-NRF2 axis, although the suppression of SEMA3A expression in AHR-knockdown NHEKs was partial, suggesting that tapinarof may also induce NRF2 activation beyond the AHR-NRF2 axis. We further examined whether the SEMA3A promoter region (−1450/+1 bp) contains DNA sequences that bind to ARNT (AHR nuclear translocator) and NRF2 (antioxidant response element: ARE). Since we found that ARNT binding sites were located at −623, −99, and −90, and AREs were located at −1244, −616, and −521, we performed a luciferase assay using constructs containing them (Figure 2A). Treatment with tapinarof (0.5 μM) for 48 h increased luciferase induction in NHEKs transfected with the −1450/+1 bp, −1030/+1 bp, −509/+1 bp, −409/+1 bp, −309/+1 bp, and −209/+1 bp constructs, which was inhibited in NHEKs transfected with the −109/+1 bp construct, suggesting that the sequence between −209 and −109 bp may contain the binding sites with ARNT and NRF2 (Figure 2A). The constructs including ARNT (−623, −99, and −90) did not affect the luciferase activity, suggesting that ARNT is unlikely to bind to these sites. We further found ARE-like loci (ARELs) at −196 (AREL1: TGAAGTTTC) and −114 (AREL2: TGAAACTGA), which possibly bind to NRF2 (Figure 2B). To test this, we analyzed luciferase induction of tapinarof-treated NHEKs transfected with constructs containing mutations in AREL1 (mut-AREL1: GATAGTTTC) and AREL2 (mut-AREL2: GATAACTGA), respectively. The increase in luciferase induction by tapinarof was reduced in NHEKs transfected with the construct containing mut-AREL2 (Figure 2C), suggesting that NRF2 may bind to AREL2. Finally, we extracted chromatin from NHEKs treated with tapinarof (0.5 μM) for 48 h and analyzed NRF2 binding to AREL2 using ChIP assay. We confirmed that NRF2 activated by tapinarof treatment bound to AREL2, not AREL1 (Figure 2D). We also observed that baicalein increased SEMA3A mRNA levels via the AHR-NRF2 axis and that luciferase induction by baicalein was decreased in NHEKs transfected with the construct lacking AREL2 as well as tapinarof (Supplementary Figure S4A–S4C). Accordingly, tapinarof upregulates SEMA3A in NHEKs and AREL2 is a critical binding site in SEMA3A’s promoter region. All data generated or analyzed during this study are included in this published article and its supplementary information files. The authors declare no conflicts of interest. Conceptualization: GT, AY, KY, MT, MKN, TI, TN; Data Curation: GT, AY, MT; Formal Analysis: GT, AY, MT; Funding Acquisition: GT; Investigation: GT; Methodology: GT, AY, KY, MT; Project Administration: GT, AY, TN; Resources: GT; Visualization: GT, AY; Writing – Original Draft Preparation: GT; Writing – Review and Editing: GT, AY, KY, MT, MKN, TI, TN. Bissonnette et al., 2021Bissonnette R. Stein Gold L. Rubenstein D.S. Tallman A.M. Armstrong A. Tapinarof in the treatment of psoriasis: A review of the unique mechanism of action of a novel therapeutic aryl hydrocarbon receptor-modulating agent.J Am Acad Dermatol. 2021; 84 (Epub Nov 3, 2020. PMID: 33157177): 1059-1067https://doi.org/10.1016/j.jaad.2020.10.085Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar, Kiseleva and Rutto, 2022Kiseleva E.P. Rutto K.V. 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Hida T. et al.SEMAphorin3A alleviates skin lesions and scratching behavior in NC/Nga mice, an atopic dermatitis model.J Invest Dermatol. 2008; 128 (Epub Jul 10, 2008. PMID: 18615113): 2842-2849https://doi.org/10.1038/jid.2008.150Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar. This research was funded by a grant from the Ministry of Health, Labour and Welfare of Japan (H30-Shokuhin-Shitei-005). Tapinarof (MedChemExpress, Monmouth Junction, NJ, USA) and baicalein (MedChemExpress) were dissolved in DMSO and stored at −80°C until use. Anti-SEMA3A antibody (ab23393) and anti-NRF2 antibody [EP1808Y] were from Abcam (Cambridge, UK), anti-Histone H3 antibody (#4620), anti-ACTB antibody (#4970), anti-AHR antibody [D5S6H], anti-rabbit IgG HRP-linked antibody (#7074), and anti-mouse IgG HRP-linked antibody (#7076) were from Cell Signaling Technology (Danvers, MA, USA) and anti-α-Tubulin antibody [B-7] was from Santa Cruz Biotechnology (Dallas, TX, USA). Normal human epidermal keratinocytes (NHEKs) purchased from Lonza (Basel, Switzerland) were grown with the KGM Gold keratinocyte growth medium bullet kit (#00192060, Lonza) at 37 °C in 5% CO2 and used within four passages. On the day of each experiment, the culture medium was changed to KGM Gold growth medium (#00192151, Lonza) containing 0.15 mM calcium supplied with bovine pituitary extract, insulin, transferrin, and epinephrine (all from Lonza). CaCl2 (Sigma-Aldrich, St. Louis, MI, USA) was used to adjust the concentration of calcium in the culture medium to 1.5 mM. Three-dimensional cultured keratinocytes (#EPI-200) and medium (EPI-100MN-HCF) were purchased from Kurabo (Osaka, Japan). Total RNA was extracted from the cultured cells with an RNeasy Mini Kit (Qiagen, Hilden, Germany) and reverse-transcribed with PrimeScript RT Reagent Kit (Takara Bio, Kusatsu, Japan). Real-time PCR was performed on CFX connect machine (Bio-Rad Laboratories, Hercules, CA, USA) using the TB Green Premix Ex Taq (Takara Bio) or TaqMan Fast Advanced Master Mix (Thermo Fisher Scientific, Waltham, MA, USA). The primers and probes were purchased from Thermo Fisher Scientific and are shown in Supplementary Table 1. mRNA expression was measured in triplicate wells and normalized using ACTB as a housekeeping gene. Small interfering RNA (siRNA) targeting AHR (s1200), NRF2 (s9492), or scrambled RNA (Silence Negative Control No. 1) (all from Thermo Fisher Scientific) was transfected into NHEKs with Lipofectamine RNAi Max (Thermo Fisher Scientific) following the manufacturer’s instructions. To prepare whole-cell lysate, cells were rinsed with ice-cold PBS and lysed with RIPA buffer supplied with PhosSTOP (Sigma-Aldrich) and protease inhibitor cocktail (Sigma-Aldrich). Protein concentration was analyzed with a BCA assay kit (Thermo Fisher Scientific) and 10 μg of protein was used for SDS-PAGE. Cytoplasmic or nuclear lysates were prepared using NE-PER nuclear and cytoplasmic extraction reagents (Thermo Fisher Scientific). Equal amounts of lysate (5 μL/lane) were applied to Blot 4-12% Bis-Tris Plus Gel (Thermo Fisher Scientific). After electrophoresis, proteins were transferred onto an Immobilon-P membrane (Merck Millipore, Burlington, MA, USA) and reacted with primary antibodies diluted in Can Get Signal (Toyobo Co., Ltd., Osaka, Japan) at room temperature for 1 h, or alternatively at 4°C overnight. Rinsed membrane was subjected to a secondary antibody reaction. Bands were detected with Super Signal West Pico (Thermo Fisher Scientific) using ChemiDoc XRS or ChemiDo Touch (both from Bio-Rad) and densitometric analysis was performed. For AHR staining, NHEKs were immobilized with ice-cold acetone. For SEMA3A, NRF2, and tubulin staining, NHEKs harvested on poly-L-lysine-coated glass were immobilized with 4% paraformaldehyde. After washing with PBS, cells were permeabilized with 0.05% Triton X-100 for 10 min. After the immobilization, cells were blocked with 5% bovine serum albumin and stained with primary antibodies at 4°C overnight. After washing three times with PBS, cells were reacted with Alexa Fluor 488 and Alexa Fluor 546 secondary antibodies (Thermo Fisher Scientific) in the dark. After washing with PBS, glass was covered with cell-mounting medium containing DAPI (4',6-diamidino-2-phenylindole) (Thermo Fisher Scientific) and images were taken with a Nikon Eclipse Ti inverted confocal microscope (Nikon Corporation, Tokyo, Japan) or SP8 LIGHTNING confocal microscope (Leica, Wetzlar, Germany). The total SEMA3A particles were counted and normalized to total cell number in the field using ImageJ software (National Institutes of Health, Bethesda, MD, USA). Three randomly selected fields were analyzed for each group considered. The mean count per cell for each condition was used for statistical analysis. A search for putative NRF2 transcription factor binding sites was performed in JASPAR (http://jaspar.genereg.net/). The promoter region of SEMA3A (−1450/+1) was amplified with PrimeSTAR Max DNA Polymerase (Takara Bio Inc.) using primers listed in Table 1, and cloned into pGL4.14 (Promega, Madison, WI, USA) using In-Fusion HD Cloning Kit (Takara Bio Inc.), in accordance with the manufacturer’s instructions. Site-directed mutagenesis to generate mutant constructs was performed with the primers listed in Supplementary Table 1. The constructs were verified using DNA sequencing by GENEWIZ (South Plainfield, NJ, USA). NHEKs were seeded at 1 × 104 per well using 96-well plates and cultured overnight. The following day, cells were transiently transfected with 0.1 μg of plasmid DNA using X-tremeGENE HP DNA transfection reagent (Roche Applied Science, Penzberg, Germany). To calculate transfection efficiency, all cells were co-transfected with the pSV-β-galactosidase control vector (Promega, Madison, WI, USA). The cells were reacted with ONE-Glo luciferase assay buffer (Promega) and luciferase activity was measured by an Infinite 200 PRO microplate reader (Tecan Group Ltd., Männedorf, Switzerland). Alternatively, cells were lysed with reporter lysis buffer (Promega) and frozen for more than 2 h at <−70 °C, following the manufacturer’s instructions. The cells were thawed at room temperature and luciferase activity was analyzed as mentioned above. To measure β-galactosidase activity, reporter lysis buffer-lysed cells were reacted with 4 mM chlorophenol red-β-d-galactopyranoside for more than 6 h and absorbance at 570 nm was used to normalize the luciferase activity. Fold luciferase induction was expressed as the ratio of the induction in mock vector-transfected cells. ChIP assays were performed with SimpleChIP Plus Enzymatic Chromatin IP Kit (Cell Signaling Technology). NHEKs were treated with 0.5 μM tapinarof or vehicle control for 24 h and fixed with 1% paraformaldehyde for 10 min at room temperature. Chromatin was sheared by the combination of enzymatic digestion by micrococcal nuclease, contained in the kit, and sonication by Bioruptor II (Sonicbio Co., Ltd., Kanagawa, Japan). A total of 5 μg of digested chromatin was reacted with 0.4 μg of anti-NRF2 antibody or rabbit control IgG overnight at 4°C with rotation, and samples were precipitated with protein A agarose. DNA was purified from input and precipitated samples and subjected to qPCR. Primers are shown in Supplementary Table 1. The culture supernatant was collected and stored immediately at −80°C. The concentration of secreted SEMA3A was measured by ELISA kit as manufacturer’s protocol (Cloud-clone, TX, USA). We employed an unpaired two-tailed t-test, and Dunnett’s or Tukey’s multiple comparison tests. All statistical analyses were performed using the JMP Pro software package (SAS Institute Japan Ltd., Tokyo, Japan). Supplementary Figure S2Tapinarof activated AHR and NRF2 in NHEKs. NHEKs were treated with vehicle or tapinarof (0.5 μM) for 1 h. AHR expression was analyzed using immunofluorescence staining of AHR (red), while DAPI (blue) was utilized for nuclear staining. The tapinarof treatment induced nuclear translocation of AHR (a). qRT-PCR analysis of CYP1A1 mRNA levels in NHEKs treated with tapinarof. CYP1A1 is a representative gene mediated by AHR activation (b and c) (Tsuji et al., 2012). NHEKs were treated with vehicle or tapinarof (0.5 μM) for 18 h. NRF2 expression was analyzed using immunofluorescence staining of NRF2 (green) and DAPI (blue). Tapinarof treatment induced nuclear translocation of NRF2 (d). qRT-PCR analysis of NQO-1 mRNA levels in NHEKs treated with tapinarof. NQO-1 is a representative gene mediated by NRF2 activation (e and f) (Tsuji et al., 2012). Western blotting analysis of nuclear AHR and NRF protein levels (g). Densitometric analysis of nuclear AHR (h) and NRF2 protein levels (i). Data are representative of experiments repeated three times with similar results (a, d, and g). Bar: 20 μm (a and d). Data are expressed as mean ± S.D.; N = 3/group. Significant difference between vehicle-treated and tapinarof-treated groups (*p < 0.05, **p < 0.01) (b, c, e, f, h and i).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Supplementary Figure S3Tapinarof upregulated SEMA3A via the AHR-NRF2 axis. NHEKs were transfected with either scramble (scramble) or siRNA of AHR (siAHR) and then treated with tapinarof (0.5 μM). NRF2 and AHR expression was analyzed using immunofluorescence staining of NRF2 (green) and AHR (red), while DAPI (blue) was utilized for nuclear staining. Tapinarof treatment induced nuclear translocation of NRF2 in scramble-transfected NHEKs, which was canceled in siAHR-transfected NHEKs (a). Transfection of siAHR abolished AHR expression (b). NHEKs were transfected with either scramble (scramble) or siRNA of AHR (siAHR) and then treated with tapinarof (0.5 μM) for 1, 3, 6, 18, and 24 h. Western blotting analysis of nuclear AHR and NRF protein levels (c). The tapinarof treatment induced an increase of nuclear NRF2 protein levels in scramble-transfected NHEKs, which was inhibited in siAHR-transfected NHEKs (c). Densitometric analysis of nuclear NRF2 protein levels (d). Data are representative of experiments repeated three times with similar results (a–c). Bar: 20 μm (a and b). Data are expressed as mean ± S.D.; N = 3/group. Significant difference between the indicated groups (#p < 0.05, ##p < 0.01) and between vehicle-treated and tapinarof-treated groups (*p < 0.05, **p < 0.01) (d).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Supplementary Figure S4Baicalein upregulated SEMA3A via AREL2. qRT-PCR analysis of SEMA3A mRNA (a and b). NHEKs were treated with vehicle, tapinarof (0.5 μM), or baicalein (10 μM) for 72 h. Both tapinarof and baicalein treatments increased SEMA3A mRNA levels. Data are expressed as mean ± S.D.; N = 3/group. A significant difference between the vehicle-treated group and tapinarof-treated group (*p < 0.05, **p < 0.01). NHEKs with either AHR or NRF2 knockdown were treated with vehicle, tapinarof (0.5 μM), or baicalein (10 μM) for 72 h. Knockdown of AHR or NRF2 canceled the increase in SEMA3A mRNA induced by treatment with baicalein as well as tapinarof (b). Luciferase assay on SEMA3A promoter activity (c). Mutation of AREL2 inhibited luciferase induction in NHEKs treated with baicalein for 48 h. Data are expressed as mean ± S.D.; N = 3/group. Significant difference between the indicated groups (*p < 0.05, **p < 0.01). a: Significant difference between scramble-transfected NHEKs treated with tapinarof and siAHR-transfected NHEKs treated with tapinarof. b: Significant difference between scramble-transfected NHEKs treated with baicalein and siNRF2-transfected NHEKs treated with baicalein (*p < 0.05, **p < 0.01).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Supplementary Table 1▪List of primers for qPCRGene symbolSpeciesForward primer (5’-3’)Reverse primer (5’-3’)ACTBHomo sapienscaccaactgggacgacatacagcctggatagcaacgAHRHomo sapienscaaatccttccaagcggcatacgctgagcctaagaactgaaagCYP1A1Homo sapienstagacactgatctggctgcaggggaaggctccatcagcatcNRF2Homo sapienstcagcgacggaaagagtatgaccactggtttctgactggatgtNQO-1Homo sapiensgaagagcactgatcgtactggcggatactgaaagttcgcagggNGFHomo sapienscatgctggacccaagctcacctgcagggacattgctctcARTNHomo sapiensatgaacactacagtggctgaggagctcccatgagtgagtacaggSEMA3A_AREL1Homo sapienscctcattatccggtgcctgtcaagacctcatggcaacactSEMA3A_AREL2Homo sapiensagtgttgccatgaggtcttgactgtattgtgcggccagagaNQO- 1_AREHomo sapiensagtggcatgcacccagggaaagggtggaagtcgtcccaagaList of probes for qPCRGene symbolSpeciesProbe #ACTBHomo sapiensHs99999903_m1SEMA3AHomo sapiensHs00173810_m1List of primers used to construct promoter assay vectorsPrimer namePrimer sequence (5’-3’)SEMA3A_prom_-1450_FwacctgagctcgctaggattcttgtaaaaatatcagctttgSEMA3A_prom_-1030_FwacctgagctcgctaggaactgattgttggaatgggSEMA3A_prom_-509_FwacctgagctcgctagccctctacatgcgtacaggSEMA3A_prom_-409_FwacctgagctcgctagtcattctgcttccccggagcSEMA3A_prom_-309_FwacctgagctcgctagcactgcagcagaccttgtSEMA3A_prom_-209_FwacctgagctcgctagtttttggagggtttgaagtttctgSEMA3A_prom_-109_FwacctgagctcgctagctgacctaaatcacctgttacctccSEMA3A_prom_+1_RvccggattgccaagctgctgcagacgctgtaggtcSEMA3A_Mut_ARE1_FwagggttgatagtttctgtgcttcagSEMA3A_Mut_ARE1_RvgaaactatcaaccctccaaaaactagcSEMA3A_Mut_ARE2_FwtatgaagataactgacctaaatcacctSEMA3A_Mut_ARE2_Rvtcagttatcttcataaatgcagacaat Open table in a new tab