Fig 2: All-trans retinoic acid downregulates the expression of OR7A17. (A,B) The expression of OR7A17 in HaCaT cells was determined via reverse transcription quantitative PCR (RT-qPCR) (A) and Western blot analysis (B). In addition, OR7A17 expression was determined in human dermal fibroblasts and human epidermal melanocytes (B). HDFa: human dermal fibroblasts, HEMn: human epidermal melanocytes. (C) After ATRA treatment for 24 h, the cells were lysed and subjected to OR7A17 luciferase reporter assays. (D) Changes in the mRNA levels of OR7A17 in HaCaT cells were observed following treatment with ATRA for 24 h. (E) Changes in OR7A17 protein levels in HaCaT cells treated with ATRA for 24 h were determined via Western blot analysis. Data are presented as the mean ± SEM of at least three independent experiments. The statistical significance of differences between groups was evaluated via one-way analysis of variance (ANOVA), followed by Tukey’s multiple-comparison test, using the GraphPad Prism 5 software. * p < 0.05 vs. control.

Fig 3: Overexpression of OR7A17 interferes with the suppressive effect of ATRA on Ca2+ influx. (A) HaCaT cells were transduced with either LvCMV-GFP (mock-transduced HaCaT cell line) or LvCMV-OR7A17 (HaCaT-OR7A17 stable cell line). The overexpression of OR7A17 in HaCaT−OR7A17 cells was validated via Western blot analysis. (B) HaCaT-OR7A17 cells and mock-transduced HaCaT cells were treated with ATRA for 24 h, and OR7A17 expression was determined via Western blot analysis. Data are presented as the mean ± SEM of more than three independent experiments. * p < 0.05 vs. control. Changes in Ca2+ influx after ATRA treatment were observed in (C) mock-transduced HaCaT cells and (D) HaCaT-OR7A17 cells. Cells were incubated with Fluo-4 NW Ca2+ reagent loading solution for 30 min at 37 °C and for another 30 min at room temperature. Fluorescence was measured immediately after ATRA treatment. The data presented are representative of at least three independent experiments. (E) Both mock-transduced HaCaT cells and HaCaT-OR7A17 cells were treated with ATRA and incubated for 24 h in an incubator at 5% CO2 and 37 °C. After 24 h of incubation, Fluo-4 NW Ca2+ reagent loading solution was added to the cells and they were incubated for 30 min at 37 °C and another 30 min at room temperature. Fluorescence was measured after the incubation. (F) Changes in Ca2+ influx after ethylenediaminetetraacetic acid (EDTA) treatment were observed in mock-transduced HaCaT cells. Cells were incubated with Fluo-4 NW Ca2+ reagent loading solution for 30 min at 37 °C and for another 30 min at room temperature. Fluorescence was measured immediately after EDTA treatment. The data presented are representative of at least three independent experiments. (G) HaCaT-OR7A17 cells were cultured and incubated for 24 h in a 5% CO2 incubator at 37 °C. After 24 h of incubation, Fluo-4 NW Ca2+ reagent loading solution was added and incubated for 30 min at 37 °C and for another 30 min at room temperature. The fluorescence was measured immediately after calcium channel inhibitor treatment. Data are representative of at least three independent experiments.

Fig 4: Schematic of OR transcriptional regulation mediating the antiproliferative effects of ATRA on human keratinocyte. OR: OR7A17.

Fig 5: OR7A17 overexpression antagonizes the antiproliferative effect of ATRA in HaCaT cells. The proliferation-promoting effect of OR7A17 overexpression was observed via (A) EdU incorporation assays and (B) CellTiter® Glo 2.0 assays. (B) The antiproliferative effect of ATRA was again confirmed in mock-transduced HaCaT cells via both (C) EdU incorporation assay and (D) CellTiter® Glo 2.0 assay. HaCaT-OR7A17 cells were treated with ATRA for 24 h as in previous experiments. (E) EdU incorporation assay and (F) CellTiter® Glo 2.0 assay were carried out, and no significant change in proliferation was observed in OR7A17-overexpressing cells. Data are presented as the mean ± SEM of at least three different independent experiments. The statistical significance of differences between groups was evaluated via one-way analysis of variance (ANOVA), followed by Tukey’s multiple-comparison test, using the GraphPad Prism 5 software. * p < 0.05 vs. control.