## 3 Results  
### Scenedensmus Deserticola JD052 Extract Increases Hair Inductivity of HDP Cells  
Microalgae fundamentally rely on photosynthesis for survival and adopt a growth condition in which they utilize light energy to drive biosynthesis while assimilating essential elements such as carbon, nitrogen, and phosphorus. This cultivation method, which leverages these processes for microalgal biomass production, is referred to as photoautotrophic cultivation [40]. To begin our analysis for the hair inductivity of photoautotrophic-cultured _S. deserticola_ JD052 extract (PE), we first assessed its effect on the potential cell viability of HDP cells using the WST-1 assay. As shown in Figure 1a, two-dimensionally (2D) cultured HDP cells, treated with PE, increased the cell viability of HDP cells up to a concentration of 100 ug/mL. HDP sphere size is related to hair shaft diameter, and the sphere formation of the HDP cells is associated with their hair-inductive properties [41]. Additionally, decreased HDP sphere sizes are observed during hair follicle miniaturization in alopecia [42,43]. Thus, we investigated whether PE increases the size and proliferative capacity of HDP spheroids compared to the DMSO-treated control (Figure 1b and Figure S1a). Consequently, PE significantly increased the size of HDP spheroids in a dose-dependent manner. We then examined the T-cell-specific transcription factor/lymphoid enhancer-binding factor (TCF/LEF) transcriptional activity after PE (0, 10, 50, and 100 ug/mL) treatment using the TOP-Flash luciferase reporter assay (Figure 1c). As expected, PE increased TCF/LEF-driven luciferase activity in 293T cells. Several reports identified that growth factors in DP cells modulate hair growth with autocrine or paracrine signaling [44]. Accordingly, we evaluated whether the HDP sphere formation is related to growth factors, including VEGF, IGF1, FGF2, and FGF7, using Western blot analysis. As shown in Figure 1d, the protein levels of intracellular VEGF, IGF1, FGF2, and FGF7 in PE-treated HDP cells were upregulated compared to the levels of the DMSO-treated control at the concentrations of 50 and 100 \(\upmu\)g/mL. WNT/\(\beta\)-catenin signaling is one of the most important signaling pathways in hair follicles [45; 46]. \(\beta\)-catenin, which regulates TCF/LEF transcription, is a key biomolecule in the hair-inductive properties of DP cells [47; 48]. Therefore, we also investigated the protein level of \(\beta\)-catenin in HDP cells. The result shown in Figure 1e demonstrated that the protein level of intracellular \(\beta\)-catenin was upregulated, but the phosphorylation level of \(\beta\)-catenin was inhibited in PE-treated HDP cells. These data indicated that _S. desericola_ JD052 extract might have effective hair-inductive properties by increasing the size of the DP spheroids and the expression of growth factor genes and \(\beta\)-catenin in HDP cells. Figure 1: The effect of PE on cell viability and hair-inductive potential in HDP cells. **(a)** HDP cells were exposed to varying concentrations of PE (0–500 \(\upmu\)g/mL) for 48 h, after which cell viability was determined using the WST-1 assay. **(b)** To assess spheroid formation, HDP cells were cultured in a 96-well ultra-low-attachment microplate and treated with PE at concentrations of 10, 50, and 100 \(\upmu\)g/mL for 48 h. The diameters of the resulting spheroids were measured, and phase-contrast microscopy was used to capture spheroid images. **(c)** In parallel, 293T cells were incubated with PE at concentrations of 50 and 100 \(\upmu\)g/mL for 24 h. The TCF/LEF luciferase reporter assay was performed to assess transcriptional activity, with luminescence quantification normalized to \(\beta\)-galactosidase activity. **(d)** The protein expression of growth factors and **(e)** \(\beta\)-catenin stabilization in HDP cells were determined using Western blot assays with specific antibodies after treatment with 50 and 100 \(\upmu\)g/mL of PE for 48 h. \(\beta\)-actin served as a loading control for total protein.