*3.3. Discrimination of Hair Growth Cycle Phases, Distribution of Sulfated GAG and GPC1 Using IR Spectral Imaging Analysis*

3.3.1. Different Phases of the Hair Growth Cycle are Discriminated by PCA of IR Spectra Based on the Variation of GAGs and Their Sulfation

Spectra used for this analysis were taken from the ORS (central zone of the HF). This zone was chosen because it is rich in HS (see Figure 3). PCA was performed on the A1, C1/C3, and T1 HF spectra in the spectral range of 1800–900 cm−<sup>1</sup> , which encompasses the protein and PG absorptions. Figure 6A shows the PCA score plot, built with the principal components PC2 and PC3 carrying 30.3% and 11.5% of the total variance, respectively. PC2 permitted clearly separating the anagen HFs from the telogen HFs. The catagen HFs were dispersed in these two groups, but in a specific manner. Interestingly, the catagen C1 HFs were in the same group as the anagen HFs, while the catagen C3 HFs were in the telogen HF group. The same pattern was observed when the PCA score plot was recalculated using the sulfate absorption (1350–1190 cm−<sup>1</sup> ) spectral range (Figure 6B; PC1 and PC2 carrying 93.2% and 3.0% of the total variance, respectively). The second and third loading vectors shown in the region of 1800–900 cm−<sup>1</sup> (Figure S3A) and the first and second loading vectors shown in the region of 1350–1190 cm−<sup>1</sup> (Figure S3B) reveal the biochemical spectral information permitting to separate the different phases of the hair growth cycle.

**Figure 6.** Discrimination of hair follicles at different phases of hair growth cycle by PCA. (**A**,**B**) PCA score plot performed on normalized mean spectra of the ORS region 4 in the 1800–900 cm−<sup>1</sup> (**A**) or 1350–1190 cm−<sup>1</sup> (**B**) range and carried out on anagen A1, catagen C1/C3, and telogen T1 hair follicles.

The PCA results, on the one hand, suggest the discriminating power of the IR method capable of differentiating HFs at different phases of the hair growth cycle *via* their spectra and, on the other hand, demonstrate that the protein, PG, GAG, and sulfated GAG content varies during the phases of the hair growth cycle.

3.3.2. GPC1 Expression Corroborated with IR Spectral Maps of Sulfated GAG Distribution, Discriminates Different Phases during the Hair Growth Cycle

In order to support the PCA results obtained in the HFs of different phases of hair growth cycle, a PCA was performed on the HF phases induced by culture. This method allowed obtaining intermediate phases such as early and intermediate anagen.

After isolation, the early anagen HFs at day 0 (D0) were analyzed by spectral imaging and Western immunoblotting. Before analysis, the intermediate anagen (D3) and catagen (D6) HFs were maintained in culture for three and six days, respectively. As previously, spectra used for PCA analysis were extracted from the ORS region.

Figure 7A displays the PCA score plot constructed with the first two principal components PC1 and PC2 carrying 95.8% and 2.7% of the total variance, respectively. The variance carried by PC2 permitted separating the early anagen D0 from the catagen D6 HFs. The intermediate anagen D3 HFs were dispersed in these two groups, but in a specific manner. The observed tendency was that intermediate anagen D3 HFs with the hair shaft seemed to be closer to the early anagen D0 HF group, while those without the hair shaft were closer to catagen D6 HFs. The biochemical spectral information is supported by the loading vectors shown in the region of 1350–1190 cm−<sup>1</sup> (Figure S3C).

**Figure 7.** Discrimination of hair follicles by PCA compared to GPC1 expression during the hair growth cycle. (**A**) PCA score plot performed on normalized mean spectra of the ORS region 4 in the 1350–1190 cm−<sup>1</sup> range and carried out on early anagen D0, intermediate anagen D3, and catagen D6 hair follicles. (**B**) GPC1 protein expression analyzed by Western immunoblotting of early anagen D0, intermediate anagen D3, and catagen D6 hair follicles. (**C**) Quantification of each GPC1 form.

This result confirms the capacity of the PCA analysis to discriminate HFs at different hair growth cycle phases based on the sulfated GAG spectral signature. Moreover, data tend to indicate that the sulfated GAG evolve throughout the hair growth cycle.

In order to confirm the difference in GAG sulfation observed by IR imaging between HFs at different phases of the hair growth cycle, a GPC1 protein expression analysis was performed on HFs in the early anagen D0, intermediate anagen D3, and catagen D6 HFs.

Protein analysis carried out on HFs at different phases of the hair growth cycle shows that the GPC1 forms (cleaved, anchored, and glycanated) changed with the phases. In particular, cleaved and glycanated forms exhibited a noticeable difference observed between D0, D3, and D6 HFs (Figure 7B). Indeed, the glycanated form of GPC1 was more abundant in the intermediate anagen D3 and catagen D6 compared to early anagen D0 HFs. Moreover, a slight decrease was observed in catagen D6 compared to intermediate anagen D3 HFs. An accumulation of the cleaved GPC1 form was observed from D0 to D6 (Figure 7C).

According to the different HF phases, differential glycation of GPC1 could be demonstrated by Western immunoblotting. These observed differences may explain the PCA grouping of HF spectra with regard to the different phases based on sulfated GAG absorption.

Altogether, the IR spectral images processed by different chemometric approaches such as PCA, *k*-means clustering, and correlation mapping, could identify the histological structures and the different phases of the hair growth cycle. In addition, these approaches corroborate immunohistochemical and biochemical analyses of the present report and from the literature.
