Figure 1.
Hair bulbs and mid-segments of hair follicles were micro-dissected from human scalp skin tissues.
(A) Pigmented (left) and unpigmented (right) whole anagenic hair follicles; (B) Each hair follicle was further excised using an ophthalmic scalpel under a stereoscope to harvest hair bulbs and mid-segments, as indicated by the arrows (left). Schematic view (right) showing the histological structures corresponding to hair bulbs and mid-segments of hair follicles. Scale bar = 1 mm.
Table 1.
Clinical and demographic characteristics of patients in this study.
Table 2.
Primers used in RT-PCR assays.
Figure 2.
Gene expression profiles of isolated hair follicles analyzed by semi-quantitative RT-PCR.
(A) Total RNA was extracted from a pool of 30–50 hair bulbs and mid-segments of hair follicle tissues. RT-PCR amplification was performed using primers specific for the molecular signature genes of the mature hair bulb melanocytes, the immature precursor cells of melanocytes, and anti-oxidant enzymes etc. as indicated in the gels and in Table 2. RT-PCR products were analyzed by electrophoresis on 1.0% agarose gels and images of PCR products are presented in reversed black and white in which the DNA band is black. PCR product sizes for each set of primers are noted in parentheses and were determined by comparison with a 100-bp DNA ladder (far left lane of each panel). (B) The intensity of each band was quantified using Image J densitometry software (NIH, Bethesda, MD, USA). The relative expression level of each targeted gene was normalized to expression of the housekeeping gene β-actin and is reported as relative expression from 3 independent experiments. *P<0.05.
Table 3.
Fold changes of gene expression in unpigmented hair follicles compared with pigmented hair follicles (qRT-PCR array analysis was performed on one subject).
Figure 3.
Expression level of catalase protein in hair follicles determined by western blotting.
Equal amounts (15 μg per lane) of each protein extract were resolved using 10% SDS-PAGE electrophoresis. Protein loading variations were determined by immunoblotting with an anti-β-actin antibody. Representative blots are shown (A). The histogram (B) shows the densitometric quantification of data with means ± SD of 3 independent experiments, *P<0.05, compared to pigmented hair follicles.
Figure 4.
Catalase enzyme activity in hair follicles estimated by spectrophotometric assay.
Catalase activity was determined spectrophotometrically using a commercial catalase analysis kit, as described in the text. Activities (unit per mg tissue protein) are expressed as means ± SD of 3 independent experiments, *P<0.05, compared to pigmented hair follicles.
Figure 5.
Hydroxyl radical-scavenging activities in hair follicles determined by an electron spin trapping ESR assay.
The effects of equal amounts of each minced tissue sample on hydroxyl radical (⋅OH) generation in the Fenton reaction was studied using the ESR method, as described in the text. (A) Representative ESR spectra of DMPO-⋅OH with the hair bulb and mid-segment tissue samples. Hydroxyl radicals were generated by the Fenton reaction (DMPO: 400 mM). (B) Histogram showing hydroxyl radical-scavenging activity, which are expressed as means ± SD of 3 independent experiments, *P<0.05, compared to pigmented hair follicles.