Fig. 2

PHGDH expression during the previous hair follicle cycle leads to increased melanin accumulation. a A region of hair was plucked from 49 day-old mice (at the second telogen) to synchronize the hair follicle cycle, and skin samples were collected at defined days thereafter. Data were collected from PHGDH^tetO;Rosa26-M2rtTA (tetO/+) or control (+/+) mice that were exposed to doxycycline (Dox) for either 2 days or 30 days prior to synchronization. Shown is a schematic of the experiment, with the red bar depicting mice exposed to doxycycline diet for 2-days before synchronization, and the blue bar depicting mice exposed to doxycycline for 30-days before synchronization. b Quantitation of the percent of early anagen hair follicles (HFs) containing any melanin granules in PHGDH^tetO;Rosa26-M2rtTA (tetO/+) or control (+/+) mice exposed to doxycycline for 2 days prior to synchronization. Data shown represent the % observed when analyzing 50 HFs per mouse from 3 mice of each genotype (c) Quantitation of the percent of early anagen hair follicles with three or more melanin granules in PHGDH^tetO;Rosa26-M2rtTA (tetO/+) or control (+/+) mice exposed to doxycycline for 2 days prior to synchronization. Data shown represent the % observed when analyzing 50 HFs per mouse from 3 mice of each genotype. No statistically significant increase in hair follicles with melanin granules were observed in (b) or (c) with p-values derived from two-tailed Fisher’s exact test. d Western blot analysis for PHGDH expression in skin from PHGDH^tetO;Rosa26-M2rtTA mice never exposed to doxycycline-containing diet (−dox) or fed a doxycycline-containing diet for 2 or 30 days as indicated. Vinculin expression is also shown as a loading control. e Quantitation of the percent of early anagen hair follicles (HFs) containing any melanin granules in PHGDH^tetO;Rosa26-M2rtTA (tetO/+) or control (+/+) exposed to doxycycline for 30 days prior to synchronization. Data shown represent the % observed when analyzing 50 HFs per mouse from 3 mice of each genotype. f Quantitation of the percent of early anagen hair follicles with three or more melanin granules PHGDH^tetO;Rosa26-M2rtTA (tetO/+) or control (+/+) mice exposed to doxycycline for 30-days prior to synchronization. Data shown represent the % observed when analyzing 50 HFs per mouse from 3 mice of each genotype. The increase in hair follicles with melanin granules shown in (e) and (f) is statistically significant with p-values derived from two-tailed Fisher’s exact test. g qPCR to assess species-specific PHGDH expression in early anagen skin isolated from PHGDH^tetO;Rosa26-M2rtTA (tetO/+) or control (+/+) mice exposed to doxycycline for 30-days prior to synchronization. An increase in human PHGDH (huPHGDH), but not mouse PHGDH (msPHGDH) expression is statistically significant with p values derived from unpaired Student’s t test. Data shown represent the mean (+/− SD)