Jag2 is the dominant ligand involved in regulating sebocyte differentiation.

(a) A representative image of the homeostatic hair follicle, including its associated sebaceous gland (SG). (b) A detailed schematic of the SG showing the outer basal stem cell layer encasing the differentiated sebocytes. The schematic also shows the gene expression in different regions of the SG. (c) Representative H&E images of SGs from mice (n = 5 each) treated with aRW, aN1, aN2 and aN1N2, 7 days post treatment. (d) Quantification of type of SG found after each treatment. The SGs were divided into three categories: normal SGs containing characteristic number of sebocytes (normal SG), SGs containing a mix of sebocytes and basal-like cells (mixed SG), and SGs containing no sebocytes and only basal-like cells (no sebocyte SG). For (c), (e) and (f), each SG panel is labeled with where the SG is normal, mixed or no sebocyte SG. p-values: aN1 = 2.02E-123, aN2 = 0.426, aN1N2 = 3.85E-114. Total n of SGs quantified per treatment: aRW = 425, aN1 = 298, aN2 = 343, aN1N2 = 217. (e) Representative H&E images of SGs from mice (n = 5 each) treated with aRW, aJ1 and aJ2, 3 days post treatment. (f) Representative H&E images of SGs from mice (n = 5 each) treated with aRW, aJ1, aJ2 and aJ1J2, 7 days post treatment. (g) Quantification of type of SG found after each treatment. 3 days: p-values: aJ1 = 0.409, aJ2 = 0.004. 7 days: p-values: aJ1 = 0.057, aJ2 = 7.61E-151, aJ1J2 = 6.23E-169. Total n of SGs quantified per treatment: 3 days, aRW = 851, aJ1 = 944, aJ2 = 760, 7 days, aRW = 491, aJ1 = 499, aJ2 = 388, aJ1J2 = 461. Chi-sqaure test used for statistical analysis. All treatments compared against aRW. Error bars represent SEM. Scale bars are 100 μm.

Notch is active in the sebaceous gland stem cells.

(a-c) Representative triple stain images for N1ICD, Notch1 and Jag2 mRNA in SGs from mice (n = 5 each) treated with aRW, 3 days post treatment. (a) Four channels showing N1ICD, N1 ISH, J2 ISH and DAPI. (b) Two channels showing N1 ISH and J2 ISH. (c) One channel showing N1ICD. (d-f) Representative triple stain images for N1ICD, Notch1 and Jag2 mRNA in SGs from mice (n = 5 each) treated with aJ1, 3 days post treatment. (d) Four channels showing N1ICD, N1 ISH, J2 ISH and DAPI. (e) Two channels showing N1 ISH and J2 ISH. (f) One channel showing N1ICD. (g) Quantification of the percentage of N1ICD positive (N1ICD+) basal stem cells in SGs from mice (n = 5 each) treated with aRW, aJ1 and aJ2, 3 days post treatment. Percentage was calculated by dividing the number N1ICD+ basal stem cells by total number of basal stem cells in each SG. (h-j) Representative triple stain images for N1ICD, Notch1 and Jag2 mRNA in SGs from mice (n = 5 each) treated with aJ2, 3 days post treatment. (h) Four channels showing N1ICD, N1 ISH, J2 ISH and DAPI. (i) Two channels showing N1 ISH and J2 ISH. (j) One channel showing N1ICD. (k) Quantification of what percentage of the N1ICD+ basal stem cells express both N1 ISH and J2 ISH, only N1 ISH or only J2 ISH. (g and k) Total n of SGs quantified per treatment: aRW = 13, aJ1 = 15, aJ2 = 15. Error bars represent SEM. Scale bars are 25 μm.

Loss of Notch activity in the SG stem cells inhibits sebocyte differentiation.

(a,b) Representative co-stain images for N1ICD and FASN in SGs from mice (n = 5 each) treated with aRW (a) and aJ2 (b), 3 days post treatment. (c,d) Representative adipophillin staining in SGs from mice (n = 5 each) treated with aRW (c) and aJ2 (d), 3 days post treatment. (e,f) Representative adipophillin staining in SGs from mice (n = 5 each) treated with aRW (e) and aJ2 (f), 7 days post treatment. (g) A representative H&E image of an SG from mice (n = 5 each) treated with aJ2, 7 days post treatment. Arrow points to a bursting sebocyte releasing sebum into the sebaceous duct. (h) Quantification of the number of cells expressing adipophilin in each SG, 3 days post treatment with aRW, aJ1 and aJ2. p-values: aJ1 = 0.749, aJ2 = 0.062. Total n of SGs quantified per treatment: aRW = 17, aJ1 = 14, aJ2 = 30. (i) Quantification of the number of cells expressing adipophilin in each SG, 7 days post treatment with aRW, aJ1, aJ2 and aJ1J2. p-values: aJ1 = 0.301, aJ2 = 4.13E-14, aJ1J2 = 4.06E-14. Total n of SGs quantified per treatment: aRW = 18, aJ1 = 19, aJ2 = 22, aJ1J2 = 21. Student’s T-test used for statistical analysis. All treatments compared against aRW. Error bars represent SEM. Scale bars are 25 μm.

Notch activity in the SG stem cells is required to prevent unregulated progenitor proliferation.

(a,b) Representative co-stain images for Lrig1 and AR in SGs from mice (n = 5 each) treated with aRW (a) and aJ2 (b), 3 days post treatment. (c) A representative co-stain image for FASN and AR in an SG from mice (n = 5 each) treated with aRW, 3 days post treatment. Arrows point to sebocytes, and arrowheads point to progenitor cells. (d) Quantification of the number of cells expressing Lrig1 in each SG, 3 days post treatment with aRW, aJ1 and aJ2. p-values: aJ1 = 0.152, aJ2 = 0.450. (e) Quantification of what percentage of the Lrig1+ cells express AR, 3 days post treatment with aRW, aJ1 and aJ2. p-values: aJ1 = 0.789, aJ2 = 0.028. (d and e) Total n of SGs quantified per treatment: aRW = 17, aJ1 = 10, aJ2 = 24. (f,g) Representative co-stain images for Lrig1 and AR in SGs from mice (n = 5 each) treated with aRW (f) and aJ2 (g), 7 days post treatment. (h) Quantification of the number of cells expressing Lrig1 in each SG, 7 days after treatment with aRW, aJ1, aJ2 and aJ1J2. p-values: aJ1 = 0.628, aJ2 = 9.06E-07, aJ1J2 = 7.88E-06. (i) Quantification of what percentage of the Lrig1+ cells express AR, 7 days after treatment with aRW, aJ1, aJ2 and aJ1J2. p-values: aJ1 = 0.117, aJ2 = 0.248, aJ1J2 = 0.091. (h and i) Total n of SGs quantified per treatment: aRW = 12, aJ1 = 12, aJ2 = 18, aJ1J2 = 16. (j,k) Representative co-stain images for Ki67 and FASN in SGs from mice (n = 5 each) treated with aRW (j) and aJ2 (k), 7 days post treatment. (l) Quantification of the number of cells expressing Ki67 in each SG, 7 days after treatment with aRW, aJ1, and aJ2. p-values: aJ1 = 0.652, aJ2 = 0.009. Total n of SGs quantified per treatment: aRW = 11, aJ1 = 12, aJ2 = 16. Student’s T-test used for statistical analysis. All treatments compared against aRW. Error bars represent SEM. Scale bars are 25 μm.

The block in sebocyte differentiation is lifted upon recovery of Notch activity.

(a) Representative H&E images of SGs from mice (n = 5 each) treated with aRW, aJ1, aJ2 and aJ1J2, 14 days post treatment. (b) Quantification of type of SG found after each treatment. The SGs were divided into three categories: normal SGs containing characteristic number of sebocytes (normal SG), SGs containing a mix of sebocytes and basal-like cells (mixed SG), and SGs containing no sebocytes and only basal-like cells (no sebocyte SG). p-values: aJ1 = 0.535, aJ2 = 2.90E-87, aJ1J2 = 1.73E-92. Chi-sqaure test used for statistical analysis. All treatments compared against aRW. Total n of SGs quantified per treatment: aRW = 485, aJ1 = 430, aJ2 = 394, aJ1J2 = 364. Scale bars are 100 μm. (c,d) Representative co-stain images for N1ICD and FASN in SGs from mice (n = 5 each) treated with aRW (c) and aJ2 (d), 7 days post treatment. (e) Quantification of the percentage of N1ICD+ cells in SGs from mice (n = 5 each) treated with aRW and aJ2, 3 and 7 days post treatment. Percentage was calculated by dividing the number N1ICD+ cells by total number of basal-like cells in each SG. p-values: for comparison between day 3 and day 7 for aRW treatment = 0.297, for comparison between day 3 and day 7 for aJ2 treatment = 1.00E-14. Total n of SGs quantified per treatment: aRW at 3days = 13, aRW at 7days = 13, aJ2 at 3days = 15, aJ2 at 7 days = 22. (f,g) Representative adipophillin staining in SGs from mice (n = 5 each) treated with aRW (f) and aJ2 (g), 14 days post treatment. (h) Quantification of the number of cells expressing adipophilin in each SG, 7 and 14 days after treatment with aRW, and aJ2. p-values: for comparison between day 7 and day 14 for aRW treatment = 0.292, for comparison between day 7 and day 14 for aJ2 treatment = 1.38E-4. Total n of SGs quantified per treatment: aRW at 7days = 18, aRW at 14days = 19, aJ2 at 7days = 22, aJ2 at 14 days = 29. (i,j) Representative co-stain images for AR and FASN in SGs from mice (n = 5 each) treated with aRW (i) and aJ2 (j), 14 days post treatment. (k) Quantification of the number of cells expressing AR in each SG, 7 and 14 days after treatment with aRW and aJ2. p-values: for comparison between day 7 and day 14 for aRW treatment = 0.273, for comparison between day 7 and day 14 for aJ2 treatment = 9.53E-09. Total n of SGs quantified per treatment: aRW at 7days = 12, aRW at 14days = 10, aJ2 at 7days = 18, aJ2 at 14 days = 23. Student’s T-test used for statistical analysis. Error bars represent SEM. Scale bars are 25 μm.