Journal: eLife

Article Title: The Jag2/Notch1 signaling axis promotes sebaceous gland differentiation and controls progenitor proliferation

doi: 10.7554/elife.98747

Figure Lengend Snippet: Figure 2. Notch is active in the sebaceous gland stem cells. (a–c) Representative triple stain images for N1ICD, Notch1, and Jag2 mRNA in sebaceous glands (SGs) from mice (n=5 each) treated with aRW, 3 d 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 d 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 d post-treatment. Percentage was calculated by dividing the number of N1ICD+ basal stem cells by the 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 d 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.

Article Snippet: DOI: https://doi.org/10.7554/eLife.98747 13 of 17 Reagent type (species) or resource Designation Source or reference Identifiers Additional information Antibody Anti- Ragweed (Mouse monoclonal) Genentech Isotype control antibody – concentrations match the maximum dose of inhibiting antibodies Antibody Anti- N1ICD (Rabbit monoclonal) Cell Signaling Cat#: 4147 Triple stain (20 ug/ ml) IF (1:500) Antibody Anti- FASN (Mouse monoclonal) BD Cat#: 610963 IF (1:100) Antibody Anti- Adipophilin (Guinea pig polyclonal) Fitzgerald Cat#: 20R- AP002 IF (1:500) Antibody Anti- Ki67 (Rabbit monoclonal) Thermo Fisher Scientific Cat#: SP6 RM9106- SO IF (1:100) Antibody Anti- Lrig1 (Goat polyclonal) R&D Systems Cat#: AF3688- SP IF (1:200) Antibody Anti- AR (Rabbit monoclonal) Abcam Cat#: ab133273 [EPR1535(2)] IF (1:250) Antibody PowerVision polyHRP anti- rabbit (Goat polyclonal) Leica Cat#: PV6119 ready to use Sequence- based reagent RNAScope LS 2.5 MurineJag2_C1 ACD Cat#: 417518 nucleotides spanning from nt 552–1480 of reference sequence NM_010588.2 Sequence- based reagent RNAScope LS 2.5 MurineNotch1_C2 ACD Cat#: 404648- C2 nucleotides spanning from nt 1153–1960 of reference sequence NM_008714.3 Sequence- based reagent RNAScope LS 2.0 MurinePPIB probe ACD Cat#: 313917 nucleotides spanning from nt 98~856 of reference sequence NM_011149.2 Sequence- based reagent RNAScope LS 2.0 DapB probe ACD Cat#: 312038 nucleotides spanning from nt 414~862 reference sequence EF191515 Commercial assay or kit RNAscope LS Multiplex Reagent Kit ACD Cat#: 322800 Commercial assay or kit Bond Epitope Retrieval Solution 2 Leica Cat#: AR9640 Commercial assay or kit Opal Polaris 7 Auto Detection Kit Akoya Cat#: NEL811001KT Continued

Techniques: Staining

Journal: eLife

Article Title: The Jag2/Notch1 signaling axis promotes sebaceous gland differentiation and controls progenitor proliferation

doi: 10.7554/elife.98747

Figure Lengend Snippet: Figure 3. Loss of Notch activity in the sebaceous gland (SG) stem cells inhibits sebocyte differentiation. (a, b) Representative co-stain images for N1ICD and fatty acid synthase (FASN) in SGs from mice (n=5 each) treated with aRW (a) and aJ2 (b), 3 d post-treatment. (c, d) Representative adipophiin staining in SGs from mice (n=5 each) treated with aRW (c) and aJ2 (d), 3 d post-treatment. (e, f) Representative adipophilin staining in SGs from mice (n=5 each) treated with aRW (e) and aJ2 (f), 7 d post-treatment. (g) A representative hematoxylin and eosin (H&E) image of an SG from mice (n=5 each) treated with aJ2, 7 d 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 d 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 d 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 were compared against aRW. Error bars represent SEM. Scale bars are 25 μm.

Article Snippet: DOI: https://doi.org/10.7554/eLife.98747 13 of 17 Reagent type (species) or resource Designation Source or reference Identifiers Additional information Antibody Anti- Ragweed (Mouse monoclonal) Genentech Isotype control antibody – concentrations match the maximum dose of inhibiting antibodies Antibody Anti- N1ICD (Rabbit monoclonal) Cell Signaling Cat#: 4147 Triple stain (20 ug/ ml) IF (1:500) Antibody Anti- FASN (Mouse monoclonal) BD Cat#: 610963 IF (1:100) Antibody Anti- Adipophilin (Guinea pig polyclonal) Fitzgerald Cat#: 20R- AP002 IF (1:500) Antibody Anti- Ki67 (Rabbit monoclonal) Thermo Fisher Scientific Cat#: SP6 RM9106- SO IF (1:100) Antibody Anti- Lrig1 (Goat polyclonal) R&D Systems Cat#: AF3688- SP IF (1:200) Antibody Anti- AR (Rabbit monoclonal) Abcam Cat#: ab133273 [EPR1535(2)] IF (1:250) Antibody PowerVision polyHRP anti- rabbit (Goat polyclonal) Leica Cat#: PV6119 ready to use Sequence- based reagent RNAScope LS 2.5 MurineJag2_C1 ACD Cat#: 417518 nucleotides spanning from nt 552–1480 of reference sequence NM_010588.2 Sequence- based reagent RNAScope LS 2.5 MurineNotch1_C2 ACD Cat#: 404648- C2 nucleotides spanning from nt 1153–1960 of reference sequence NM_008714.3 Sequence- based reagent RNAScope LS 2.0 MurinePPIB probe ACD Cat#: 313917 nucleotides spanning from nt 98~856 of reference sequence NM_011149.2 Sequence- based reagent RNAScope LS 2.0 DapB probe ACD Cat#: 312038 nucleotides spanning from nt 414~862 reference sequence EF191515 Commercial assay or kit RNAscope LS Multiplex Reagent Kit ACD Cat#: 322800 Commercial assay or kit Bond Epitope Retrieval Solution 2 Leica Cat#: AR9640 Commercial assay or kit Opal Polaris 7 Auto Detection Kit Akoya Cat#: NEL811001KT Continued

Techniques: Activity Assay, Staining, Expressing

Journal: eLife

Article Title: The Jag2/Notch1 signaling axis promotes sebaceous gland differentiation and controls progenitor proliferation

doi: 10.7554/elife.98747

Figure Lengend Snippet: Figure 2. Notch is active in the sebaceous gland stem cells. (a–c) Representative triple stain images for N1ICD, Notch1, and Jag2 mRNA in sebaceous glands (SGs) from mice (n=5 each) treated with aRW, 3 d 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 d 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 d post-treatment. Percentage was calculated by dividing the number of N1ICD+ basal stem cells by the 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 d 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.

Article Snippet: For Immunohistochemistry, we used N1ICD (Cell Signaling, 4147), at 20 ug/ml working concentration.

Techniques: Staining

Journal: eLife

Article Title: The Jag2/Notch1 signaling axis promotes sebaceous gland differentiation and controls progenitor proliferation

doi: 10.7554/elife.98747

Figure Lengend Snippet: Figure 3. Loss of Notch activity in the sebaceous gland (SG) stem cells inhibits sebocyte differentiation. (a, b) Representative co-stain images for N1ICD and fatty acid synthase (FASN) in SGs from mice (n=5 each) treated with aRW (a) and aJ2 (b), 3 d post-treatment. (c, d) Representative adipophiin staining in SGs from mice (n=5 each) treated with aRW (c) and aJ2 (d), 3 d post-treatment. (e, f) Representative adipophilin staining in SGs from mice (n=5 each) treated with aRW (e) and aJ2 (f), 7 d post-treatment. (g) A representative hematoxylin and eosin (H&E) image of an SG from mice (n=5 each) treated with aJ2, 7 d 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 d 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 d 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 were compared against aRW. Error bars represent SEM. Scale bars are 25 μm.

Article Snippet: For Immunohistochemistry, we used N1ICD (Cell Signaling, 4147), at 20 ug/ml working concentration.

Techniques: Activity Assay, Staining, Expressing

Journal: eLife

Article Title: The Jag2/Notch1 signaling axis promotes sebaceous gland differentiation and controls progenitor proliferation

doi: 10.7554/eLife.98747

Figure Lengend Snippet:

Article Snippet: Antibody , Anti-N1ICD (Rabbit monoclonal) , Cell Signaling , Cat#: 4147 , Triple stain (20 ug/ml) IF (1:500).

Techniques: Control, Staining, Sequencing, RNAscope, Multiplex Assay