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. Author manuscript; available in PMC: 2021 May 30.
Published in final edited form as: Nat Mater. 2020 Nov 30;20(4):548–559. doi: 10.1038/s41563-020-00849-5

Extended Data Figure 4: Validation of LOX overexpression in doxycycline (DOX) treated MMTV-rtTA+/−; TetO_mLox+/− mice.

Extended Data Figure 4:

(a-c) Representative dot plots (n = 2) of GFP expression in mouse mammary epithelial cells analyzed by flow cytometry. GFP and mLOX are encoded on the same mRNA transcript with independent translation initiation sites. (a) GFP expression is induced in DOX treated MMTV-rtTA+/−; TetO_mLox+/− mice. (b-c) GFP expression is not induced in control mice lacking the MMTV-rtTA promoter (b) or control mice not treated with DOX (c). To select for mammary epithelial cells were gated out cells positive for anti-mouse CD45-APC, anti-mouse CD31-APC, anti-mouse Ter119-APC antibodies. (d) Quantification of Lox gene expression in whole mouse mammary gland by RT-qPCR in control MMTV-rtTA−/−; TetO_mLox+/− mice (n = 2) and Lox overexpressing MMTV-rtTA+/−; TetO_mLox+/− (n = 2) mice. (e) Western blot of whole tumor lysate from control (n = 4) and epithelial LOX overexpressing (n = 6) PyMT tumors. Scatter plots with mean ± SEM quantify optical density of each band normalized to E-cadherin. Western blot for these lysates was run once. Statistical analyses were performed using two-tailed Mann-Whitney U test Pro-LOX **p = 0.0095, Cleaved LOX *p = 0.0381. All n values represent biologically independent mouse tissue specimens.