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Fig. 5. Senescent fibroblast-produced factors stimulate epithelial branching. Collagen-embedded organoids were cultured in the presence of conditioned medium from presenescent (Presen) or senescent (Sen) mBF. The conditioned medium was either unsupplemented (-) or preincubated with HGF blocking antibody (anti-HGF), an MMP-3 blocking peptide (MMP-3i) or an MMP-2 inhibitor (MMP-2i). Error bars show s.e.m. of triplicate wells. (A) Average size (Total), branch length (Branches) and core area (Core) of organoids cultured with conditioned media lacking or containing an HGF blocking antibody or MMP-3i. (B) Average number of primary (1ary), secondary (2ary) and tertiary (3ary) branches in the organoids analyzed in A. (C) Organoids in A were analyzed for average primary and secondary branch lengths in the presence of HGF blocking antibody or MMP-3i, and expressed as change relative to presenescent or senescent-derived conditioned medium lacking antibody or inhibitor. The asterisks indicate a statistically significant reduction in branch length by HGF neutralization, compared to medium lacking HGF antibody, as determined by a Student's t-test. Branching length in presenescent and senescent conditioned media was affected similarly by HGF neutralization. (D) Average number of epithelial cells per organoid, quantified by DAPI fluorescence of nuclei. Asterisks indicate a statistically significant change in proliferation, as determined by a Student's t-test. (E) Percentage of organoids having primary (1' Only) or higher level (secondary and tertiary; Side Branching) branches in the absence or presence of MMP-2i. (F) Casein zymography of conditioned medium from presenescent and senescent mBF. A 50 kDa marker and MMP-3 are indicated. Densitometry was performed on the reverse image to determine the relative expression; the signal from presenescent conditioned medium was set arbitrarily at 1.
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