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. Author manuscript; available in PMC: 2014 Dec 1.

Published in final edited form as: Breast Cancer Res Treat. 2013 Nov 12;142(3):489–503. doi: 10.1007/s10549-013-2741-5

A) Representative confocal photomicrographs (63X) of HGF (green) co-stained with fibroblast marker - smooth muscle actin (SMA, red) in normal mammary and tumor. Nucleus (blue) is stained with DAPI. B) HGF was quantified in conditioned media at indicated time points from mono-cultures of 4T1 basal like epithelial cells, as well as primary normal associated fibroblasts (NAF) and cancer associated fibroblasts (CAF) isolated from lean (10%-fed mice) and obese (60%-fed mice) for n=3 experiments. *P = 0.0001 vs. all other groups; + vs. all other groups (P=0.001); # vs. 10% NAF (P=0.0001), 60% NAF (P=0.005), and 10% CAF (P=0.043). C) phospho-c-Met and total c-Met expression in 4T1 cells was determined using Western immunoblot analysis after 15 min treatment with conditioned media (CM) from primary 60% CAFs or recombinant mouse HGF (50 ng/ml). Loading control is non-specific band from c-Met immunoblot. D) Proliferation index measured using BrdU incorporation is shown for n = 3 experimental groups. * vs. HGF (50 ng/ml) (P = 0.024); # vs. 60%-CAF with 10%-NAF (P = 0.003) and 60% NAF (P = 0.02). E) Fluorescently labeled 4T1 cells were co-cultured with NAFs or CAFs isolated from lean (10% diet) and obese (60% diet) mice, with or without HGF neutralizing antibody during the 24 hour co-culture period and the 12 hour scratch test. Representative photomicrographs of wound migration assay at 0h (time point of scratch) and 12h (end of study). F) Percent wound closure is shown for n = 3 experiments. * vs. control (no anti-HGF) for 10% CAF (P=0.0001) and 60% CAF (P=0.0004). + vs. 10% NAF (P=0.0002) and vs. 60%-NAF (P=0.006) controls. # vs. all groups (P = 0.0001).

A) Representative confocal photomicrographs (63X) of HGF (green) co-stained with fibroblast marker - smooth muscle actin (SMA, red) in normal mammary and tumor. Nucleus (blue) is stained with DAPI. B) HGF was quantified in conditioned media at indicated time points from mono-cultures of 4T1 basal like epithelial cells, as well as primary normal associated fibroblasts (NAF) and cancer associated fibroblasts (CAF) isolated from lean (10%-fed mice) and obese (60%-fed mice) for n=3 experiments. *P = 0.0001 vs. all other groups; + vs. all other groups (P=0.001); # vs. 10% NAF (P=0.0001), 60% NAF (P=0.005), and 10% CAF (P=0.043). C) phospho-c-Met and total c-Met expression in 4T1 cells was determined using Western immunoblot analysis after 15 min treatment with conditioned media (CM) from primary 60% CAFs or recombinant mouse HGF (50 ng/ml). Loading control is non-specific band from c-Met immunoblot. D) Proliferation index measured using BrdU incorporation is shown for n = 3 experimental groups. * vs. HGF (50 ng/ml) (P = 0.024); # vs. 60%-CAF with 10%-NAF (P = 0.003) and 60% NAF (P = 0.02). E) Fluorescently labeled 4T1 cells were co-cultured with NAFs or CAFs isolated from lean (10% diet) and obese (60% diet) mice, with or without HGF neutralizing antibody during the 24 hour co-culture period and the 12 hour scratch test. Representative photomicrographs of wound migration assay at 0h (time point of scratch) and 12h (end of study). F) Percent wound closure is shown for n = 3 experiments. * vs. control (no anti-HGF) for 10% CAF (P=0.0001) and 60% CAF (P=0.0004). + vs. 10% NAF (P=0.0002) and vs. 60%-NAF (P=0.006) controls. # vs. all groups (P = 0.0001).