Figure 2

LF4 deficiency in bone marrow was associated with significantly decreased numbers of fibrocytes and myofibroblasts in metastatic lung. (a) The peripheral blood monocytes (PBMCs) from the chimeric mice as described in Figure 1 were isolated using Histoplaque (Sigma-Aldrich) according to the manufacturer’s instructions. EGFP-expressing PBMCs were stained and gated by biotin-conjugated mouse Collagen Type I alpha1 antibody (COL1A1; Rockland, Gilbertsville, PA, USA; left) and further analyzed using CD45 (clone 30-F11, eBiosciences) and CD11b antibodies (right) by flow cytometry. Representative dot plots were shown (n=8, P<0.05). (b) Lung-infiltrating immune cells were stained with biotin-conjugated mouse COL1A1, rabbit anti-mouse α-SMA (Millipore, Billerica, MA, USA), CD11b and CD45 antibodies and then examined using flow cytometry. Quantifications of the total number of COL1A1⁺CD45⁺CD11b⁺EGFP⁺ cells (left) and CD11b⁺α-SMA⁺EGFP⁺ cells (right) in lung were shown based on cell counting and flow cytometry data. (c and d) Representative immunofluorescence staining of lung tissues with rabbit anti-mouse COL1A1 (Rockland; 1:200, scale bars=100 μm) and α-SMA (1:200, scale bars=50 μm). COL1A1⁺EGFP⁺ and α-SMA⁺EGFP⁺ double positive cells (yellow arrows) within dotted lines encircling metastatic lesions were counted for every five high power fields (HPF) and compared. Mean±s.e.m., *P<0.05, ^**P<0.01, ^***P<0.001.