Figure 5.

Effects of SPP1 and LGALS3 on normal human fibroblasts. Effects of rhSPP1 and rhLGALS3 on proliferation of NHDFs. NHDFs were cultured in the absence or presence of 500 ng/ml rhSPP1, 1000 ng/ml rhLGAL3 or 5 ng/ml rhTGF-β1 (as positive control) for 24 and 48 h. (a) Evaluation of cell growth by Trypan Blue exclusion assay after 24 and 48 h of treatment. Results are shown as number of cells, starting from 5000 cells per sample, as reported in ‘Materials and Methods’ section. Values are reported as mean±standard error of the mean (s.e.m.); n=6. *P<0.05 versus untreated control. (b) Effect of an anti-SPP1 (i) and anti-LGALS3 (ii) neutralizing antibody on SPP1-induced NHDFs cell growth. NHDFs were treated with rhSPP1 (i) or rhLGALS3 (ii) in the absence or presence of anti-SPP1 neutralizing antibody (2.5 μg/ml, bi) or anti-LGALS3 neutralizing antibody (10 μg/ml, bii) or an isotype-matched antibody for 24 h. Cell growth was evaluated by Trypan Blue exclusion assay. For each sample the fold of increase in cell counts after 24 h of culture is shown as normalized to the number of cells plated (t₀). Values are reported as mean±s.e.m. (n=6). (c) Expression levels of COL1A1, an extracellular matrix-related gene involved in fibrosis, measured by qRT-PCR after 24 h of culture in the presence or absence of rhSPP1, rhLGALS3 or rhTGF-β1 and are reported as RQ (mean±s.e.m.; n=3) respect to the untreated control samples, set as calibrator. COL1A1, collagen type 1 alpha 1; NHDFs, normal human dermal fibroblasts; n=number of experiments; rh, recombinant human.