Figure 6.

Effects of SPP1 and LGALS3 on PMF mesenchymal stromal cells. PMF-derived MSCs were cultured in the absence or presence of 500 ng/ml rhSPP1, 1000 ng/ml rhLGAL3 or 5 ng/ml rhTGF-β1 (used as positive control) for 48 h. (a) Cell proliferation was monitored by Trypan Blue exclusion assay after 48 h of treatment. Results are shown as number of cells, starting from 1.6 × 10⁴ cells per sample. Values are reported as mean±standard error of the mean (s.e.m.). The results come from three independent experiments. *P<0.05; **P<0.01 versus untreated control. (b) Effect of an anti-SPP1 (i) and anti-LGALS3 (ii) neutralizing antibody on MSCs cell proliferation. PMF MSCs 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 48 h. Cell proliferation was evaluated by Trypan Blue exclusion assay. For each sample the fold of increase in cell counts after 48 h of culture was normalized to the number of cells plated at t₀. Values are reported as mean±s.e.m. (n=5). *P<0.05. (c) Expression levels of the fibrotic marker COL1A1 in PMF MSCs were measured by qRT-PCR after 48 h of culture with rhSPP1, rhLGALS3 or rhTGF-β1 and are reported as RQ (mean±s.e.m.; n=5) respect to the untreated control sample, set as calibrator. *P<0.05; **P<0.01. COL1A1, collagen type 1 alpha 1; MSCs, mesenchymal stromal cells; rh, recombinant human; n, number of experiments.