Fig. 6

NTPDase3 gene silencing using lenti-shRNA promotes mineralization of Pm BM-MSC cultures allowed to grow for 35 days in an osteogenic-inducing medium. Cells were exposed for 24 h to a lenti-shRNA encoding to a sequence (TL313202VD) designed to silence NTPDase3 or to a scramble sequence applied at multiplicities of infection 3 (MOI 3). Confocal microscopy images shown in panel A confirm that NTPDase3 immunoreactivity transiently decreases 7-days after exposure of Pm BM-MSC cultures to a lenti-shRNA encoding to the TL313202VD sequence, but not when the cells were treated with the scramble sequence. Blue dots represent nuclei stained with DAPI. Experiments were performed in parallel keeping unaltered the settings of the confocal microscope throughout the documentation procedure (see Materials and Methods). The scale bar is 50 µm. Panel Bi shows the per cent variation of the ALP activity of Pm BM-MSC cultures on days 7 and 21 corrected for cells growth/viability (MTT assay) obtained after treatment with the lenti-shRNA encoding to TL313202VD or the scramble sequence (MOI 3) (100%; 0.6 ± 0.1 and 7.1 ± 3.7 nmol/min/MTT at days 7 and 21, respectively). Panel Bii shows the per cent variation of the total mineralized area of Pm BM-MSCs at culture day 35 after treatment with the lenti-shRNA encoding to TL313202VD or the scramble sequence (MOI 3) (100%; 13,714.1 ± 3657.6 µm²). Panel Biii shows the Alizarin Red staining denoting extracellular matrix mineralization and bone nodule formation of BM-MSC cultures (red-brownish spots) in a Pm woman; the scale bar is 50 µm. Boxes and whiskers represent pooled data from four Pm women (69 ± 7 years old); three to four replicas were made per individual. In panel Bi, *P < 0.05 (one-way ANOVA with Sidák’s multiple comparison test, single pooled variance) represents significant differences; in panel Bii, ***P < 0.01 (two-tailed unpaired t-test) represent significant differences