Figure 2

Effect of varying concentrations and combnations of HIV1 proteins on MSC osteogenesis. MSCs were cultured in a 96-well plate format and induced to differentiate in the presence and absence of 10–100 ng/ml of HIV REV, gp120, p55-gag, and TAT. Untreated, differentiated cells were included as controls. Cells were harvested at 15 days post induction of differentiation, fixed and stained for either calcium deposition (alizarin red) or alkaline phosphatase (BCIP/NBT). Plates were first photographed, then the stain extracted, and the degree of staining quantified spectrophotometrically and normalised to cell number. Fig 2A; calcium staining normalised to cell number for diferentiating MSCs (diff con), and differentiating MSCs exposed to 10–100 ng/ml HIV gp120, p55-gag, REV and TAT proteins, data expressed as percentage of untreated control ± SEM. Fig 2B; ALP staining normalised to cell number, for diferentiating MSCs (diff con), and differentiating MSCs exposed to 10–100 ng/ml HIV gp120, p55-gag, REV and TAT. proteins, data expressed as percentage of untreated control ± SEM. Differentiating MSCs were also incubated with various combinations of HIV1 proteins (all proteins at 100 ng/ml), and there affects on calcium deposition and alkaline phosphatase staining assayed and quantified as described. Fig 2C; Fig 2A; calcium staining normalised to cell number for differentiating MSCs (diff control), and differentiating MSCs exposed to either 100 ng/ml REV/p55, 100 ng/ml p55/REV/TAT, 100 ng/ml p55/REV/TAT/gp120, 100 ng/ml p55/gp120, or 100 ng/ml REV/TAT proteins, data expressed as percentage of untreated control ± SEM. Fig 2D; ALP staining normalised to cell number, for differentiating MSCs (diff con), and differentiating MSCs exposed to either 100 ng/ml REV/p55, 100 ng/ml p55/REV/TAT, 100 ng/ml p55/REV/TAT/gp120, 100 ng/ml p55/gp120, or 100 ng/ml REV/TAT proteins, data expressed as percentage of untreated control ± SEM. All data is the mean of several independent experiments. (Significance determined at; *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.005 relative to undifferentiated control, ^p ≤ 0.05, ^^p ≤ 0.01, ^^^p ≤ 0.005 relative to differentiated control).