Figure 6.

IGF-1R and IR levels and its isoforms (IR-A and IR-B) in differentiating PMSCS are regulated by oxygen tension and IGFs. PMSCs were cultured for 14 days in nondifferentiation or osteogenic differentiation conditions containing 2% FBS in the presence or absence of 100 ng/mL of IGF-1 or IGF-2 in room air (20% O₂) or low oxygen levels (1% O₂). Treatments were stopped after 3, 7, and 14 days. Immunoblots were used to detect levels of (a) IGF-1R and IR in the absence of IGFs over time. Quantification of immunoblots, shown in Figure S3, shows the IGF-1 or IGF-2 effect on (b) IGF-1R and (c) IR over the three days. Levels were normalized to β-actin, a protein loading control (two-way ANOVA, P < 0.05, N = 3). X indicates significance between different days without IGFs; # indicates significance of IGF addition compared with no IGFs in the same day. By end-point PCR, mRNA levels of IR-A versus IR-B were measured and a ratio was calculated and normalized to total IR in (d) undifferentiated day 0 PMSCs, (e) differentiation without IGFs, (f) differentiation with IGF-1, and (g) differentiation with IGF-2 (two-way ANOVA, P < 0.05, N = 3). ∗ indicates significance between room air and low oxygen tension; lowercase letter (a, b) indicates significance between time points within room air condition, uppercase letter (A) indicates within low oxygen tension.