Fig 2: CAFs but not IGF-1 increase PAI-1 expression in cancer cells(A) MDA-MB-231 spheroids were culture alone or in presence of CAFs or IGF-1 [50 nM] for 72 h and assayed for SERPINE1 (PAI1) expression by RT-PCR. (B) MDA-MB-231 cell spheroid were cultured alone or in presence of CAFs, IGF-1 [50 nM] and/or Y27632 [10 µM] for 72 h and immunoblotted for PAI-1 and ß-actin as loading control. HUVECs were used as a positive control for PAI-1 expression. (C–D) Effect of PAI-1 inhibition on scattering and invasion of MDA-MB-231 cells in a collagen gel. MDA-MB-231 spheroids cultured with or without CAF2 in a collagen gel over a 7-day period in absence or in presence of 10 µM of geodin. (E) Effect of PAI-1 inhibition on RhoA pathway activation in MDA-MB-231 cells: MDA-MB-231 cells were cultured alone or in presence of CAF2 in absence or presence of 10 µM of geodin and assayed for RhoA activation by RhoA-GTP pulldown assay and immunobloted for phospho-MLC (Thr18/Ser19). Data expressed as mean ± SEM. *p = 0.05, **p = 0.01, ***p = 0.001, ****p = 0.0001.

Fig 3: Metabolic profiling of IGFBP-1-KO mice. (A) Plasma was taken from IGFBP- 1-KO mice and wildtype litter mate controls, ELISA data confirmed IGFBP-1 knockdown (IGFBP-1-KO 0 ± 0 v Wt 5.69 ± 3.93 ng/mL). (B) Body mass was no different between groups (IGFBP-1-KO 22.8 ± 0.9 v Wt 23.1 g ± 0.9). (C) There was no difference in organ weights of IGFBP-1-KO mice when compared with wild-type litter mate control mice when normalized to body weight (Heart Wt 0.55 ± 0.04 v IGFBP-1-KO 0.56 ± 0.04) (Liver Wt 4.9 ± 0.4 v IGFBP-1-KO 5.07 ± 0.04) (Spleen Wt 0.4 ± 0.08 v IGFBP-1-KO 0.39 ± 0.03) (Kidney Wt 0.68 ± 0.05 v IGFBP-1-KO 0.6 ± 0.07) (WAT Wt 0.85 ± 0.07 v IGFBP-1-KO 0.9 ± 0.2). (D) Random blood glucose was no different between groups (IGFBP-1-KO 10.4 ± 0.5 v Wt 10 ± 0.5). (E) Fasting blood glucose was no different between groups (IGFBP-1-KO 5.1 ± 0.1 v Wt 5.6 ± 0.4). (F) Change in blood glucose levels after an IP injection of glucose, showed no difference in glucose handling, also shown by area under the curve analysis in G (IGFBP-1-KO 41.3 ± 1.8 v Wt 41.6 AU ± 0.7). (H) Change in blood glucose levels after an IP injection of insulin, showed no difference in insulin sensitivity, also shown in area under the curve analysis in I: (IGFBP-1-KO 24.8 ± 3.1 v Wt 23.5 AU ± 1.9). (J) Random plasma insulin was no different between groups (IGFBP-1-KO 0.89 ± 0.18 v Wt 0.67 ± 0.24). (K) Change in blood glucose levels after an IP injection of IGF-I shows that IGFBP-1-KO mice was not significantly different between the groups. Data are presented as mean ± SEM. N = 6 to 8 per group unless otherwise stated. (*P ≤ 0.05).

Fig 4: Detection of transgene (TGF-β, IGF-I) overexpression in human OCDs following implantation of rAAV-transduced hMSC aggregates. Aggregates were cotransduced with rAAV TGF-β/IGF-I, TGF-β/lacZ, IGF-I/lacZ or transduced with rAAV lacZ and implanted in human OCDs as described in Figure 1 and in the Materials and Methods. Samples were processed after 21 days to detect the expression of TGF-β and of IGF-I by immunohistochemistry (magnification ×10; insets showing the surrounding cartilage in the region immediately adjacent to the implanted pellets at magnification × 20; representative data). Scale bars: 100 µm, insets: 50 µm.

Fig 5: Study design. Human bone marrow-derived mesenchymal stromal cells MSCs were prepared as high-density aggregates (2 × 105 cells/aggregate) in defined chondrogenic medium overnight, transduced with the various rAAV shuttles (TGF-ß/IGF-I, TGF-ß/lacZ, IGF-I/lacZ: 40 µL each vector/aggregate; lacZ: 80 µL vector/aggregate), and directly applied to human osteochondral defects (OCDs; 6-mm diameter) for further culture in defined chondrogenic medium for 21 days when the OCDs were processed to monitor transgene expression and for biochemical, immunohistochemical/histological, and real-time RT-PCR.