Fig 2: IGFBP2 expression of vASCs. (A) SDS-PAGE analysis is shown of the proteins precipitated from conditioned media of vASCs or ASC.B6 cell culture, stained with Coomassie Brilliant Blue R-250. (B) IGFBP2 protein was detected by Western blotting experiment from the cell lysate of vASCs at passage number p3 and p16, and ASC.B6 cell cultures or conditioned media of vASCs p3 and ASC.B6 cell cultures. β-actin was used as loading control. (C) Recombinant IGFBP2 (1 μg/ml) inhibited the proliferation of 4T1 cells in the presence of ASC.B6 at a ratio of 2.5:1 in Transwell inserts. The bars show the mean ± SD from three independent experiments, the statistical analysis was t-test with P-values set at: *P < 0.05.

Fig 3: Reduced PPARA expression in the AEC2 cells from fibrotic lung regions of patients with IPF(A) Western blot for the expression of PPARα and β-actin in MLE-12 cells exposed to absence or presence of bleomycin at 4 h.(B) Non-targeting or Ppara siRNA-transduced MLE-12 cells were exposed to absence or presence of bleomycin treatment at 4 h. Western blot for the expression of PPARα and IGFBP2. β-Actin served as internal control. Data are representative of minimum of 3 independent experiments.(C) Ppara mRNA expression in the primary AEC2 cells isolated from aged mice subjected to low-dose bleomycin challenge after 14 days. Eukaryotic 18S rRNA was used as an endogenous control (n = 5 WT saline; n = 5 WT bleomycin).(D) Representative multicolor color immunohistochemistry of lung sections from aged WT mice 28 days after bleomycin injury. Green color indicates SPC expression; brown color indicates PPARα expression. Scale bars, 10 μm (n = 5 WT saline; n = 5 WT bleomycin).(E) Quantification of percentages of double-positive cells for SPC and PPARα in the lungs of aged WT mice subjected to low-dose bleomycin after 28 days.(F) Ppara mRNA expression was determined by qPCR in the primary AEC2 cells of patients with IPF (n = 21) compared with HP (n = 5) or COPD (n = 9).(G) Representative multicolor immunohistological staining of PPARA and SPC. Arrows indicate examples of SPC-positive and PPARA-positive cells. Staining was performed with lung sections from 2 healthy controls and 2 patients with IPF.(H) Quantification of percentages of double-positive cells for SPC and PPARA in the fibrotic lung regions of patients with IPF and donor (healthy) controls. Data are mean ± SEM. NS, not significant; ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001, one-way ANOVA with Tukey’s post-hoc test (for multiple group comparisons) or Student’s unpaired two-tailed t test (for two group comparisons).

Fig 4: Stable transduction with Igfbp2 lentivirus vector decreased P21 expression and β-galactosidase activity in vitro(A) Mock-virus- and Igfbp2 lentivirus-transduced MLE-12 cells in the absence or presence of hypoxia treatment at 4 h. Western blot for the expression of IGFBP2 and P21. β-Actin served as internal control.(B) Western blot for the expression of IGFBP2 and P21 in the cytosolic and nuclear fractions of Igfbp2 lentivirus-transduced MLE-12 cells in the absence or presence of hypoxia treatment at 4 h. α-Tubulin and histone-3 served as internal controls.(C) Western blot for the expression of IGFBP2, P21, and phosph-H2AX in Igfbp2 lentivirus-transduced MLE-12 cells in the absence or presence of cigarette smoke treatment (100 μg/mL).(D) Western blot for the expression of IGFBP2, P21, and phospho-H2AX in Igfbp2 lentivirus-transduced MLE-12 cells in the absence or presence of bleomycin (10 μg/mL). β-Actin served as internal control.(E) Bar graph showing the β-galactosidase activity of MLE-12 cells pretreated with ATZ for 1 h and subjected to hypoxia for 96 h.(F) Bar graph showing the β-galactosidase activity of MLE-12 cells treated with bleomycin for 48 h. Data are representative of minimum of 3 independent experiments. Data are mean ± SEM. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗∗p < 0.0001, one-way ANOVA with Tukey’s post-hoc test.

Fig 5: IGFBP2 deficiency increases P21 expression in response to fibrotic stimuli in vitro(A) Western blot for the expression of IGFBP2, P21, and phospho-H2AX in MLE-12 cells pretreated with atazanavir (ATZ; 20 μg/mL) for 1 h and exposed to hypoxia treatment for 72 h.(B) Western blot for the expression of IGFBP2, P21, and phospho-H2AX in MLE-12 cells exposed to absence or presence of chronic exposure to bleomycin (two-hit model; 10 μg/mL).(C) Western blot for the expression of IGFBP2 and P21 in MLE-12 cells exposed to absence or presence of hypoxia treatment at 4 h. β-Actin served as an internal control.(D) Western blot for the expression of IGFBP2 and P21 in the cytosolic and nuclear fractions of MLE-12 cells that were exposed to absence or presence of hypoxia treatment. α-Tubulin and histone-3 served as internal controls.(E) Western blot for the expression of IGFBP2 and P21 in MLE-12 cells exposed to absence or presence of cigarette smoke treatment (100 μg/mL).(F) Non-targeting or Igfbp2 siRNA-transduced MLE-12 cells were exposed to absence or presence of hypoxia treatment at 4 h. Western blot for the expression of IGFBP2 and P21.(G) Non-targeting or Igfbp2 siRNA-transduced MLE-12 cells were challenged with absence or presence of bleomycin exposure (10 μg/mL). Western blot for the expression of IGFBP2, P21, and phospho-H2AX in MLE-12 cells subjected to bleomycin exposure at 4 h. β-Actin served as an internal control. Data are representative of minimum of 3 independent experiments.