Journal: Advanced Science

Article Title: Alkylating Agent–Induced FANCL Impairment Damages Pulmonary Arterial, Venous, and Microvascular Endothelial Cells and Predisposes to PAH/PVOD

doi: 10.1002/advs.78081

Figure Lengend Snippet: MMC or CDDP Treatment Induces Progressive DNA Damage Accumulation in Pulmonary Vascular Endothelium. (A–H) Immunoblots and analyzed graphs showing the γ‐H2AX protein expression in the lung tissue of rats treated with MMC (3 mg/kg, once weekly) for 2 or 4 weeks (A–D), and CDDP (4 mg/kg, once weekly) for 2 or 4 weeks (E–H). n = 6–12 rats per group. Protein expression levels were calculated as the ratio of the target protein band intensity to that of β‑actin and were normalized to the mean values of the respective 2‑week or 4‑week NC groups, expressed as fold change. (I, J) Immunohistochemical staining for the FA core factors (FANCL and FANCD2) and the DNA damage marker γ‐H2AX in lung sections from rats treated with MMC (3 mg/kg, once weekly) or CDDP (4 mg/kg, once weekly) for 2 or 4 weeks, followed by quantification of the percentage of positive cells within the pulmonary vasculature. n = 6 rats per group. (K, L) Immunofluorescence staining for the endothelial cell marker vWF and the DNA damage marker γ‐H2AX in lung sections from rats treated with MMC (3 mg/kg, once weekly) or CDDP (4 mg/kg, once weekly) for 4 weeks, followed by correlation analysis between γ‐H2AX fluorescence intensity within vWF‑positive regions and vessel wall area percentage. n = 6 rats per group, with 10–25 vessels analyzed per group. Data are presented as mean ± SD. Differences between two groups were analyzed by t ‐test for C (FANCD2, FANCL), D (all panels), G (FANCD2, γ‐H2AX), H (all panels), and L (CDDP), Mann‐Whitney U‐test for C (γ‐H2AX), D (PCNA), G (FANCL), and L (MMC), or one‐way ANOVA with Tukey's honestly significant difference (Tukey's HSD) post hoc test for J. A value of P < 0.05 is considered statistically significant, n.s. indicates no statistical significance. Scale bar = 50 µm in I and 25 µm in K. NC: normal control.

Article Snippet: Primary antibodies for fibronectin (FN, #15613‐I‐AP, Proteintech, Rosemont, IL, USA), Twist (ab50887, Abcam, Cambridge, MA, USA), Vimentin (#5741, Cell Signaling Technology, Danvers, MA, USA), Snail (#ab53519, Abcam, Cambridge, MA, USA), ZO‐1 (21773‐1‐AP, proteintech, Wuhan, China), VEGFR2 (9698, CST, Boston, USA), Slug (YM8357, Immunoway, California, USA), proliferating cell nuclear antigen (PCNA, ab29, Abcam, Cambridge, MA, USA), CD31 (AF3628, R&D Systems, Minneapolis, MN, USA), Cleaved caspase3 (#9664, Cell Signaling Technology, Danvers, MA, USA), Bax (#2772S, Cell Signaling Technology, Danvers, MA, USA), B‐cell lymphoma/leukemia 2 (Bcl‐2, 13–8800, Thermo Fisher Scientific, Rockford, IL, USA), γ‐H2AX (#05‐635, EMD Millipore/Merck, USA), α‐smooth muscle actin (α‐SMA, ab124964 and ab7817, Abcam, Cambridge, MA, USA), FANCL (LS‐C807939, LifeSpan BioSciences, Seattle, WA, USA), FANCD2 (NB100‐182, Novus Biologicals, USA), general control nonderepressible 2 (GCN2, #46041, Cell Signaling Technology, Danvers, MA, USA), and β‐actin (66009‐1‐Ig, Proteintech, Rosemont, IL, USA) were used for immunoblotting, immunofluorescence (IF), and immunohistochemistry (IHC) staining.

Techniques: Western Blot, Expressing, Tissue, Immunohistochemical staining, Staining, Marker, Immunofluorescence, Analysis, Fluorescence, MANN-WHITNEY, Control