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

Journal: Blood Advances

Article Title: Inhibition of NAMPT targets DNA damage response to sensitize alkylating chemotherapy in TP53 mutant mantle cell lymphoma ∗

doi: 10.1182/bloodadvances.2025016765

Figure Lengend Snippet: Inhibition of NAMPT function via KPT-9274 inhibits tumor growth in vivo. (A) NOG mice were injected subcutaneously with Mino cells. After detection of the tumor, the mice were randomized and treated orally with either KPT-9274 or the vehicle for 5 consecutive days per week for 3 weeks. Tumor volume was evaluated via caliper measurement. Differences between the 2 groups were evaluated using the standard t test. (B) Tumor cells collected from mice were lysed in radio-immunoprecipitation assay buffer, and the whole-cell lysate was subjected to WB analysis and probed with antibodies against Cl-PARP, FANCD2, RAD51, and γ-H2AX. GAPDH was used as the loading control. ns > .05; ∗∗ P ≤ .01; ∗∗∗ P ≤ .001; ∗∗∗∗ P ≤ .0001.

Article Snippet: Western blotting (WB) was performed to evaluate the expression levels of total protein and phospho-specific isoforms using the following antibodies: FANCD2 (Santa Cruz Biotechnology, sc-20022), RAD51 (Santa Cruz Biotechnology, sc-398587), PBEF (Santa Cruz Biotechnology, sc-166946), cleaved Caspase3 (Cell Signaling Technology, 9664s), PARP (Cell Signaling Technology, 9532s), cleaved PARP (Cell Signaling Technology, 5625s), γ-H2AX (Ser139; Cell Signaling Technology, 9718s), p-CHK1 (Ser345; Cell Signaling Technology, 2341s), p53 (Cell Signaling Technology, 2527s), p-ATR (Ser428; Cell Signaling Technology, 2853s), p-ATM (Ser1981; Cell Signaling Technology, 4526s), and p-CHK2 (Thr68; Cell Signaling Technology, 2197s).

Techniques: Inhibition, In Vivo, Injection, Radio Immunoprecipitation, Control

Journal: Blood Advances

Article Title: Inhibition of NAMPT targets DNA damage response to sensitize alkylating chemotherapy in TP53 mutant mantle cell lymphoma ∗

doi: 10.1182/bloodadvances.2025016765

Figure Lengend Snippet: Inhibition of NAMPT function via KPT-9274 inhibits tumor growth in vivo. (A) NOG mice were injected subcutaneously with Mino cells. After detection of the tumor, the mice were randomized and treated orally with either KPT-9274 or the vehicle for 5 consecutive days per week for 3 weeks. Tumor volume was evaluated via caliper measurement. Differences between the 2 groups were evaluated using the standard t test. (B) Tumor cells collected from mice were lysed in radio-immunoprecipitation assay buffer, and the whole-cell lysate was subjected to WB analysis and probed with antibodies against Cl-PARP, FANCD2, RAD51, and γ-H2AX. GAPDH was used as the loading control. ns > .05; ∗∗ P ≤ .01; ∗∗∗ P ≤ .001; ∗∗∗∗ P ≤ .0001.

Article Snippet: Western blotting (WB) was performed to evaluate the expression levels of total protein and phospho-specific isoforms using the following antibodies: FANCD2 (Santa Cruz Biotechnology, sc-20022), RAD51 (Santa Cruz Biotechnology, sc-398587), PBEF (Santa Cruz Biotechnology, sc-166946), cleaved Caspase3 (Cell Signaling Technology, 9664s), PARP (Cell Signaling Technology, 9532s), cleaved PARP (Cell Signaling Technology, 5625s), γ-H2AX (Ser139; Cell Signaling Technology, 9718s), p-CHK1 (Ser345; Cell Signaling Technology, 2341s), p53 (Cell Signaling Technology, 2527s), p-ATR (Ser428; Cell Signaling Technology, 2853s), p-ATM (Ser1981; Cell Signaling Technology, 4526s), and p-CHK2 (Thr68; Cell Signaling Technology, 2197s).

Techniques: Inhibition, In Vivo, Injection, Radio Immunoprecipitation, Control