Journal: EMBO Reports

Article Title: Arginine methylation-dependent METTL14-SMN interaction regulates RNA m 6 A homeostasis

doi: 10.1038/s44319-025-00590-7

Figure Lengend Snippet: ( A ) Inhibiting PRMT activity or knockdown of SMN expression reduces m 6 A deposition on DNA repair genes. MeRIP-qPCR was performed to detect the levels of m 6 A on the transcripts of several DNA repair genes. RNA was extracted from control, MS023-treated, and SMN knockdown HeLa cells followed by immunoprecipitation using an m 6 A antibody. * P = 0.0151, * P = 0.0144, * P = 0.0225, * P = 0.0386, * P = 0.0165, * P = 0.0272, *** P = 0.0005, ** P = 0.0073, ** P = 0.0047, *** P = 0.0008, *** P = 0.0001, *** P = 0.0007, ** P = 0.0012, from left to right. ( B ) The levels of m 6 A on the transcripts of several DNA repair genes were reduced in SMA patient-derived fibroblasts. Similar to ( A ), RNA extracted from SMA patient-derived fibroblasts was subjected to MeRIP-qPCR analysis. * P = 0.0131, * P = 0.0225, * P = 0.0466, * P = 0.0125, * P = 0.0285, ** P = 0.0018, * P = 0.0194, * P = 0.0245, * P = 0.0217, * P = 0.0110, ** P = 0.0025, ** P = 0.0011, ** P = 0.0013, from left to right. ( C ) Inhibiting PRMT activity or knockdown of SMN expression reduces the protein levels of several DNA repair genes. Western blot analysis was performed on total cell lysates from HeLa cells treated with MS023 or transfected with siRNA targeting SMN. ( D ) Knockdown of SMN dampens Mitomycin C (MMC)-induced FANCD2 expression. HeLa cells were transfected with either control siRNA (siControl) or SMN-specific siRNA (siSMN) and subjected to a dosage-dependent MMC treatment for 72 h. The total cell lysates were subjected to western blot analysis using indicated antibodies. ( E ) Knockdown of SMN has a marginal effect on FANCD2 mRNA expression. The RNA expression of FANCD2 was analyzed in HeLa cells treated as described in ( D ) by using RT-qPCR. *** P = 0.0008, **** P < 0.0001, * P = 0.0168, from left to right. ( F ) Knockdown of SMN dampens MMC-induced m 6 A deposition on FANCD2 transcripts. MeRIP-qPCR was performed to compare the levels of m 6 A on FANCD2 in HeLa cells treated as described in ( D ). *** P = 0.0006, ** P = 0.0014, *** P = 0.0002, from left to right. ( G ) The protein levels of several DNA repair genes were reduced in SMA patient-derived fibroblasts. Western blot analysis was performed on total cell lysates from SMA patient-derived fibroblasts. ( H ) SMN loss sensitizes fibroblasts to DNA damage induced cell death. MMC and Cisplatin induced cell death on SMA patient-derived fibroblasts were visualized by bright field microscopy. ( I ) Similar to ( H ), the viability of SMA patient-derived fibroblasts were quantitively measured using CCK-8 assay under increasing dosage of MMC and Cisplatin. In ( A , B , E , F ), data from three independent replicates were analyzed by Student’s t test and shown as mean ± SD. *** P = 0.0002, * P = 0.0133, ** P = 0.0040, from top to bottom. .

Article Snippet: Rabbit anti-FANCD2 antibody , Novus Biologicals , NB100-182SS.

Techniques: Activity Assay, Knockdown, Expressing, Control, Immunoprecipitation, Derivative Assay, Western Blot, Transfection, RNA Expression, Quantitative RT-PCR, Microscopy, CCK-8 Assay

Journal: Journal of Clinical Investigation

Article Title: Spontaneous abrogation of the G2 DNA damage checkpoint has clinical benefits but promotes leukemogenesis in Fanconi anemia patients

doi: 10.1172/jci43836

Figure Lengend Snippet: Figure 1 Attenuation of G2 arrest — a new phenotype in FA. (A) Cell cycle analysis of PHA-stimulated PBLs from a classical FA patient (FA), an attenuated FA patient (ATT), a revertant (REV; somatic mosaic), and a healthy control (Ctrl). The arrow indicates the typical MMC-induced G2 arrest of clas- sical FA cells, and the asterisk designates the G2 checkpoint abrogation in the attenuated cells; as expected, revertant cells demonstrated no G2 arrest. (B) Cell cycle analysis of primary fibroblasts (fibro) from the same patients, showing G2 arrest (arrow) and confirming the constitutive FA phenotype and dissociation with PBLs in attenuated and revertant patients. Horizontal bars in A and B indicate the G1 and G2 cell cycle phases (M1 and M2, respectively). (C) Immunoblot analysis showed that attenuated FA PBLs lacked the large monoubiquitinated 162-kDa isoform of FANCD2 (asterisk), like classical FA cells and unlike revertant cells; primary fibroblasts retained the FA phenotype. (D) Attenuated cells still have a high number of MMC-induced chromosomal breaks, like classical FA cells and unlike revertant cells (original magnification, ×630). Arrows indicate the chromosome breaks. Breaks scoring are shown in Supplemental Figure 1. (E) Classification of the FA patients as having classical, revertant, and attenuated PBL phenotypes and ordering by class age: fewer than 5 years (n = 9; 9.5%), 5–9 years (n = 31; 32.6%), 10–14 years (n = 15; 15.8%), 15–19 years (n = 11; 11.6%), 20–24 years (n = 10; 10.5%), 25–29 years (n = 9; 9.5%), and more than 30 years (n = 10; 10.5%). For the clarity of the figure, 2 patients whose fibroblasts were later found to be intermediate for G2 arrest are not shown (see text).

Article Snippet: Antibodies were purchased against CHK1, CHK2, p53, ATM, CDC25A, actin (all from Santa Cruz Biotechnology Inc.), FANCD2 (Novus Biologicals), ATR (Cell Signaling), BRCA1 (Calbiochem), and vinculin (Abcam).

Techniques: Cell Cycle Assay, Control, Western Blot

Journal: Journal of Clinical Investigation

Article Title: Spontaneous abrogation of the G2 DNA damage checkpoint has clinical benefits but promotes leukemogenesis in Fanconi anemia patients

doi: 10.1172/jci43836

Figure Lengend Snippet: Figure 4 Inhibition of CHK1 and ATR, but not CHK2, ATM, or BRCA1, mimics the attenuated phenotype in FA cells. (A) Cell cycle analysis of MMC- induced G2 arrest in HeLa cells transfected with combinations of siRNAs (si) against luciferase (control), FANCD2, and the checkpoint genes CHK1, CHK2, ATR, ATM, and BRCA1, as indicated. FANCD2-silenced HeLa cells displayed a strong MMC-induced G2 arrest and were used as FA-like cells for convenience in cotransfection experiments. The immunoblot shows the knockdown of different proteins for each siRNA. Arrows and asterisks indicate typical FA G2 arrest and its abrogation, respectively. All data were obtained in at least 3 independent experiments for each targeted gene with consistent results. (B) Abrogation of MMC-induced G2 arrest by the CHK1 inhibitor SB-218078 (CHK1i) but not by the CHK2 inhibitor C3742 (CHK2i) in FA and non-FA EBV cells. DMSO was used as control, and all data were obtained in 3 independent experiments with consistent results. The arrows indicate the MMC-induced G2 arrest, and the asterisk designates the G2 checkpoint abrogation. Horizontal bars in A and B indicate the G1 and G2 cell cycle phases (M1 and M2, respectively).

Article Snippet: Antibodies were purchased against CHK1, CHK2, p53, ATM, CDC25A, actin (all from Santa Cruz Biotechnology Inc.), FANCD2 (Novus Biologicals), ATR (Cell Signaling), BRCA1 (Calbiochem), and vinculin (Abcam).

Techniques: Inhibition, Cell Cycle Assay, Transfection, Luciferase, Control, Cotransfection, Western Blot, Knockdown

Journal: PLoS ONE

Article Title: Intrinsic Radiosensitivity and Cellular Characterization of 27 Canine Cancer Cell Lines

doi: 10.1371/journal.pone.0156689

Figure Lengend Snippet: (A) Western blot analysis of DNA-PKcs (460 kDa), FANCD2 (165 kDa) and RAD51 (37 kDa). β-actin (42 kDa) expression was used as a control. Each expression band was estimated from molecular weight. (B) Band intensity of western blot. (C, D) Representative images for RAD51 foci (C) and FANCD2 foci (D) co-localized with gamma-H2AX in Moresco cells.

Article Snippet: The primary antibodies used in this study were the mouse anti-DNA-PKcs monoclonal antibody (Ab-4; Neomarkers, Fremont, CA), the rabbit anti-Rad51 polyclonal antibody (H-92; Santa Cruz Biotechnology Inc., Santa Cruz, CA), the rabbit anti-FANCD2 polyclonal antibody (NB100-182; Novus Biologicals, Littleton, CO) and the mouse monoclonal beta-actin antibody (Abram 8226; Abcam, Cambridge, MA).

Techniques: Western Blot, Expressing, Control, Molecular Weight

Journal: eLife

Article Title: LIN37-DREAM prevents DNA end resection and homologous recombination at DNA double-strand breaks in quiescent cells

doi: 10.7554/eLife.68466

Figure Lengend Snippet:

Article Snippet: Antibody , Anti-FANCD2 (Rabbit monoclonal) , R and D Systems , MAB93691 , WB (1:1000).

Techniques: Recombinant, Plasmid Preparation, Genome Wide, CRISPR, Clone Assay, Flow Cytometry, Staining, Sequencing, Software, Microscopy