a300 110a  (Bethyl)

Journal: International Journal of Molecular Sciences

Article Title: ESCO2 Interacts with TRF1/2 and Facilitates Telomere Maintenance

doi: 10.3390/ijms27062635

Figure Lengend Snippet: ESCO2 deficiency results in telomere abnormalities. ( A ) HeLa1.2.11 cells transfected with indicated siRNAs were analyzed by Q-FISH using a FITC-labeled telomere PNA probe. DAPI was used to stain the nuclei. The histograms show the distribution of relative telomere length presented as fluorescence intensity (TFU, telomere fluorescence unit); the red lines mark the mean telomere signal intensity. n indicates the total number of telomere signals detected. Error bars indicate standard errors. *** p < 0.001. ( B ) Examples of telomere abnormalities from ( A ) observed in a telomere FISH assay (upper panel). The incidence of telomere abnormalities in cells lacking ESCO1 or ESCO2 is shown in the bottom panel. ( C ) Sankey diagram showing KEGG pathway analysis of high-confidence proteins associated with ESCO2. Pathway enrichment was performed based on high-confidence ESCO2-interacting proteins, and the results are visualized as a Sankey diagram to illustrate the functional distribution across different KEGG pathways. ( D ) Chord diagram showing KEGG pathway analysis of DNA replication- and repair-related pathways and their associated proteins. Diagram visualizes the relationship between key proteins and their corresponding pathways involved in DNA replication and repair. ( E ) Selected lists of ESCO2-associated proteins analyzed by mass spectrometry. ( F ) Western blot performed to determine ESCO2 and BLM siRNA-knockdown efficiency in HeLa1.2.11 cells. ( G ) Representative images of metaphase telomere FISH in cells from ( G ) (upper panel). The incidence of telomere abnormalities is shown in the bottom panel.

Article Snippet: The antibodies used in this study were: 53BP1 (NB100-904; Novus Biologicals, Littleton, CO, USA), Flag (F3165; Sigma, St. Louis, MO, USA), BLM (A300-110A, Bethyl, Boston, MA, USA), ESCO2 (ab86003; Abcam, Cambridge, UK), Myc (sc-40; Santa Cruz Biotechnology, Santa Cruz, CA, USA), and vinculin (V9131; Sigma-Aldrich, St. Louis, MO, USA).

Techniques: Transfection, Labeling, Staining, Fluorescence, Functional Assay, Mass Spectrometry, Western Blot, Knockdown

Journal: Nucleic Acids Research

Article Title: The SMC5/SMC6 complex is critical for resolving R-loop-induced transcription–replication conflicts

doi: 10.1093/nar/gkaf1537

Figure Lengend Snippet: SMC5/6 is required for recruitment of BLM/TOP3A/RMI (BTRR) complex to TRC. ( A, B, C, D ) U2OS WT and SETX -KO cells with or without RNASEH1 expression were subjected to PLA analysis showing colocalization between TOP3A and FANCD2 (A), TOP3A and SMC5-Flag (B left), TOP3A and R-loops (B right), BLM and SMC5-Flag (C left), RMI1 and SMC5-Flag (C right), BLM and R-loops (D left), and RMI1 and R-loops (D right). ( E ) U2OS SETX -KO cells were infected with SMC5 shRNA or vector. Three days after infection, cells were used for PLA analysis showing colocalization between BLM and R-loops (left), TOP3A and R-loops (middle), and RMI1 and R-loops (right). ( F ) U2OS WT cells were infected with shRNA to knock down indicated genes. Three days after infection, cells were used for PLA analysis showing colocalization between SMC5-Flag and R-loops (S9.6). ( G ) U2OS WT cells were infected with shRNA to knock down indicated genes. Three days after infection, cells were subjected to PLA analysis showing colocalization between replication (PCNA) and transcription (pPOLR2A).

Article Snippet: PCNA (sc-56, Santa Cruz), POLR2A [p Ser2] (NB100-1805), FLAG (F1804, Sigma–Aldrich), BLM (sc-365753, Santa Cruz), TOP3A (this work), RMI1 (14630-1-AP, Proteintech), TOP2A (20233-1-AP, Proteintech), SMC6 (sc-365742, Santa Cruz), FLAG (AE004, Abclonal), FANCD2 (NB100-182SS, Novus Biologicals), SETX (NB100-57542, Novus Biologicals), S9.6 antibody (ENH001, Kerafast), S9.6 antibody (Kf-Ab01137-23.0, Kerafast).

Techniques: Expressing, Infection, shRNA, Plasmid Preparation, Knockdown

Journal: Nucleic Acids Research

Article Title: The SMC5/SMC6 complex is critical for resolving R-loop-induced transcription–replication conflicts

doi: 10.1093/nar/gkaf1537

Figure Lengend Snippet: FANCD2 activation depends on BTRR. ( A ) U2OS WT and SETX -KO cells with or without RNASEH1 expression were used for immunostaining with antibody against FANCD2. ( B ) SMC6-Flag-expressing U2OS WT and SETX -KO cells, with or without RNaseH1 expression, were subjected to PLA analysis showing colocalization between SMC6-Flag and FANCD2. ( C ) U2OS WT and SETX -KO cells with or without RNASEH1 expression were subjected to PLA analysis showing colocalization between BLM and FANCD2. ( D ) U2OS SETX -KO cells were infected with shRNA to knock down indicated genes. Three days after infection, cells were used for immunostaining with an antibody against FANCD2. ( E ) U2OS WT cells were infected with shRNA to knock down indicated genes. Three days after infection, cells were subjected to PLA analysis showing colocalization between FANCD2 and R-loops (S9.6). ( F ) U2OS WT cells were infected with FANCD2 shRNA or vector. Three days after infection, cells were subjected to PLA analysis showing colocalization between SMC5-Flag and BLM (left), SMC5-Flag and TOP3A (middle), and SMC5-Flag and RMI1 (right).

Article Snippet: PCNA (sc-56, Santa Cruz), POLR2A [p Ser2] (NB100-1805), FLAG (F1804, Sigma–Aldrich), BLM (sc-365753, Santa Cruz), TOP3A (this work), RMI1 (14630-1-AP, Proteintech), TOP2A (20233-1-AP, Proteintech), SMC6 (sc-365742, Santa Cruz), FLAG (AE004, Abclonal), FANCD2 (NB100-182SS, Novus Biologicals), SETX (NB100-57542, Novus Biologicals), S9.6 antibody (ENH001, Kerafast), S9.6 antibody (Kf-Ab01137-23.0, Kerafast).

Techniques: Activation Assay, Expressing, Immunostaining, Infection, shRNA, Knockdown, Plasmid Preparation