α blm  (Santa Cruz Biotechnology)

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

Journal: Communications Biology

Article Title: BLM and FANCJ role in the response to G-quadruplex-dependent telomeric replicative stress

doi: 10.1038/s42003-025-09367-z

Figure Lengend Snippet: a The diagram shows the workflow on a timeline: on the day after cell seeding, cells were treated with RHPS4 (0.2 and/or 0.5 μM). At 48 h after treatment, cells were again treated with RHPS4 (0.2 and/or 0.5 μM) or harvested and counted. At 72 h after the first treatment, the cell viability was checked, and finally at 96 h after the first treatment, the cells were harvested and processed. b Representative images showing U251MG cells stained by immunofluorescence using anti-BLM (red signals), and anti-TRF1 or anti-TRF2 (green signals) antibodies. Merged images allow visualization of colocalizing dots (yellow signals). Yellow arrows indicate BLM and TRF1-TRF2 colocalizations. c Quantification of the colocalizations between BLM and TRF1 or TRF2 proteins in U251MG cell line upon RHPS4 treatment (0.2 and 0.5 μM). d , e Sensitivity of U251MG and BLM −/− cell lines to RHPS4 concentrations ranging from 0.01 to 2 μM (0.01; 0.125; 0.25; 0.5; 1; 2 μM), evaluated 96 h after the first treatment. Mitomycin C (MMC) was used as a positive control at concentrations of 0.1; 0.5; 1; 2; 5 μg/ml. The Sulforhodamine B (SRB) cytotoxicity assay showed that RHPS4 sensitivity was unchanged in U251MG and BLM −/− (IC50 was 0.56 μM in both cell lines). f Short-term cell proliferation in untreated cells, U251MG and BLM −/− , and in RHPS4-treated cells (0.5 μM) as evaluated 48 and 96 h after the first treatment. g Long-term cell proliferation in U251MG and BLM −/− untreated and RHPS4-treated cells (0.5 μM). Scale bars represent 5 μm. * p < 0.05, ** p < 0.01 (two-way ANOVA; n = 3). Error bars denote the standard deviation of the mean.

Article Snippet: Successively, samples were processed for immunolabeling with mouse monoclonal anti-BLM (B-4 clone, sc-365753, Santa Cruz Biotechnology) and rabbit polyclonal anti-FANCJ (#B1310, Sigma-Aldrich) antibodies.

Techniques: Staining, Immunofluorescence, Positive Control, Cytotoxicity Assay, Standard Deviation

Journal: Communications Biology

Article Title: BLM and FANCJ role in the response to G-quadruplex-dependent telomeric replicative stress

doi: 10.1038/s42003-025-09367-z

Figure Lengend Snippet: a Representative images of U251MG and siFANCJ cells immunostained using anti-BLM and anti-FANCJ antibodies (red and green signals, respectively). b The graph shows the frequency of BLM (red circles), FANCJ (green circles), and colocalization (orange circles) foci in untreated and RHPS4-treated U251MG, siSCR, and siFANCJ cells. * p < 0.05, **** p < 0.0001 (ordinary one-way ANOVA; n = 3). c Western blot showing BLM protein levels in U251MG and siFANCJ cells. d Western blot representative image and densitometric analysis of FANCJ protein level in U251MG and BLM −/− cells. * p < 0.05 (Unpaired t -test) ( n = 3). e Representative PLA images of BLM −/− , U251MG, and RHPS4-treated U251MG cells. BLM −/− cells were used as controls. f Quantification of the PLA signal was significantly higher in RHPS4-treated than in untreated cells, indicating that the treatment induced an increase in FANCJ–BLM interaction. * p < 0.05, **** p < 0.0001 (ordinary one-way ANOVA, Dunnett’s post-test; n = 3). Error bars denote the standard deviation of the mean.

Article Snippet: Successively, samples were processed for immunolabeling with mouse monoclonal anti-BLM (B-4 clone, sc-365753, Santa Cruz Biotechnology) and rabbit polyclonal anti-FANCJ (#B1310, Sigma-Aldrich) antibodies.

Techniques: Western Blot, Standard Deviation