Journal: Nature Communications

Article Title: Bloom helicase mediates formation of large single–stranded DNA loops during DNA end processing

doi: 10.1038/s41467-022-29937-7

Figure Lengend Snippet: a Western blots of extracts from U2OS (SA-GFP and DR-GFP) cells depleted for BLM and EXO1 were reconstituted with the indicated genotypes and immune-stained with anti-BLM and anti-Tubulin antisera, as indicated. b Bar graph showing results for the SA-GFP reporter assay in U2OS cells with the indicated genotypes; data points represent %SSA. c Bar graph showing results for the DR-GFP reporter assay in U2OS cells with the indicated genotypes; data points represent %HR. In b and c data points reflect the mean ± SEM for N = three independent experiments.

Article Snippet: NC membranes were blocked using 5% dry milk in TBST (Tris-buffered saline with 0.1% Tween® 20 detergent) and stained with primary antibodies: anti-BLM (Santa Cruz Biotechnology, Cat. No.: SC-365753; used at a 1:2000 dilution; or Bethyl, Cat. No.: A300-100A; used at a 1:1000 dilution), anti-EXO1(Bethyl, Cat. No.: A302-639A; used at a 1:1000 dilution), anti-FLAG (M2) (Sigma Cat. No.: F1804; used at a 1:1000 dilution), anti-Actin (CST, Cat. No.: 12262S; used at a 1:3000 dilution), anti-GFP (Invitrogen, Cat. No.: A-6455; used at a 1:4000 dilution), or Alpha–Tubulin–HRP (Cell Signaling Technology, Cat. No.: CST 11H10; used at a 1:5000 dilution), as indicated, O/N at 4 °C, washed with TBST buffer 3 times for 10 min each and incubated with appropriate secondary antibody (anti-mouse IgG HRP, Invitrogen Cat. No.: A16078; used at a 1:4000 dilution; or anti-rabbit IgG Fc HRP, Cat. No.: A16116; used at a 1:4000 dilution) for 1 h at RT.

Techniques: Western Blot, Staining, Reporter Assay

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: Membranes were then incubated at 4 °C ON with the following primary antibodies: rabbit polyclonal anti-BLM (#A300-110A, Bethyl) (1:500); rabbit polyclonal anti-FANCJ (#B1310, Sigma-Aldrich) (1:1000), and mouse monoclonal anti-Vinculin (#V9131, Sigma-Aldrich) (1:1000).

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: Membranes were then incubated at 4 °C ON with the following primary antibodies: rabbit polyclonal anti-BLM (#A300-110A, Bethyl) (1:500); rabbit polyclonal anti-FANCJ (#B1310, Sigma-Aldrich) (1:1000), and mouse monoclonal anti-Vinculin (#V9131, Sigma-Aldrich) (1:1000).

Techniques: Western Blot, Standard Deviation