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: The slides were then incubated in blocking buffer (1% BSA dissolved in 1X PBS [w/v]) and then incubated overnight (ON) at 4 °C with the primary antibodies (rabbit polyclonal anti-BLM (#A300-110A, Bethyl) (1:100); mouse monoclonal anti-TRF1 (4E4 clone, GTX70304, GeneTex) (1:20) or mouse monoclonal anti-TRF2 (9F10 clone, sc-47693, Santa Cruz Biotechnology) (1:100)).

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

Journal: iScience

Article Title: Human SKI component SKIV2L regulates telomeric DNA-RNA hybrids and prevents telomere fragility

doi: 10.1016/j.isci.2024.111096

Figure Lengend Snippet:

Article Snippet: Mouse monoclonal anti-TRF2 (Clone 4A794) , Millipore , Cat#05-521; RRID: AB_2303145.

Techniques: Virus, Recombinant, Protease Inhibitor, Reverse Transcription, Blocking Assay, Mass Spectrometry, SYBR Green Assay, Flow Cytometry, Imaging, Mutagenesis, Cell Cycle Assay, shRNA, Software

Journal: Frontiers in Cell and Developmental Biology

Article Title: Loss of FAM111B protease mutated in hereditary fibrosing poikiloderma negatively regulates telomere length

doi: 10.3389/fcell.2023.1175069

Figure Lengend Snippet: FAM111B -deficient cells have shorter telomeres. (A) Widefield images of chromosome spreads and (B) interphase cells from wild-type (WT) and FAM111B knockout cells stained with fluorescent probe against telomeric repeats. Scale bar 20 μm. (C) Quantification of individual TeloFISH foci intensity in metaphase spreads and interphase cells. Green bars show averages of N = 3, with at least 30 metaphase spreads or 60 cells scored per sample in each experiment. (D) Widefield images of wild-type (WT) and FAM111B −/- U2OS cells stained with TRF2 antibodies. Scale bar 20 μm. (E) Quantification of TRF2 foci intensity in U2OS wild-type (WT) FAM111B negative U2OS cells. Green bars show averages of N = 3 experiments, where 400 cells was scored per sample in each experiment. (F) Quantification of TRF2 intensities in U2OS cells over-expressing empty vector (EV) or FLAG-tagged FAM111B variants wild-type (WT), protease-dead (PD) or HFP mutant (Q430P, QP), respectively. Green bars show averages of N = 3 experiments, with at least 50 cells per sample scored in each experiment. K-W test was used in C, E and (F) .

Article Snippet: Primary antibodies were as follows: mouse anti-human BLM (sc-365753, Santa Cruz Biotechnology) at 1:250 (IF), mouse anti-BrdU (347580, Becton Dickinson) at 1:20 (Flow cytometry), rabbit anti-human FAM111B (HPA038637, Atlas Antibodies) at 1:1,000 (IB and IF), mouse anti-human Histone H3 (10799, Abcam) at 1:1,000 (IB), mouse anti-human Lamin A/C (sc-376248, Santa Cruz Biotechnology) at 1:1,000 (IB and IF), rabbit anti-human Lamin B1 (12987-1-AP, Proteintech Group) at 1:1,000 (IB and IF), rabbit anti-human NUP42 (16587-1-AP, Proteintech Group) at 1:1,000 (IB), mouse anti-human PCNA (sc-56, Santa Cruz Biotechnology) at 1:1,000 (IF), rabbit anti-human SEC13 (15397-1-AP, Proteintech Group) at 1:1,000 (IB), mouse anti-human TRF2 (NB100-56506, Novus Biologicals) at 1:1,000 (IF), mouse anti-human αTubulin (T6074, Sigma) at 1:10,000 (WB) or 1:1,000 (IF).

Techniques: Knock-Out, Staining, Expressing, Plasmid Preparation, Mutagenesis