Fig 2: Binding of hnRNP E1 to telomeric DNA.(A, B) Agarose gel showing fractionation of ChIP-PCR products generated by telomere primers (tel1 and tel2) from mouse cell line NMuMG. M, DNA molecular weight markers; Control: no antibody control, FLAG: FLAG antibody used to pull-down FLAG-tagged hnRNP E1, Trf2:Trf2 antibody used to pull-down Trf2. (C) Autoradiogram of electrophoretic mobility shift assay showing hnRNP E1 binding to mutants of TEL DNA C that contain mutations in four or five first “C”s of CCC repeats present in a 33 base deoxyoligonucleotide with five repeats of “CCCTAA.” (D) Immunofluorescence showing i-motifs in a single nucleus of A549 and E1KD cells. Fig 2J shows multiple nuclei.

Fig 3: Matched ADRN and MES cell lines show prominent differences in telomere protein expression and localization.a and b Western analysis of cell lineage, DNA sensing, and telomere-related factors in three matched ADRN and MES cell lines. The LA1-55N (ADRN) and LA1-5S (MES) pair, as well as the LA1-66N (ADRN) and LA1-6S (MES) pair, were both derived from LAN-1, a MYCN-amplified NB tumor. The SH-SY5Y (ADRN) and SH-EP1 (MES) pair was derived from a non-MYCN-amplified tumor. ADRN and MES cell line names are displayed in blue and green, respectively, in all figures in this study. c and d IF-FISH analysis of TRF1 and TRF2 in the indicated cell lines. Prominent examples of peri-nuclear staining of TRF2 in LA1-55N are marked with white arrows.

Fig 4: Recruitment of 53BP1 and TRF2 to fs NIR laser-induced DNA lesions. (A) EGFP-53BP11220-1711 and TRF2-YFP cell lines were subjected to NIR laser microirradiation with the input power ranging from 20 mW to 155 mW (corresponding to ˜7 × 1010 W/cm2 to 5.41 × 1011 W/cm2 peak irradiance) as indicated. EGFP-53BP11220-1711 and TRF2-YFP recruitment was monitored for 15 min and 6 min p.i., respectively. The number of the cells with the recruitment and the number of cells examined are shown at the top of each histogram. Detailed microirradiation energy and irradiance calculations versus the laser input power (the power of the laser beam entering the microscope) are shown in Table 2. (B) Time course fluorescence measurement of the damage-site recruitment of EGFP-53BP11220-1711 and TRF2-YFP. PtK2 cells stably expressing EGFP-53BP11220-1711 or TRF2-YFP were damaged with either low (60 mW input power with peak irradiance of ˜2.1 × 1011 W/cm2; upper panel) or high (100 mW input power with peak irradiance of 3.49 × 1011 W/cm2; lower panel) dose of NIR laser microirradiation as indicated. For EGFP-53BP11220-1711, four and three cells were followed for 30 min for 60 mW and 100 mW damage, respectively. For TRF2-YFP, four cells each were followed for 4 min. The fluorescent intensity at the damage site was divided by that in the nucleoplasm and was subtracted by one for normalization. (C) Similar experiments as in (B) were performed following EGFP-53BP11220-1711 recruitment, and live fluorescent images of one representative cell each at indicated p.i. time points are shown. Time 0:00 refers to pre-laser condition. The irradiation site is indicated by a black line at time 0:00 and white arrowheads. Consistent results were obtained with 12 and 8 cells examined for the low (60 mW) and high (100 mW) irradiation condition, respectively. Scale bar = 10 µm. (D) Similar experiments as in (C) for TRF2-YFP. Consistent results were obtained with 11 and 12 cells examined for the low (60 mW) and high (100 mW) irradiation condition, respectively. Scale bar = 10 µm.

Fig 5: ZBTB48 binds telomeres via its ZnF11 domain in vitro and in vivo Domain structure of wild-type (WT) ZBTB48 containing a N-terminal BTB domain and 11 zinc fingers (ZnF) at the C-terminus of the 688-amino-acid protein. Note that ZnF2 is degenerate. Below is a schematic of the deletion construct lacking ZnF1-10 used in (B).Sequence-specific DNA pull-downs with either telomeric (TTAGGG) or a control sequence (GTGAGT) for FLAG-ZBTB48 WT, point mutants for the ten functional zinc fingers and a domain deletion construct for ZnF1-10.Sequence-specific DNA pull-downs for FLAG-ZBTB48 WT and ZnF11 point mutant for telomeric and subtelomeric variant repeat sequences (green) and their respective scrambled controls (blue).Co-localization analysis of endogenous ZBTB48 and TRF2 in U2OS cells by immunofluorescence (IF) staining. A representative image illustrating the co-localization between ZBTB48 (green) and TRF2 (red) as a marker for telomeres is shown with DAPI (blue) used as a nuclear counterstain. The quantification of frequency of co-localization events (right) was done after 3D reconstruction of the acquired z-stacks (n = 104 cells). The average value is indicated by a red bar.IF stainings for exogenous FLAG-ZBTB48 WT and point mutants for ZnF10 and ZnF11 in U2OS cells. The same analysis as in (D) was performed and average co-localization frequencies are shown (n = 24–33 cells). Error bars indicate standard deviations, and P-values are based on Student's t-test.Co-localization analysis between ZBTB48 (green) and PML (red) as a marker for PML bodies analogous to (D) in U2OS cells (n = 102 cells).Data information: (D–F) Co-localization events are indicated by white arrows. Scale bars represent 5 µm.Source data are available online for this figure.