Fig. 5. TERRA G-quadruplex structure is required for LSD1 phase separation and ALT functions.

Representative DIC images (a) and turbidity assay (b) of LSD1^ΔIDR (20 μM) with the addition of different RNAs (20 μM). Turbidity measurements are shown as mean ± SEM (n = three independent experiments, one-way ANOVA). c Representative images of fluorescent LSD1^ΔIDR (50 μM) with 12× TERRA (25 μM) pretreated with G-quadruplex binding ligand, N-methyl mesoporphyrin IX, (NMM) (n= three independent experiments). d In vitro EMSA of 8X TERRA GG(UUAGGG)₈U (+ = 2 μM) alone and in the presence of LSD1 (+ = 10 μM and ++ = 20 μM) and NMM (+ = 200 μM). The 0.75% agarose gel contained Syber Safe dye (10,000:1, Invitrogen) and was performed under the following conditions (60 V, 1 h, 4 °C). The fluorescence data were visualized at 520 nm using an Amersham Imager 600 (AI600) instrument (n = two independent experiments). e Representative images and f quantification of telomere clustering (DNA FISH) after treatment of NMM (200 nM, 24 h) in FokI stable cell line (n = 77 cells for DMSO, n = 80 cells examined for NMM over two independent experiments, two-tailed unpaired t test). g Representative images and h quantification of telomere clustering (DNA FISH) after co-transfection of eDHFR-DHX36, Halo-TRF1 and mCh-FokI with or without TFH treatment in U2OS cells (n = 96 cells for -TFH, n = 99 cells examined for +TFH over three independent experiments, two-tailed unpaired t test). DHX36 are indicated by antibody-based immunostaining. i Representative images and j quantification of telomere clustering (DNA FISH) after co-transfection of polyC with mCh-FokI in U2OS cells (n = 69 cells for -polyC, n = 84 cells for +polyC over three independent experiments, two-tailed unpaired t test). Error bars are mean ± SEM. N.S., not significant. Source data are provided as a Source Data file.