Fig. 1: MYC induces Z-DNA formation through the FACT complex.

a IF results showing the localization and expression levels of MYC and Z-NA. HA-tagged MYC was overexpressed in U2OS cells and stained with HA antibodies 3 days after transfection. Z-NA was detected using Z22 antibodies. Nuclei were stained with DAPI. Scale bar, 10 µm. b Z22 fluorescence intensity quantification results for each cell in (a). The data were represented by the scatter dots and mean values (WT, N = 50 cells, HA-MYC, N = 65 cells). p value was determined by unpaired two-sided t-test. The exact p values were indicated in the figure. Source data are provided as a Source Data file. c The correlation between HA-MYC and Z22 fluorescence intensity among each cell (N = 50 cells) in (a). Data were analyzed using simple linear regression (two-sided test), with no adjustments for multiple comparisons. The exact p values were indicated in the figure. Source data are provided as a Source Data file. d IF results showing the Z-NA signals after treatment with various nucleases. U2OS cells expressing HA-MYC for 3 days were fixed, permeabilized, and then treated with 0.2 U/μL DNase I, 1 mg/mL RNase A, and 0.2 U/μL RNase H, respectively, at 37 °C for 3 h. The efficiencies of DNase I, RNase H, and RNase A treatments were verified by DAPI, S9.6, and Acridine Orange staining, respectively. Cells treated with PBS served as the negative control. Scale bar, 10 µm. e Z22 fluorescence intensity quantification results of each cell in (d). The data were represented by the scatter dots and mean values (WT, N = 51 cells; PBS control for DNAse I treatment, N = 52 cells; DNAse I, N = 52 cells; PBS control for RNAseH treatment, N = 64 cells; RNAseH, N = 54 cells; PBS control for RNAseA treatment, N = 64 cells; RNAseA, N = 63 cells). p value was determined by unpaired two-sided t-test. The exact p values were indicated in the figure. Source data are provided as a Source Data file. f IF results showing the MYC and Z-NA signals in H526 (low MYC expression), H211 and H82 cells (high MYC expression) of human SCLC lines. Scale bar, 10 µm. g Z22 fluorescence intensity quantification results for each cell in (f). The data were represented by the scatter dots and mean values (H526, N = 53 cells; H211, N = 52 cells; H82, N = 53 cells). p value was determined by unpaired two-sided t-test. The exact p values were indicated in the figure. Source data are provided as a Source Data file. h IF results showing the MYC and Z-NA signals in RPS1 (low MYC expression), RPP-mTmG and RPP-GSDME-KO cells (high MYC expression) of mouse SCLC lines. Scale bar, 10 µm. i Z22 fluorescence intensity quantification results for each cell in (h). The data were represented by the scatter dots and mean values (RPS1, N = 53 cells; RPP-mTmG, N = 55 cells; RPP-GSDME-KO, N = 55 cells). p value was determined by unpaired two-sided t-test. The exact p values were indicated in the figure. Source data are provided as a Source Data file. j Z22 fluorescence intensity quantification results of U2OS cells treated with different RNAP II inhibitors. Cells expressing HA-tagged MYC were treated with 1 μM BMH−21, 1 μM Flavopiridol, 1 μM THZ1, 1 μg/mL Actinomycin-D, and 10 μg/μL α-amanitin for 6 h. The data were represented by the scatter dots and mean values (Vector, N = 61 cells; DMSO, N = 57 cells; BMH-21, N = 69 cells; Flavopiridol, N = 52 cells; THZ1, N = 67 cells; Actinomycin-D, N = 74 cells; α–amanitin, N = 68 cells). p value was determined by unpaired two-sided t-test. The exact p values were indicated in the figure. Source data are provided as a Source Data file. k IF results showing the Z-DNA signals in HA-MYC overexpressing cells 3 days after SSRP1 or SPT16 depletion. NT non-target control. Scale bar, 10 µm. l Z22 fluorescence intensity quantification results for each cell in (k). The data were represented by the scatter dots and mean values (WT, N = 59 cells; shNT shSSRP1-1, N = 69 cells; shSSRP1-2, N = 60 cells; shSPT16-1, N = 60 cells; shSPT16-2, N = 61 cells). p value was determined by unpaired two-sided t-test. The exact p values were indicated in the figure. Source data are provided as a Source Data file. m SSRP1 bound with MYC and SPT16. FLAG-tagged SSRP1 was purified by FLAG beads in HEK293T cells. Proteins from input and IP samples were analyzed by Western blotting using the indicated antibodies. The assay was performed in three independent biological replicates with similar results. Source data are provided as a Source Data file. n Endogenous MYC bound with SSRP1 and SPT16. MYC antibody was used to pull down endogenous MYC and bound FACT proteins in HEK293T cells. Proteins from input and IP samples were analyzed by Western blotting using the indicated antibodies. The assay was performed in three independent biological replicates with similar results. Source data are provided as a Source Data file. o IF results showing DAPI and GFP merged signals after GFP_1-10-tagged MYC and GFP₁₁-tagged SSRP1 or SPT16 were expressed in U2OS cells together or respectively. Scale bar, 10 µm. The assay was performed in three independent biological replicates with similar results.