Extended Data Fig. 8: Loss of INTS3 impairs uridine-rich small nuclear RNA processing and blocks myeloid differentiation.
From: Coordinated alterations in RNA splicing and epigenetic regulation drive leukaemogenesis
a, Schematic of snRNA processing site and qPCR primers for detecting cleaved or uncleaved snRNA. b, qPCR (top; n = 3; mean ± s.d.; a two-sided Student’s t-test) and representative western blot of INTS3 in HL-60 cells transduced with shRNAs targeting human INTS3 (bottom, representative results from three biologically independent experiments). c–e, s, t, qPCR results of U2 (c, s) and U4 (d, t) snRNAs in isogenic HL-60 cells and U7 snRNA in murine cells from Extended Data Fig. 6n (e). Ratio of uncleaved/total snRNAs expression was compared (n = 3, mean ratio ± s.d.; one-way ANOVA with Tukey’s multiple comparison test; the largest P values calculated among 2 × 2 comparisons of two components from different groups are shown. For example, P values were calculated from the following four comparisons; bars 1 versus 3, 2 versus 3, 1 versus 4, 2 versus 4). f, Schematic of the U7 snRNA–GFP reporter. g, v, Flow cytometry analysis of 293T cells transduced with U7 snRNA-GFP reporter and IDH2, SRSF2 and INTS3 constructs as labelled on the right (representative results from three biologically independent experiments are shown). h, w, Quantification of per cent GFP− and GFP+ 293T cells (n = 3 biologically independent experiments, mean percentage ± s.d.; one-way ANOVA with Tukey’s multiple comparison test; P values are shown as in c). i, l, y, Flow cytometry analysis of CD11b expression in isogenic HL-60 cells after ATRA treatment for two days (representative results from three biologically independent experiments are shown). j, m, z, Quantification of percentages of CD11b+ HL-60 cells over time (n = 3; mean percentage ± s.d.; two-way ANOVA with Tukey’s multiple comparison test). k, n, Cytomorphology of isogenic HL-60 cells after ATRA treatment for two days (Giemsa staining; scale bar, 10 μm; original magnification, ×400; representative results from three biologically independent experiments are shown). o, p, qPCR of Ints3 (o) (mean ± s.d.; Kruskal–Wallis tests with uncorrected Dunn’s test) and western blot of INTS3 (p) in Ba/F3 cells transduced with shRNAs targeting mouse Ints3. q, r, Representative cytomorphology (q) and immunophenotype (r) of colony cells at the sixth colony. Normal BMMNCs were used as a control (the percentage listed represent the percent of cells within live cells; representative results from three biologically independent experiments are shown). u, x, Western blot of proteins extracted from HL-60 cells (u) assayed in s-t and y-z and 293T cells (x) assayed in v and w (representative results from three biologically independent experiments). *P < 0.05, **P < 0.01, ***P < 0.001, #P < 0.05, ##P < 0.01, ###P < 0.001.
