Figure 9. Protein aggregates in... | Rockefeller University Press

Figure 9.

$Protein aggregates in SETX-deficient cells are driven by RNA POL1 and non-coding RNAs from the rDNA loci. (A) Levels of IGS ncRNAs were measured in U2OS control or SETX knock-out cells with mock or BMH-21 (1 μM) treatment, as indicated, by RT-qPCR, normalized by 7SK RNA. Error bars indicate standard deviation. *, **, ***, and **** indicate P < 0.05, 0.01, 0.001, and 0.0001, respectively, by two-tailed t test assuming unequal variance between control cells with BMH-21 or mock treatment, and between SETX knock-out cells with BMH-21 or mock treatment. (B) Detergent-resistant aggregates were isolated from control or SETX knock-out U2OS cells with mock or BMH-21 (1 μM) treatment as indicated. Abundance of PSMD2, TDP-43, and PSMD8 in lysate and aggregate fractions are shown by Western blotting. (C) Levels of PSMD2, TDP-43, and PSMD8 in aggregate fractions were quantified in three independent experiments of B and normalized by lysate; shown relative to control cells. Error bars indicate standard error. *, **, ***, and **** indicate P < 0.05, 0.01, 0.001, and 0.0001 by two-tailed t test assuming unequal variance; ns = not significant. (D and E) Levels of IGS ncRNAs were measured in U2OS shSETX or SETX knock-out cells, respectively, with Adm Cas13d expression and either control vector (NT) or Cas13d gRNA expression by RT-qPCR, normalized by 7SK RNA. (F and G) Detergent-resistant aggregates were isolated from shSETX or SETX knock-out U2OS cells with Adm Cas13d expression and either control vector (NT) or Cas13d gRNA expression. PSMD2, TDP-43, and PSMD8 in lysate and aggregate fractions are shown by Western blotting. (H and I) Levels of PSMD2, TDP-43, and PSMD8 in aggregate fractions were quantified in three independent experiments of F and G, respectively, normalized by lysate; shown relative to control cells. Error bars and P values as in C. (J) Model showing non-coding RNA expression from the nucleolus in SETX-deficient cells with associated protein aggregates (nucleolar sequestration). Source data are available for this figure: SourceData F9.$

Protein aggregates in SETX-deficient cells are driven by RNA POL1 and non-coding RNAs from the rDNA loci. (A) Levels of IGS ncRNAs were measured in U2OS control or SETX knock-out cells with mock or BMH-21 (1 μM) treatment, as indicated, by RT-qPCR, normalized by 7SK RNA. Error bars indicate standard deviation. *, **, ***, and **** indicate P < 0.05, 0.01, 0.001, and 0.0001, respectively, by two-tailed t test assuming unequal variance between control cells with BMH-21 or mock treatment, and between SETX knock-out cells with BMH-21 or mock treatment. (B) Detergent-resistant aggregates were isolated from control or SETX knock-out U2OS cells with mock or BMH-21 (1 μM) treatment as indicated. Abundance of PSMD2, TDP-43, and PSMD8 in lysate and aggregate fractions are shown by Western blotting. (C) Levels of PSMD2, TDP-43, and PSMD8 in aggregate fractions were quantified in three independent experiments of B and normalized by lysate; shown relative to control cells. Error bars indicate standard error. *, **, ***, and **** indicate P < 0.05, 0.01, 0.001, and 0.0001 by two-tailed t test assuming unequal variance; ns = not significant. (D and E) Levels of IGS ncRNAs were measured in U2OS shSETX or SETX knock-out cells, respectively, with Adm Cas13d expression and either control vector (NT) or Cas13d gRNA expression by RT-qPCR, normalized by 7SK RNA. (F and G) Detergent-resistant aggregates were isolated from shSETX or SETX knock-out U2OS cells with Adm Cas13d expression and either control vector (NT) or Cas13d gRNA expression. PSMD2, TDP-43, and PSMD8 in lysate and aggregate fractions are shown by Western blotting. (H and I) Levels of PSMD2, TDP-43, and PSMD8 in aggregate fractions were quantified in three independent experiments of F and G, respectively, normalized by lysate; shown relative to control cells. Error bars and P values as in C. (J) Model showing non-coding RNA expression from the nucleolus in SETX-deficient cells with associated protein aggregates (nucleolar sequestration). Source data are available for this figure: SourceData F9.

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