Figure 5.
Lysine malonylation increases ribosomal RNA expression
(A) WB with subcellular fractions of HEK293T cells. Ct, cytosol; Mt, mitochondria; Nu, nucleus.
(B) Representative image of immunofluorescence staining and confocal imaging of ACC (red), fibrillarin (FBL, green), and DAPI (blue) in HeLa cells. Scale bar: 10 μm. White dotted arrow area in the overlay (Merge) image was sampled for fluorescence distribution analysis in (C).
(C) The distribution of fluorescence signals of ACC (red), FBL (green), and DAPI (blue) along the dotted arrow in the overlay image in (B) was plotted to show the relative localizations of ACC, FBL, and DAPI.
(D) Representative image of immunofluorescence staining and wide-field imaging of FBL (green) and DAPI (blue) in HeLa cells using a 40x objective. Inset shows nucleolar areas identified (circled with red lines) using a machine learning algorithm.
(E) Nucleolar percentage area (ratio of nucleolar area to nuclear area) was quantified in HeLa cells transfected with empty vector (Vec) or KAT2A expressing plasmid (KAT2A OE) 24 h before DMSO or orlistat treatment (25 μM) for another 24 h. N = 1201–2265. ∗∗∗∗p < 10−4 using unpaired Student’s t test.
(F) Nucleolar percentage area was quantified in HEK293T cells expressing MeCP2-KRAB-dCas9 with scramble guild RNA (sgNC) or two different gRNAs targeting SIRT5 promoter (sgSIRT5#1 and #2). N = 1352–3054. ∗∗∗∗p < 10−4 using unpaired Student’s t test.
(G) qPCR examination of ribosomal RNA levels in HeLa cells after DMSO or orlistat treatment for 24 h. rRNA expression levels were normalized to β-actin expression level. ∗∗∗∗p < 10−4 using Sidak multiple comparisons test following two-way ANOVA.
(H) qPCR examination of ribosomal RNA levels in sgKAT2A, sgSIRT5 K562 cells upon vec or KAT2A overexpression (O/E). rRNA expression levels were normalized to β-actin expression level. ∗∗∗p < 0.001 using Sidak multiple comparisons test following two-way ANOVA.