Fig. 6: SETD8 deletion impairs nucleolar structure and function.
From: SETD8 inhibition targets cancer cells with increased rates of ribosome biogenesis
A Representative images of the colocalization of SETD8HA (green) and UBF (red) in U2OS cells, which was lost upon treatment with C23. Scale bar (white) indicates 2.5 μM. DAPI (blue) was used to stain DNA. B WB illustrating the loss of SETD8 expression in Setd8lox/− mESC 24 h after exposure to 4-OHT (1 μM). Targeted deletion was mediated by the expression of a tamoxifen inducible Cre recombinase (CreER). β-ACTIN levels are shown as a loading control. C Immunofluorescence images from UBF (green) in Setd8+/− and Setd8lox/− mESC 24 h after exposure to 4-OHT (1 μM). DAPI (blue) was used to stain DNA. Scale bar (white) indicates 2.5 μM. D HTM-dependent quantification of UBF nuclear levels, from the experiment defined in C. Dashed lines indicate median values. E Immunofluorescence images of EU levels (green) in Setd8+/− and Setd8lox/− mESC 24 h after exposure to 4-OHT (1 μM). EU was added for the last 30 min before fixation. DAPI (blue) was used to stain DNA. Scale bar (white) indicates 2.5 μM. F HTM-dependent quantification of EU levels per nucleus, from the experiment defined in E. Dashed lines indicate median values. G Representative TEM images from Setd8+/− and Setd8lox/− mESC 24 h after exposure to 4-OHT (1 μM). Whereas cells that preserved SETD8 expression lacked nucleolar alterations with several small fibrillar centers (FC), depletion of SETD8 induced severe nucleolar abnormalities, including prominent nucleolar segregation, appearance of both giant fibrillar centers (GFC) and intranucleolar vacuoles (V) and nucleolar fragmentation, as seen with SETD8 inhibitors. Scale bar (white) indicates 2 μm. Additional examples are provided in Supplementary Fig. S4D. n.s. non-significant; ***P < 0.001; t-test.
