Extended Data Fig. 3: ELL3 deficieny leads to spindle abnormality in human oocytes.
From: Maternal ELL3 loss-of-function leads to oocyte aneuploidy and early miscarriage
a, Western blot showing the specificity of the ELL3 polyclonal antibody against the N-terminal region of ELL3 (pAb) using WT, ELL3 KO and ELL3 KO HCT 116 cells with GFP-ELL3 expression. Red asterisks pointing to the distinct bands corresponding to ELL3 or GFP-ELL3. Throughout this study, unless otherwise explicitly stated, this ELL3 pAb was used for IF, Western blot and IP experiments. b, Schematic diagram of ELL3 TRIM-Away assay in human oocytes. c, Western blot showing the specificity of the ELL3 monoclonal antibody (mAb) using WT, ELL3 KO and ELL3 KO HCT 116 cells with GFP-ELL3 expression. Red asterisks pointing to the distinct bands corresponding to ELL3 or GFP-ELL3. The ELL3 mAb was used in TRIM-Away experiments. d, Representative images showing ELL3 TRIM-Away efficiency, assayed by the validated ELL3-pAb, and spindles and chromosomes in control and ELL3 TRIM-Away human oocytes. Green, microtubules; blue, DNA. Scale bar, 5 μm. e, Spindle abnormality rate in control (n = 27) and ELL3 TRIM-Away (n = 29) human oocytes. f, Immunostaining showing ELL3 KD efficiency in human oocytes. Scale bar, 5 μm. g, PB1 extrusion rate in control (n = 24) and ELL3 KD (n = 27) human oocytes. h, Representative images showing chromosome spreading of control, ELL3, ELL3 D150H, ELL3 R217C, ELL3 E251D, ELL3 T323A, and ELL3 T323 overexpressing mouse MII oocytes. The number x/y marked on the lower right corner of each picture indicating that x oocytes are aneuploid among the y examined oocytes. i, PB1 extrusion rate in control (n = 54), ELL3 (n = 56), ELL3 D150H (n = 84), ELL3 R217C (n = 72), ELL3 E251D (n = 70), ELL3 T323A (n = 74), and ELL3 T323 (n = 73) overexpressing mouse oocytes. j, Representative images showing spindle morphologies in control, ELL3 WT, ELL3 R217C and ELL3 E251D overexpressing mouse oocytes. Scale bar, 10 μm. Data are presented as mean ± s.e.m. (g,i). Data are from three independent experiments (e,g,i). N indicated the cumulative number of independent oocytes and independent experiments are denoted by dots (g,i). Statistical analysis was performed using a two-tailed, unpaired Student’s t-test (g,i) or a two-sided, fisher’s exact test (e). ns, not significant.
