Extended Data Fig. 6: GEM2 recruitment to endogenous Ki-67, Nup96 and seipin has little effect on cellular phenotype.
From: Genetically encoded multimeric tags for subcellular protein localization in cryo-EM
a, Mitotic chromosomes (stained with SiR-DNA) after induction of Ki-67 GEM-labelling with rapalog treatment for the indicated time. Ki-67 knock-out (KO) cells serve as a control for aberrant mitotic chromosome coalescence upon Ki-67 impairment36. b, Mitotic chromosome area measurements in GEM-expressing cells, n = 139, 154, 158, 165, 188 cells per treatment (left to right), 2 experiments. Lines indicate median. ***P < 0.0001, Kruskall-Wallis test followed by Dunn’s test, compared to 0 h treatment. c, Chromosome area at 60 min treatment as a function of mean GEM fluorescence intensity at chromosomes, n = 255 cells, 2 experiments. d, Mitotic chromosomes (DNA, magenta) and endogenous mEGFP-Ki-67 signal (green) upon transfection with control siRNA (siCont.) and Ki-67 siRNA (siKi-67) in comparison with Ki-67 KO cells. Cells were transfected with 0.05–5 pmol siRNAs for partial knockdown. e, Chromosome area as a function of mean mEGFP-Ki-67 fluorescence intensity on chromosomes. n = 156 (siRNA Cont.), 459 (siRNA Ki-67), 150 (Ki-67 KO) cells, 2 experiments. f, Importin β binding domain (IBB)-mCherry localization after induction of Nup96 GEM-labelling for the indicated time. Non-doxycycline-induced cells, thus not expressing GEM or adaptor protein, were included as a control. Impairment of nuclear pore integrity results in redistribution of IBB to the cytoplasm89. g, IBB-mCherry intensity ratio (nucleus/cytoplasm), n = 41, 40, 42, 42, 39 cells per treatment (left to right), 2 experiments. Lines indicate median. h, IBB-mCherry intensity ratio at 12 h rapalog treatment time as a function of total GEM intensity on Nup96 in the same cells. i, Lipid droplets (LDs, stained with LD540) after induction of seipin GEM-labelling for the indicated time. Cells were treated with oleic acid during the final hour of rapalog treatment to induce LD biogenesis. Seipin KO cells serve as a control for mean LD size reduction upon seipin impairment38. Contrast is adjusted in insets for comparison with small LDs of seipin KO cells. j, Mean LD size per cell, n = 594, 610, 639, 738, 335 cells per treatment (left to right), 2 experiments. Lines indicate median. ***P < 0.0001, Kruskall-Wallis test followed by Dunn’s test, compared to 0 h treatment. k, Mean LD size at 12 h rapalog treatment time as a function of mean GEM intensity at LDs in the same cells. Dashed line indicates the mean LD size of control cells.
