Saha-Shah A , Esmaeili M , Sidoli S , Hwang H , Yang J , Klein PS , Garcia BA .

R01 AI118891 NIAID NIH HHS , R01 GM110174 NIGMS NIH HHS , P01 CA196539 NCI NIH HHS , R01 GM111816 NIGMS NIH HHS , R35 GM131810 NIGMS NIH HHS , R01 HL141759 NHLBI NIH HHS , R01 GM115517 NIGMS NIH HHS

	Figure 1 (A) Schematic showing the work-flow for single cell proteomics study, (B) scanning electron micrograph of a micropipette showing end-on view, pipette sputter coated with 4 nm iridium for image quality. (C) Optical micrograph of a micropipette showing sideon view, (D) optical micrograph showing a micropipette puncturing the embryo. (E) A plot showing number of proteins detected from single Xenopus laevis blastomeres at various stages of development. The cell sizes decrease progressively from stage 1 (single cell stage) to 7 (128 cell stage). n=3replicates were used for the study (F) A correlation plot showing log2 transformed and normalized protein quantification data from blastomeres (ventral, 8 cell stage) sampled with pipette (in y-axis) and physically separated by dissection (x-axis),
	Figure 2. (A) A violin plot depicting the relative standard deviation in protein measurements (normalized iBAQ) across replicates belonging to same female and various females. Dorsal 1 (D1) and dorsal 2 (D2) cells from three females were used for the study. Median of each violin plot is listed in the figure. A time course plot showing decrease in abundance of vitellogenin A2 (B) and tubulin (C) with embryonic development. We see a sharp decrease in abundance of both proteins from 4 to 8 cell stage. Each trace in the plot represents a particular cell during 8 cell stage and its precursors at earlier stages. Each point in the plot (b and c) depicts one measurement, biological replicates are plotted separately (n=3 for 1- and 2 cell stage, n=4 for 4- and 8-cell stage).
	Figure 3. (A) Optical micrograph of an embryo at the 4-cell stage showing pigment asymmetry; the least pigmented cell is labeled dorsal 1 (D1) and the most pigmented cell is labeled ventral 2 (V2), scale: 0.5 mm (B) Significant differences in protein expression between two ventral cells and dorsal-ventral cells at the 4-cell stage is depicted in the volcano plot. (C) Volcano plots showing differences in protein composition between dorsal2 and ventral1 cell at both 4- and 8-cell stage. (D) A histogram depicting the number of differentially expressed proteins between cell types. Principal component analysis of proteome data at 4 (E) and 8 (F) cell stage, respectively.
	Figure 4. (A) A volcano plot showing differential localization of proteins across animal and vegetal hemispheres of embryos at the 8-cell stage (n=3). Marked in red are components of the 20S core particle that were found to be enriched in the animal hemisphere. In yellow are proteins enriched in the vegetal hemisphere and in green (also marked by arrow) is protein Hsc70 which is equally distributed across the animal and vegetal hemispheres. Enrichment of 20S CP in the animal hemisphere (B) and DDX4/VASA in the vegetal hemisphere (C) was further validated by immunofluorescence staining in sections of 8-cell embryos. Scale: 250 µm Equal distribution of Hsc70 protein was further validated by western blotting of protein extracted from individual animal vs. vegetal halves dissected from intact embryos (D). Western blot image from 6 embryos (lower panel) was scanned and quantitated by ImageJ, and fraction in each animal hemisphere relative to total for each embryo is plotted in the upper panel. l. The mean was found to be 0.52 with an error of 0.09 as depicted in the figure.

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