Absolute Copy Numbers of β-Actin Proteins Collected from 10,000 Single Cells.

Beiyuan Fan,Lixing Liu,Xiufeng Li,Dong Men,Jian Chen,Shanshan Cao,Junbo Wang,Deyong Chen

doi:10.3390/mi9050254

Abstract

Semi-quantitative studies have located varied expressions of β-actin proteins at the population level, questioning their roles as internal controls in western blots, while the absolute copy numbers of β-actins at the single-cell level are missing. In this study, a polymeric microfluidic flow cytometry was used for single-cell analysis, and the absolute copy numbers of single-cell β-actin proteins were quantified as 9.9 ± 4.6 × 105, 6.8 ± 4.0 × 105 and 11.0 ± 5.5 × 105 per cell for A549 (ncell = 14,754), Hep G2 (ncell = 36,949), and HeLa (ncell = 24,383), respectively. High coefficients of variation (~50%) and high quartile coefficients of dispersion (~30%) were located, indicating significant variations of β-actin proteins within the same cell type. Low p values (≪0.01) and high classification rates based on neural network (~70%) were quantified among A549, Hep G2 and HeLa cells, suggesting expression differences of β-actin proteins among three cell types. In summary, the results reported here indicate significant variations of β-actin proteins within the same cell type from cell to cell, and significant expression differences of β-actin proteins among different cell types, strongly questioning the properties of using β-actin proteins as internal controls in western blots.

Highlights

As housekeeping proteins, β-actins are obligatory parts of cell cytoskeletons, playing important roles in the maintenance of cellular shapes, migrations, and signal transductions [1]
Β-actins are obligatory parts of cell cytoskeletons, playing important roles in the maintenance of cellular shapes, migrations, and signal transductions [1]. Due to their constitutive expressions, β-actin proteins are commonly used as internal controls in western blots, based on the assumptions of constant expressions from cell to cell and sample to sample
Recent studies indicate varied expressions of β-actin proteins; the use of β-actin proteins as internal controls is under question [2,3]

Summary

Introduction

Β-actins are obligatory parts of cell cytoskeletons, playing important roles in the maintenance of cellular shapes, migrations, and signal transductions [1]. In 2016, Chen et al reported differences of β-actin proteins in the submandibular glands of male and female mice [10]. All of these previous data about the expressions of β-actin proteins were obtained from western blots. The previously reported data were derived from population studies, which cannot be used to address questions of whether there exist different expressions of β-actin proteins from cell to cell even within the same cell type. In order to address this issue, in this study, absolute copy numbers of β-actin proteins were obtained, leveraging a recently reported polymeric microfluidic flow cytometry [11]. The data reported here may be used as references for future studies of β-actin proteins

Materials

Working Flowchart

Device Operation and Data Processing

Data Analysis

Results