Journal: bioRxiv

Article Title: A phosphorylation-controlled switch confers cell cycle-dependent protein relocalization

doi: 10.1101/2024.06.05.597552

Figure Lengend Snippet: (A) Four stable cell lines were generated via lentiviral transduction that express similar levels of 2xMARS-nGFP and varying levels of GFP, which was under control of either a PGK or CMV promoter (the three CMV-GFP-expressing lines were isolated by FACS, sorting for different levels of GFP expression). Shown are representative Western blots showing GFP (asterisk denotes a longer CMV-GFP product potentially arising from an upstream alternative start codon) and 2xMARS-nGFP (mCherry blot) expression levels in these cell lines alongside blots of purified GFP and mCherry protein standards. (B) Quantification of GFP to 2xMARS-nGFP molar ratios in HeLa cells stably expressing 2xMARS-nGFP and various levels of GFP from (A). (C-D) Imaging analysis of GFP recruitment to the PM during mitosis at different stoichiometries of GFP to 2xMARS-nGFP determined by quantitative image analysis. (C) Quantification of the PM to cytosolic fluorescence ratios of GFP as a readout for measuring GFP recruitment levels by 2xMARS-nGFP. For PGK-GFP: n=8 cells for low GFP expression, n=4 cells for medium expression, and n=7 cells for high expression. For CMV-GFP cell lines, n=10 cells each for low, medium, and high GFP expression level. Cells analyzed came from two independent experiments (one-way ANOVA with Tukey post hoc multiple comparisons test). Note: CMV-GFP cells were sorted by FACS to generate three separate cell lines with low, medium, and high GFP expression, and images were acquired for each cell line. For PGK-GFP cells, the low, medium, and high cells were all from a single cell line and are categorized based on GFP fluorescence intensity during imaging analysis by ImageJ post-acquisition. (D) Representative micrographs for HeLa cells stably expressing 2xMARS-nGFP and varying levels of GFP analyzed in (C). Brightness of the GFP channel was adjusted post-acquisition for better visualization of the weaker fluorescence signals for PGK-GFP and CMV-GFP low cells. (E) Quantification of mean fluorescence intensity in the cytosol during interphase as a means to determine GFP expression levels in PGK-GFP low, medium, high cells and CMV-GFP low cells (n = 40 cells from two independent experiments). Compared to CMV-GFP low cells, PGK-GFP low cells exhibited an average of 7.6x lower expression, PGK-GFP medium cells exhibited ∼2.2x lower expression, and PGK-GFP-high cells exhibited ∼1.1x higher expression. Converting to GFP : 2xMARS-nGFP molar ratio in these cells, the estimated ratios are 1.8 (low), 6.2 (medium), and 15.1 (high). (F-G) Ectopic expression of MARS and 2xMARS-nGFP did not negatively impact rates of cell division and proliferation. (F) Representative growth curves for wild-type (WT) HeLa cells and those stably expressing MARS and 2xMARS-nGFP (n=2 technical replicates for each curve). Raw data (faded-color curves with error bar) acquired by IncuCyte Live-Cell Analysis System were fitted using GraphPad Prism (Exponential growth curve model). Fitted curves are shown as solid lines. (G) Doubling time of the three cell lines calculated from the fitted growth curves (n = 3 biological replicates, one-way ANOVA with Tukey post hoc multiple comparisons test).

Article Snippet: Confluency data within the exponential growth phase were plotted and fitted for doubling time calculation using the Exponential growth curve model in GraphPad Prism.

Techniques: Stable Transfection, Generated, Transduction, Control, Expressing, Isolation, Western Blot, Purification, Imaging, Fluorescence, Cell Analysis