Fluorescence Microplate-Based Cycloheximide Chase Assay: A Technique to Monitor the Degradation Kinetics of Fluorescent Nuclear Misfolded Proteins

Overview

This video describes an in vitro fluorescence-based microplate assay to study the degradation kinetics of a fluorescently-labeled misfolded luciferase mutant protein expressed in the nuclei of transfected mammalian cells. The assay involves the treatment of cells expressing the fluorescent mutant protein with a translation inhibitor and a proteasome inhibitor to assess the proteasome-mediated degradation of the misfolded protein.

Protocol

1. ​Preparation of Reagent

1. Prepare low-fluorescence DMEM medium for assays using a microplate fluorescence reader. Mix 25 mM glucose, 0.4 mM glycine, 0.4 mM arginine, 0.2 mM cysteine, 4.0 mM glutamine, 0.2 mM histidine, 0.8 mM isoleucine, 0.8 mM leucine, 0.8 mM lysine, 0.2 mM methionine, 0.4 mM phenylalanine, 0.4 mM serine, 0.8 mM threonine, 0.078 mM tryptophan, 0.4 mM tyrosine, 0.8 mM valine, 1.8 mM CaCl₂, 0.81 mM MgSO₄, 5.33 mM KCl, 44.0 mM NaHCO₃, 110 mM NaCl, 0.9 mM NaH₂PO₄. Adjust the pH of the solution using HCl or NaOH to pH 7.4. Sterilize the medium through filtration.
   NOTE: This medium contains components of the standard DMEM medium with high glucose except that Fe(NO₃)₃, vitamins, and Phenol Red are omitted. Phenol Red, riboflavin, and pyridoxal in regular DMEM culture medium significantly interfere with the detection of fluorescence signal. Culture medium without those components is crucial for successful live cell GFP imaging. The medium is stable for 12 months when stored refrigerated.

2. Real-time Degradation Assay of NLS-luciferase-GFP Using Fluorescence Microplate Reader

1. Seed approximately 1 x 10⁴ HeLa cells into black 96-well tissue culture plates with transparent bottom. After O/N culturing, a confluence of 50-70% is reached at the time of transfection.
   NOTE: 60 µl of medium containing completely suspended HeLa cells are seeded directly into each well. Do not add additional medium or rock plate back and forth after seeding, otherwise, cells may be distributed unevenly.
2. Transfect HeLa cells with 0.05-0.1 µg of NLS-luciferase-GFP/pRK5 plasmid into each well using transfection reagent according to manufacturer's instruction. Make a master transfection mix containing DNA and transfection reagent and aliquot it for each well. Three wells with cells are not transfected with DNA, which serves as control for background fluorescence signal for each treatment condition.
   NOTE: An alternative way to reduce transfection variation is to transfect cells before seeding. However, a large portion of cells transfected with misfolded proteins fail to attach or show reduced viability using this method. It is likely that some cells may not stand the stress caused by trypsin digestion when expressing toxic misfolded proteins. As a result, the overall fluorescent signal is significantly reduced.
3. 20-24 hr after transfection, examine the live cells under an inverted fluorescent microscope for GFP expression with excitation wavelength 450-490 nm.
4. Remove the medium by vacuum aspiration. Add approximately 200 µl 1x PBS to each well and then aspirate it to remove residual amount of DMEM medium.
5. Add 60 µl of low-fluorescence DMEM medium with 5% FBS and 50 µg/ml CHX. To examine proteasomal degradation, include additional proteasome inhibitor MG132 (10 µM) in one set of samples. Set triplicate of wells for each treatment/condition.
   NOTE: MG132 treatment is included as controls for proteasomal degradation because it is crucial to rule out other factors that can cause drop of fluorescence signal, including cell death and fluorescence quenching.
6. Measure the fluorescence signal of GFP immediately on a fluorescent plate reader after adding CHX.
   NOTE: The software measurement settings are shown in Table 1.
7. Read the plate every hour for up to 8-10 hr. After reading, return the plate back to cell culture incubator.
8. Export the data as spreadsheet file. Use mean value of multi-reads for each well as fluorescence intensity. Normalize values of each data point to the mean values of Time 0 in the respective groups. Plot the normalized fluorescence intensity over time as shown in Figure 1A and 1B, and Figure 2, right panels.
9. Perform statistical analysis of degradation rates between two conditions using two-way ANOVA with repeated measures.
   NOTE: In this analysis, "fluorescence intensity" is the dependent variable whereas "treatment conditions" and "time" are the two factors. Instead of comparing data at individual time points, two-way ANOVA with repeated measures analyzes the difference between the two treatment groups over the entire time course.

Table 1: Measurement settings on fluorescence microplate reader.

Plate definition file		GRE96fb
Measurement mode		Fluorescence Intensity Bottom
Wavelength	Excitation wavelength	485 nm
	Excitation bandwidth	9 nm
	Emission wavelength	535 nm
	Emission bandwidth	20 nm
Number of flashes		5
Gain		Manual 200
Integration time		20 µs
Multiple reads per well	Type	Circle (filled)
	Size	5×5
	Border	400 µm

Representative Results

Figure 1: Fluorescent microplate-based assay for NLS-LucDM-GFP degradation. HeLa cells seeded on 96 well plates were transfected with 0.05 µg (A) or 0.1 µg (B) NLS-LucDM-GFP and treated with CHX in the presence or absence of MG132. (A and B) Fluorescence intensities of wells were measured at the indicated time points (Mean values ± standard deviations, n = 3). Panels on the left show the original fluorescence reading, whereas the panels on the right show the values normalized to readings at Time 0 in the respective treatment groups. The difference between the groups treated with and without MG132 was analyzed by two-way ANOVA with repeated measures. P values between the two treatment groups are indicated. (C) Wells were examined before or after 9 hr CHX treatment, or after CHX and MG132 treatment, by fluorescence microscope. To better show both cells with bright aggregated NLC-LucDM-GFP and those with dim diffused protein, gamma value of 1.5 was applied to all the images. Scale bar = 100 µm.

Figure 2: A reduced degradation rate of NLS-LucDM-GFP in PML knockdown cells. HeLa cells previously treated with control or PML siRNA were seeded into 96 well plates. Degradation of NLS-LucDM-GFP was analyzed as described in Figure 1 (two-way ANOVA; p <0.0001).

Materials

Name	Company	Catalog Number	Comments
Dulbecco's Modified Eagle Medium	Life Technologies	11995-092
Fetal Bovine Serum	Life Technologies	10082147
Lipofectamine 2000	Life Technologies	11668019
MG132	Sigma-Aldrich	M8699
Amino acids	Sigma-Aldrich		Amino acids are used for making low fluorecence culturing medium
Cycloheximide	Sigma-Aldrich	C7698
Olympus IX-81 Inverted Fluorescence Microscope	Olympus	IX71/IX81
96 Well Black TC Plate w/ Transluscent Clear Bottom	Sigma-Greiner	89135-048
Fluorescence Bottom Plate Reader Infinite 200® PRO	TECAN	Infinite 200® PRO
Prism 5	GraphPad		Statistical analysis software