Materials and Reagents

10 cm culture dish (Corning, catalog number: 430167 )
Gloves (VWR International, catalog number: 82026 )
Protective clothing (VWR International, catalog number: 414004-444 )
Eyewear (VWR International, catalog number: 89187-984 )
Human tumor cell lines: H1299 (ATCC, catalog number: ATCC^® CRL-5803^TM )
Dulbecco's Modified Eagle's Medium (DMEM) (high glucose with L-glutamine) (Corning, catalog number: 10-013-CV )
Phosphate-Buffered Saline (PBS) (Corning, catalog number: 21-040-CV )
Fetal bovine serum (FBS) (ATLANTA BIOLOGICALS, catalog number: S11150 )
Thymidine (Sigma-Aldrich, catalog number: T9250 )
Propidium Iodide (PI) (Thermo Fisher Scientific, catalog number: P3566 )
Antibodies
1. Anti-Cyclin A (Abcam, catalog number: ab38 )
2. Anti-Cyclin D (Santa Cruz Biotechnology, catalog number: sc-753 )
3. Anti-β-Actin (Santa Cruz Biotechnology, catalog number: sc-58673 )
Tris-HCl, pH 8.0 (Thermo Fisher Scientific, catalog number: 15568025 )
NaCl (Sigma-Aldrich, catalog number: S9888 )
NP-40 (Abcam, catalog number: ab142227 )
EDTA (Thermo Fisher Scientific, catalog number: 15576028 )
β-Mercaptoethanol (Sigma-Aldrich, catalog number: M6250 )
EBC cell lysis buffer (see Recipes)
Electrophoresis running buffer (see Recipes)
Transfer buffer (see Recipes)

Equipment

Cell culture incubator (VWR International, model: 98000-368 )
Flow cytometry system (BD, model: FACSLyric )
X-RAY Film processor (Konica Minolta Healthcare Americas, model: SRX-101A )

Procedure

Plate H1299 cells at 20-30% confluence in a 10 cm culture dish (2 x 10⁶-3 x 10⁶ cells per dish) containing 10 ml of Roswell Park Memorial Institute (RPMI) 1640 Medium supplemented with 10% Fetal Bovine Serum (FBS).
Incubate cells at 37 °C overnight.
Add thymidine to a final concentration of 2 mM.
Culture cells in a tissue culture incubator at 37 °C for 18 h.
Remove thymidine by washing cells through addition of 10 ml pre-warmed 1x PBS and discard PBS.
Add 10 ml of pre-warmed fresh medium and incubate for 9 h in a tissue culture incubator at 37 °C.
Add second round of thymidine to a final concentration of 2 mM.
Culture cells at the tissue culture incubator for another 18 h at 37 °C.
Cells are now in G1/S boundary.
Release cells by washing with pre-warmed 1x PBS and incubating cells in pre-warmed fresh media. Cells are collected at 0, 2, 6, 8, 10, 12, 14, 24 h for analysis of cell cycle by DNA staining using PI, or analysis of protein by Western blot using cyclin A, cyclin D and β-Actin antibodies (Figure 1).

Figure 1. G1/S phase synchronized H1299 cells enter into normal cell cycle progression after release into fresh medium. A. Cell cycle profiles at indicated time points after release following double thymidine block. B. Expression levels of Cyclin A, Cyclin B and β-actin in cells at indicated time points after release.

Data analysis

Cell cycle was analyzed by flow cytometry with Flowjo software (Figure 1A). Cyclin A, Cyclin B and β-actin were detected by Western blotting (Figure 1B). Data are the representative of three independent experiments.

Notes

Dissolve thymidine in PBS and make 100 mM stock solution.
The time points for distinct cell cycle phase are dependent on the cell cycle progression time of different cell lines.
Propidium Iodide (PI) is a mutagen. Gloves, protective clothing, and eyewear should be worn.

Recipes

EBC cell lysis buffer
50 mM Tris-HCl pH 7.6-8.0
120 mM NaCl
0.5% NP-40
1 mM EDTA
1 mM Na₃VO₄
50 mM NaF
1 mM β-Mercaptoethanol
Electrophoresis running buffer
25 mM Tris-HCl pH 8.3
192 mM glycine
0.1% SDS
Transfer buffer
25 mM Tris-HCl pH 8.3
192 mM glycine
10% methanol
Cell culture medium
Roswell Park Memorial Institute (RPMI) 1640 Medium
10% Fetal Bovine Serum (FBS)

Acknowledgments

This work was supported by NIH/NCI grants R01CA193828, R01CA136534 and R01CA200905 (to X. Deng), by the Winship Research Pathology and Integrated Cellular Imaging shared resource and the Emory Comprehensive Glycomics Core (ECGC) supported by the Winship Cancer Institute of Emory University (P30CAJ 38292), by the Winship Fashion a Cure Research Scholar Award (to X. Deng), a philanthropic award provided by the Winship, and by the Winship Endowment Fund (to XD). This protocol was adapted from our previous work (Chen et al., 2018).

Competing interests

The authors have declared that no conflict of interest exists.

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