Journal: BMC Cancer

Article Title: Acetylation of PGK1 at lysine 323 promotes glycolysis, cell proliferation, and metastasis in luminal A breast cancer cells

doi: 10.1186/s12885-024-12792-8

Figure Lengend Snippet: Effects of PGK1-323 K acetylation on metastasis in T47D cells. A Wound healing assay in T47D cells transfected with WT-PGK1 and non-acetylatable PGK1-K323R mutant, monitored at 0-, 24-, and 48-h post-scratch. B Quantitative analysis of wound closure from Fig. 7A. C Transwell invasion assay for T47D cells expressing either WT-PGK1 or PGK1-K323R, evaluated after 48 h of culture. D Quantitative results of the invasion assay from Fig. 7C. E Wound healing assay in T47D cells transfected with the non-acetylatable PGK1-K323R mutant or the acetylation-mimicking PGK1-323Q mutant, monitored at 0-, 24-, and 48-h post-scratch. F Quantitative analysis of wound closure from Fig. 7E. G Transwell invasion assay for T47D cells expressing either PGK1-K323R or PGK1-323Q, evaluated after 48 h of culture. H Quantitative results of the invasion assay from Fig. 7G. I Western blot analysis assessing the levels of metastasis-related markers (MMP9, MMP2) and epithelial-to-mesenchymal transition (EMT) markers (E-cadherin, N-cadherin, Vimentin) in T47D cells expressing WT-PGK1 or PGK1-K323R, with β-actin as the loading control. J Quantitative evaluation of protein expression levels from Fig. 7I. K Western blot analysis comparing the expression of metastasis and EMT markers in T47D cells transfected with PGK1-K323R or PGK1-K323Q, using β-actin for loading control. L Quantification of protein expression from Fig. 7K. WT-PGK1 refers to the wild-type PGK1; PGK1-K323R denotes a mutant variant where lysine is substituted with arginine, inhibiting acetylation; PGK1-K323Q represents a mutant designed to simulate acetylation at lysine 323

Article Snippet: Related antibodies and dilution ratio were as follows: PGK1 (Santa Cruz, sc-130335, 1:1000), Pan-acetylation (Cell Signaling Technology, 9441, 1:1000), β-actin (Cell Signaling Technology, 4970, 1:1000), FLAG (Sigma, F7425, 1:1000), PCNA(Cell Signaling Technology, 13,110, 1:1000), MMP2 (Cell Signaling Technology, 40,994, 1:1000), MMP9 (Cell Signaling Technology, 13,667, 1:1000), E-cadherin (Proteintech, 20,874–1-AP, 1:2000), N-cadherin (Proteintech, 22,018–1-AP), Vimentin (Proteintech, 10,366–1-AP), HAT1 (PTM Biotechnology, PTM-5195, 1:1000), p300 (Cell Signaling Technology, 70088S, 1:1000), Sirtuin3 (Cell Signaling Technology, 5490S, 1:1000).

Techniques: Wound Healing Assay, Transfection, Mutagenesis, Transwell Invasion Assay, Expressing, Invasion Assay, Western Blot, Control, Variant Assay