Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: YY2/PHGDH axis suppresses tumorigenesis by inhibiting tumor cell de novo serine biosynthesis.

doi: 10.1016/j.biopha.2023.115006

Figure Lengend Snippet: Fig. 1. YY2 is a novel regulator of de novo serine biosynthesis. (A–B) Viabilities of HCT116 (A) and MHCC-97H (B) cells overexpressing YY2. (C–D) Colony formation potentials of HCT116 (C) and MHCC-97H (D) cells overexpressing YY2. Representative images and quantification results (n = 6) are shown. (E) Colony formation potentials of HCT116 cells transfected with indicated amounts of YY2 overexpression vector. Representative images and quantification results (n = 6) are shown. (F) KEGG enrichment of differentially expressed genes with P-value < 0.05 in HCT116 cells overexpressing YY2 obtained from RNA-sequencing. (G–H) Intracellular serine level in YY2-overexpressed (G) and YY2-knocked out (H) HCT116 and MHCC-97H cells. Wild-type HCT116 cells or cells transfected with pcCon were used as controls. Total protein was used for normalizing intracellular serine levels. Quantification data are shown as mean ± SD (n = 3, unless otherwise indicated). Ser (-): cells cultured in medium without serine for 48 h; pcCon: pcEF9-Puro; YY2KO: YY2-knocked out cells; ** P < 0.01.

Article Snippet: YY2-knocked out HCT116 (HCT116YY2null) and MHCC-97H (MHCC-97HYY2null) cells were established using CRISPR/ Cas9 method as described previously using vectors targeting YY2 (HCP301990-CG04–3–10-a, target site: GAT GGC AAT TGG ATC TAC GG; HCP301990-CG04–3–10-b, target site: TAG CCC GTG TTC GTG AAG AG; HCP301990-CG04–3–10-c, target site: TCC GTC GGA ATG TCC TCC AT; Gene Copoiea, Rockville, MD) [16].

Techniques: Transfection, Over Expression, Plasmid Preparation, RNA Sequencing, Cell Culture

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: YY2/PHGDH axis suppresses tumorigenesis by inhibiting tumor cell de novo serine biosynthesis.

doi: 10.1016/j.biopha.2023.115006

Figure Lengend Snippet: Fig. 2. YY2 regulation on tumor cell serine metabolism is crucial for its tumor suppressive effect. (A) Schematic diagram showing de novo serine metabolism. (B–C) Viabilities of HCT116YY2null (B) and MHCC-97HYY2null (C) cells. (D–E) Viabilities of HCT116 (D) and MHCC-97H (E) cells overexpressing YY2. (F–G) Colony for mation potentials of HCT116YY2null cells (F) and HCT116 cells overexpressing YY2 (G). Representative images and quantification data (n = 6) are shown. Wild-type HCT116 cells or cells transfected with pcCon were used as controls. Quantification data are shown as mean ± SD (n = 3, unless otherwise indicated). Ser (+) and (-): cells cultured in medium with or without serine for 48 h, respectively; pcCon: pcEF9-Puro; YY2KO: YY2-knocked out cells; ** P < 0.01.

Article Snippet: YY2-knocked out HCT116 (HCT116YY2null) and MHCC-97H (MHCC-97HYY2null) cells were established using CRISPR/ Cas9 method as described previously using vectors targeting YY2 (HCP301990-CG04–3–10-a, target site: GAT GGC AAT TGG ATC TAC GG; HCP301990-CG04–3–10-b, target site: TAG CCC GTG TTC GTG AAG AG; HCP301990-CG04–3–10-c, target site: TCC GTC GGA ATG TCC TCC AT; Gene Copoiea, Rockville, MD) [16].

Techniques: Transfection, Cell Culture

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: YY2/PHGDH axis suppresses tumorigenesis by inhibiting tumor cell de novo serine biosynthesis.

doi: 10.1016/j.biopha.2023.115006

Figure Lengend Snippet: Fig. 5. YY2 regulates tumor cells de novo serine biosynthesis in a p53- independent manner. (A–B) PHGDH mRNA (A) and protein (B) expression levels in HCT116p53null cells over expressing YY2, as determined using qRT-PCR and western blotting, respec tively. (C–E) Intracellular serine (C), NADH/NAD+ (D), and NADPH/NADP+

Article Snippet: YY2-knocked out HCT116 (HCT116YY2null) and MHCC-97H (MHCC-97HYY2null) cells were established using CRISPR/ Cas9 method as described previously using vectors targeting YY2 (HCP301990-CG04–3–10-a, target site: GAT GGC AAT TGG ATC TAC GG; HCP301990-CG04–3–10-b, target site: TAG CCC GTG TTC GTG AAG AG; HCP301990-CG04–3–10-c, target site: TCC GTC GGA ATG TCC TCC AT; Gene Copoiea, Rockville, MD) [16].

Techniques: Expressing, Quantitative RT-PCR, Western Blot

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: YY2/PHGDH axis suppresses tumorigenesis by inhibiting tumor cell de novo serine biosynthesis.

doi: 10.1016/j.biopha.2023.115006

Figure Lengend Snippet: Fig. 6. YY2 directly binds to PHGDH promoter and suppresses its transcriptional activity. (A) Schematic diagram of PHGDH promoter reporter vectors with or without the predicted YY2 binding site (PHGDH-luc and PHGDHdel-luc, respectively). (B) Relative luciferase activities of PHGDH-luc and PHGDHdel-luc in HCT116 cells overexpressing YY2. (C–D) Binding capacity of YY2 to the predicted region in PHGDH promoter, as examined using ChIP assay with an anti-Flag antibody followed by PCR and qRT-PCR. The predicted YY2 binding site in the promoter region of PHGDH and the location of primer set used for PCR are shown. Anti-histone H3 antibody was used as a positive control. (E–F) Schematic diagram (E) and relative luciferase activity of PHGDHmut-luc in HCT116 cells overexpressing YY2 (F). Mutated base pairs are indicated in red. (G) Ribbon structure of human YY2 zinc-finger domains (amino acid residues 256–372) as predicted by AlphaFold Protein Structure Database. (H) Schematic diagram showing the zinc-finger domains with two cysteine-to-alanine mutations in mutant YY2 overexpression vectors (YY2m1, YY2m2, YY2m3, and YY2m4). (I) Relative luciferase activities of PHGDH-luc in HCT116 cells overexpressing YY2 mutants. (J–K) PHGDH mRNA (J) and protein (K) expression level in HCT116 cells overexpressing YY2 mutants. Cells transfected with pcCon were used as control. β-actin was used for qRT-PCR normalization and as western blotting loading control. Quantification data are shown as mean ± SD (n = 3). pcCon: pcEF9-Puro; ** P < 0.01; NS: not significant.

Article Snippet: YY2-knocked out HCT116 (HCT116YY2null) and MHCC-97H (MHCC-97HYY2null) cells were established using CRISPR/ Cas9 method as described previously using vectors targeting YY2 (HCP301990-CG04–3–10-a, target site: GAT GGC AAT TGG ATC TAC GG; HCP301990-CG04–3–10-b, target site: TAG CCC GTG TTC GTG AAG AG; HCP301990-CG04–3–10-c, target site: TCC GTC GGA ATG TCC TCC AT; Gene Copoiea, Rockville, MD) [16].

Techniques: Activity Assay, Binding Assay, Luciferase, Quantitative RT-PCR, Positive Control, Mutagenesis, Over Expression, Expressing, Transfection, Control, Western Blot

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: YY2/PHGDH axis suppresses tumorigenesis by inhibiting tumor cell de novo serine biosynthesis.

doi: 10.1016/j.biopha.2023.115006

Figure Lengend Snippet: Fig. 8. Schematic diagram showing the regulatory mechanism of YY2 on PHGDH-mediated de novo serine biosynthesis.

Article Snippet: YY2-knocked out HCT116 (HCT116YY2null) and MHCC-97H (MHCC-97HYY2null) cells were established using CRISPR/ Cas9 method as described previously using vectors targeting YY2 (HCP301990-CG04–3–10-a, target site: GAT GGC AAT TGG ATC TAC GG; HCP301990-CG04–3–10-b, target site: TAG CCC GTG TTC GTG AAG AG; HCP301990-CG04–3–10-c, target site: TCC GTC GGA ATG TCC TCC AT; Gene Copoiea, Rockville, MD) [16].

Techniques: