Journal: Cell Host & Microbe

Article Title: Aspergillus fumigatus hijacks human p11 to redirect fungal-containing phagosomes to non-degradative pathway

doi: 10.1016/j.chom.2023.02.002

Figure Lengend Snippet:

Article Snippet: HCP216549-SG01-3 for generation of p11-KO cell line , GeneCopoiea , Cat# HCP216549-SG01-3.

Techniques: Virus, Recombinant, Cell Culture, Protease Inhibitor, Transfection, SYBR Green Assay, Mass Spectrometry, CyQUANT Assay, LDH Cytotoxicity Assay, cDNA Synthesis, Purification, Gene Expression, Construct, Control, Transformation Assay, Cloning, Plasmid Preparation, Expressing, Software, Imaging, Blocking Assay, Red Blood Cell Lysis, Membrane

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:

Journal: mSphere

Article Title: Host Lipid Transport Protein ORP1 Is Necessary for Coxiella burnetii Growth and Vacuole Expansion in Macrophages

doi: 10.1128/msphere.00104-23

Figure Lengend Snippet: Generation of ORP1-null HeLa cell line. (A) Schematic representation of ORP1 isoforms ORP1L and ORP1S, showing location targeted by sgRNA used in CRISPR-Cas9 generation of ORP1-null cells. ANK, ankyrin repeat domains; FFAT, two phenylalanines in an acidic tract motif; ORD, ORP-family cholesterol binding domain. (B) ORP1 knockout was confirmed by Western blotting. Absence of ORP1L (108 kDa) and ORP1S (50 kDa) was demonstrated in ORP1-null HeLa cells. GAPDH (35 kDa) was used as a loading control.

Article Snippet: Human ORP1-targeted sgRNA Cas9 plasmids were obtained from GeneCopoeia (HCP291905-SG01-3-B, 3× sgRNA expression clones targeting OSBPL1A; accession number NM_018030.4 ; Rockville, MD, USA).

Techniques: CRISPR, Binding Assay, Knock-Out, Western Blot

Journal: eLife

Article Title: TIGAR deficiency enhances skeletal muscle thermogenesis by increasing neuromuscular junction cholinergic signaling

doi: 10.7554/eLife.73360

Figure Lengend Snippet: ( A ) Representative images of α-bungarotoxin immunofluorescent labeling of nicotinic acetylcholine receptor clusters in the extensor digitorium longus (EDL) muscle from wild type (WT) and whole-body Tigar knockout (TKO) mice. ( B ) Quantification of the number of nicotinic acetylcholine receptor clusters following 15 min exposure at 4°C (WT n = 17, TKO n = 13). These data represent the average of over six mice in each group of mean ± SD (unpaired t -test, two-tailed, **p=0.0035). ( C ) Acetylcholine levels in the gastrocnemius muscle of WT and TKO male (n = 7) mice at room temperature or following 1 hr at 4°C. ( D ) Acetylcholine levels in the gastrocnemius muscle of Chat Cre and chTKO male (n = 6) mice at room temperature or following 1 hr at 4°C. ( E ) Representative immunoblots of TIGAR and actin proteins from two scrambled sgRNA and two Tigar sgRNA knockout Sh-SY5Y cell lines. ( F ) Acetylcholine concentrations in the medium of scrambled and TKO SH-SH5Y neuroblastoma cells. ( G ) m2 acetylcholine enrichments in cells labeled with d -glucose-1,2- 13 C 2. ( H ) m2 acetylcholine enrichments in cells labeled with U- 13 C 6 d -glucose. ( I ) m1 glutamate ( m/z 128, C2-C4 fragment) enrichment in the medium of scrambled and TKO SH-SY5Y human neuroblastoma cells labeled with d -glucose-[1,2]- 13 C2 . Statistical analyses are described in ‘Method details,’ and the data are presented as the mean ± SD. *p<0.05, ***p<0.001, ****p<0.0001. Figure 6—source data 1. The culture SH-SY5Y cells were collected from both scrambled and whole-body Tigar knockout (TKO) cells, and 30 μg of the cell lysates were used for TIGAR (30 kDa) immunoblotting analysis as described in the ‘Immunoblotting’ section. The left four lanes of the raw image (lysate from neuroblastoma cells) were used for to confirm the efficiency of TIGAR protein loss in the SH-SY5Y neuroblastoma TKO cells. The right four lanes of the raw image represent the TIGAR immunoblotting of the cell lysates from 7-day differentiated neuroblastoma cells. A detailed description of the raw images is shown in . Figure 6—source data 2. The culture SH-SY5Y cells were collected from both scrambled and whole-body Tigar knockout (TKO) cells, and 30 μg of the cell lysates were used for actin β (42 kDa) immunoblotting analysis as described in the ‘Immunoblotting’ section. The left four lanes of the raw image (lysate from neuroblastoma cells) were used for to show actin β protein in the SH-SY5Y neuroblastoma cells. The right four lanes of the raw image represent the actin β immunoblotting of the cell lysates from 7-day differentiated neuroblastoma cells. A detailed description of the raw images is shown in .

Article Snippet: The CRISPR 3xsgRNA/Cas9 all-in-one expression clone targeting Tigar (NM_020375.2) (Cat# HCP215394-CG04-3) and scrambled sgRNA control for pCRISPR-CG04 (Cat# CCPCTR01-CG04-B) were purchased from GeneCopoeia (Rockville, MD).

Techniques: Labeling, Knock-Out, Two Tailed Test, Western Blot

Journal: eLife

Article Title: TIGAR deficiency enhances skeletal muscle thermogenesis by increasing neuromuscular junction cholinergic signaling

doi: 10.7554/eLife.73360

Figure Lengend Snippet:

Article Snippet: The CRISPR 3xsgRNA/Cas9 all-in-one expression clone targeting Tigar (NM_020375.2) (Cat# HCP215394-CG04-3) and scrambled sgRNA control for pCRISPR-CG04 (Cat# CCPCTR01-CG04-B) were purchased from GeneCopoeia (Rockville, MD).

Techniques: Knock-Out, Recombinant, Expressing, Control, Sequencing, Enzyme-linked Immunosorbent Assay, Plasmid Preparation, Lysis, Blocking Assay, Electron Microscopy, Software