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: Nucleic Acids Research

Article Title: Reducing the inherent auto-inhibitory interaction within the pegRNA enhances prime editing efficiency

doi: 10.1093/nar/gkad456

Figure Lengend Snippet: Small RNA-seq analysis of different pegRNA species bound to the Prime editor in HEK293T cells. ( A ) Schematic for the small RNA-seq library preparation. Briefly, HEK293T cells were transfected with plasmids encoding one of two effectors (SpCas9 or PEmax), and one guide RNA (sgRNA, pegRNA or epegRNA). Cells were harvested after 2 days, crosslinked and then lysed for total RNA isolation. To sequence the bound pegRNA or epegRNA population, the SpCas9 or PEmax protein (containing 3xHA-tag) was immunoprecipitated then crosslinking was reversed to purify the bound RNA. This was followed by 3’ DNA adapter ligation (3’ adapter contains 15 bp UMI sequence) to the purified RNA, cDNA synthesis and two rounds of PCR to add sequencing adapters. The final library was deep sequenced and analyzed. A detailed protocol is present in the methods section. ( B ) Bulk or effector-bound RNA species present from each treatment group. ‘Bulk’ indicates sequencing of the sgRNA/pegRNA present in the cell without IP pulldown to examine the sgRNA/pegRNA 3’ sequence lengths irrespective of whether it is bound to SpCas9 or PEmax. The length of the PBS in the pegRNA (7 or 13 nt) is indicated in the name. Small RNAs were categorized into six species based on the length of 3’ truncation: full-length pegRNA, pegRNA with truncated but potentially functional PBS (≥7 nt remaining), pegRNA with truncated likely insufficient PBS (<7 nt), pegRNA with truncated RTT, and pegRNA with truncated sgRNA scaffold. Abundance of each RNA species was calculated based on UMIs incorporated into the 3’ adaptor from the small RNA-seq library (see for IGV plots). ( C ) RNP-mediated PE3 editing efficiencies in mCherry reporter cell line with different ratio of pegRNA:nicking sgRNA. The amount of PEmax protein (50 pmol) and pegRNA (200 pmol; IDT) was held constant while increasing the amount of nicking sgRNA (IDT) delivered by electroporation. Frequency of mCherry positive cells was quantified by flow cytometry 72 h following treatment. One-way ANOVA was used to compare all the groups for each graph, PE2 was used as a control column for multiple comparisons. ns stands for P > 0.05, * indicates P ≤ 0.05,** indicates P ≤ 0.01, and **** indicates P ≤ 0.0001 (also see Supplementary table). (D, E) RNP-mediated PE3 editing efficiencies at the specified positions for ( D ) FANCF (+5 G to T) and ( E ) HEK4 (+5 G to T) loci in HEK293T cells. The amount of PEmax protein (50 pmol) and pegRNA (200 pmol; IDT) was held constant while increasing the amount of nicking sgRNA (from IDT) delivered by electroporation. Editing efficiency reflects the frequency of sequencing reads from amplicon deep sequencing that contain the intended edit or others (indels and imprecise prime editing) among all sequencing reads. Values and error bars reflect mean ± s.d. of n = 3 independent biological replicates. One-way ANOVA was used to compare the intended edit across all the groups for each graph, PE2 was used as a control column for multiple comparisons. ns indicates P > 0.05, * indicates P ≤ 0.05,** indicates P ≤ 0.01, and **** indicates P ≤ 0.0001 (also see Supplementary table).

Article Snippet: Backbone plasmids used for pegRNA and sgRNA cloning are available from Addgene (#122089).

Techniques: RNA Sequencing, Transfection, Isolation, Sequencing, Immunoprecipitation, Adapter Ligation, Purification, cDNA Synthesis, Functional Assay, Electroporation, Flow Cytometry, Control, Amplification

Journal: Molecular cell

Article Title: CRISPR-Mediated Base Editing Enables Efficient Disruption of Eukaryotic Genes through Induction of STOP Codons

doi: 10.1016/j.molcel.2017.08.008

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Plasmid: B270 (containing sgRNA targeting ATP1A1 + empty sgRNA-expressing cassette) , This paper , Available in Addgene.

Techniques: Virus, Subcloning, Recombinant, Transfection, DNA Extraction, PCR Cloning, Cloning, Plasmid Preparation, Software

Journal: Science translational medicine

Article Title: Precise genomic editing of pathogenic mutations in RBM20 rescues dilated cardiomyopathy

doi: 10.1126/scitranslmed.ade1633

Figure Lengend Snippet: (A) ABE correction of the R636Q mutation using sgRNA (blue) and ABEmax-VRQR-SpCas9. On-target: A6 (red). Bystander: A14 and A20 (green). Silent: A4, A13, and A19 (brown). (B) Percentage of adenine-to-guanine editing determined by deep sequencing of cDNA from hearts of R636Q/R636Q mice (6 weeks after ABE correction). Data are expressed as means ± SEM (n = 3). (C) Fractional shortening at 4 and 8 weeks after ABE correction in WT, R636Q/+, R636Q/R636Q, and corrected mice. Data are expressed as means ± SEM (n = 6 per group). Two-way ANOVA with Tukey’s multiple comparisons test was performed. ****P < 0.0001. (D) H&E staining of four-chamber hearts (12 weeks after ABE correction). Scale bar, 1 mm. LV, left ventricle. (E) Kaplan-Meier survival curve of all groups (n = 16 per group). Log-rank (Mantel-Cox) test was performed. ****P < 0.0001 for R636Q/R636Q versus other groups. (F) Immunohistochemistry showing the translocation of RBM20 in CMs of WT, R636Q/+, R636Q/R636Q, and corrected mice (12 weeks after ABE correction). cTnT (red), RBM20 (green), and DAPI (blue). Scale bar, 10 μm. (G) Quantification of RBM20 localization before and after correction in R636Q/R636Q mice. Data are expressed as means ± SEM (n = 4 per genotype). Two-way ANOVA with Bonferroni’s multiple comparisons test was performed. ****P < 0.0001.

Article Snippet: The N-terminal and C-terminal regions of ABEmax-VRQR-SpCas9 were extracted from CMV_Npu-ABEmax N-terminal (Addgene plasmid no. 137173) ( 52 ) and hu6 HGPS sgRNA expression and ABE7.10max VRQR C-terminal AAV vectors (Addgene plasmid no. 154430) ( 28 ) from D. Liu’s laboratory, respectively.

Techniques: Mutagenesis, Sequencing, Staining, Immunohistochemistry, Translocation Assay