Journal: Cell Biology and Toxicology

Article Title: CD36 inhibition reduces non-small-cell lung cancer development through AKT-mTOR pathway

doi: 10.1007/s10565-024-09848-7

Figure Lengend Snippet: CD36 expression is positively correlated with cell proliferation and migration in vitro . A–F A549 cells were transfected with pCMV or pCMV-CD36 plasmid, and NCI-H520 cells were transfected with CasRX or CasRX-CD36 plasmid for 12 h in serum-free medium and then cultured in complete medium for another 24 h. Cells were collected for determination of cell viability ( A ), cell cycle ( B , C ), migration ( D ), and apoptosis ( E ) by MTT assay, FACS, wound healing test, and Annexin V-FITC/PI staining, respectively. Protein expression of CD36, CDH1, PCNA, vimentin, Bcl-2, and BAX was determined by Western blot with density quantitative analysis ( F ). G , H NCI-H520 cells were transfected with CasRX or CasRX-CD36 plasmid for 12 h and then cultured in complete medium for 24 h, followed by FFAs (150 µM) treatment for 24 h. Cells were collected for determination of cell viability ( G ) and apoptosis ( H ). Mean ± SEM; * p < 0.05; ** p < 0.01; *** p < 0.001; A , G : n = 6; B – F , H : n = 3

Article Snippet: CasRx gRNA cloning backbone (pXR003, #109053, Addgene) was digested with BbsI and then ligated with annealed oligo duplex using T4 ligase.

Techniques: Expressing, Migration, In Vitro, Transfection, Plasmid Preparation, Cell Culture, MTT Assay, Staining, Western Blot

Journal: Cell Biology and Toxicology

Article Title: CD36 inhibition reduces non-small-cell lung cancer development through AKT-mTOR pathway

doi: 10.1007/s10565-024-09848-7

Figure Lengend Snippet: The effects of pitavastatin on cell proliferation, migration, and apoptosis are related to CD36 expression. A549 cells were transfected with pCMV or pCMV-CD36 plasmid, and NCI-H520 cells were transfected with CasRX or CasRX-CD36 plasmid for 12 h and then cultured in complete medium for 24 h, followed by received pitavastatin (5 µM) treatment for 24 h. Cells were collected for determination of cell viability ( A ), cell cycle ( B , C ), migration ( D ), and apoptosis ( E ). Protein expression of CD36, PCNA, Bcl-2, and BAX was determined by Western blot with density quantitative analysis ( F , G ). Mean ± SEM; * p < 0.05; ** p < 0.01; *** p < 0.001; A : n = 6; B – F : n = 3; Pita, pitavastatin

Article Snippet: CasRx gRNA cloning backbone (pXR003, #109053, Addgene) was digested with BbsI and then ligated with annealed oligo duplex using T4 ligase.

Techniques: Migration, Expressing, Transfection, Plasmid Preparation, Cell Culture, Western Blot

Journal: Cell Biology and Toxicology

Article Title: CD36 inhibition reduces non-small-cell lung cancer development through AKT-mTOR pathway

doi: 10.1007/s10565-024-09848-7

Figure Lengend Snippet: The reduction effects of pitavastatin on tumor progression are regulated by CD36/AKT/mTOR pathway. A–E A549 ( A ) and NCI-H520 ( B ) cells were treated with 150 µM FFAs or 5 µM pitavastatin plus FFAs for 24 h. A549 cells were transfected with pCMV or pCMV-CD36 plasmid for 12 h ( C ); NCI-H520 cells were transfected with CasRX or CasRX-CD36 plasmid ( D , E ) for 12 h and then cultured in complete medium for 24 h, followed by treatment with 5 µM pitavastatin ( C , D ) or 150 µM FFAs ( E ) for 24 h. Protein expression of p-AKT, AKT, p-mTOR, and mTOR was detected by Western blot. F , G Tumor paraffin sections collected from Fig. A ( F ) or Fig. A ( G ) were performed IHC staining to detect the expression of p-AKT and p-mTOR with MD quantified by ImageJ software. H–L A549 cells were transfected with pCMV or pCMV-CD36 plasmid for 12 h and then cultured in complete medium for 24 h, followed by received LY294002 (10 µM) treatment for 24 h. Cells were collected for determination of cell viability ( H ) and apoptosis ( I , J ). Protein expression of CD36, vimentin, PCNA, BAX, p-AKT, AKT, p-mTOR, and mTOR was determined by Western blot ( K , L ). Mean ± SEM; * p < 0.05; ** p < 0.01; *** p < 0.001; A – E , I – L : n = 3; F , G : n = 5; H : n = 6; Pita, pitavastatin; LY, LY294002

Article Snippet: CasRx gRNA cloning backbone (pXR003, #109053, Addgene) was digested with BbsI and then ligated with annealed oligo duplex using T4 ligase.

Techniques: Transfection, Plasmid Preparation, Cell Culture, Expressing, Western Blot, Immunohistochemistry, Software

Journal: Methods in molecular biology (Clifton, N.J.)

Article Title: Analysis of Nucleoporin Function Using Inducible Degron Techniques

doi: 10.1007/978-1-0716-2337-4_9

Figure Lengend Snippet: Design of gRNAs for targeting the NUP153 gene. (a) Position and sequences of gRNAs targeting last exon of NUP153 gene. SpCas9 cut sites on genomic DNA are indicated with blue arrows, gRNA sequences are highlighted in orange, PAM sequences are shown in yellow. Note, PAM sequence should be present only in genomic DNA. (b) Cloning of gRNA sequences with added CACC and CAAA overhangs into pX330 vector. gRNA-1 requires additional G nucleotide in the beginning of gRNA sequence to initiate expression of gRNA from U6 promoter. Note that the gRNA-9 starts from a G nucleotide and does not require additional G nucleotide

Article Snippet: 3.1. gRNA Cloning We used the pX330 vector (Addgene, 42230) to drive simultaneous expression of S. pyogenes Cas9 (SpCas9) protein and guide RNA (gRNA) sequence in the same cell.

Techniques: Sequencing, Cloning, Plasmid Preparation, Expressing

Journal: Nature methods

Article Title: CRISPR-assisted detection of RNA-protein interactions in living cells.

doi: 10.1038/s41592-020-0866-0

Figure Lengend Snippet: Fig. 1 | CARPID identifies lncRNA XIST-associated proteins in living cells. a, Scheme of the CARPID workflow. Biotin is represented by red circles marked with ‘B’. b, Volcano plot of XIST-associated proteins identified by CARPID. Significantly enriched proteins are shown as orange dots. Proteins previously confirmed to interact with XIST are shown in the orange font (n = 3 and 9 independent experiments for control and XIST group, respectively). Two previously uncharacterized RBPs of XIST identified and validated in this study—TAF15 and SNF2L—are labeled in blue. n.s., nonsignificant. c, Top, Western blot of TAF15 in input and streptavidin immunoprecipitation samples of control (C) and three XIST gRNA sets (L1, L2 and L3). Bottom, immunoFISH images of XIST and TAF15 in HEK293T cells. The white boxed region on the left is magnified and shown on the right. Three independent experiments were carried out with similar results, and a representative result is shown. d, Validation of XIST–TAF15 interaction using RIP (mean ± s.e.m., n = 3 independent experiments, two-sided paired Student’s t-test). XIST: P1, P2 and P3 represent qPCR primer pairs enriched regions covering corresponding XIST gRNA pairs targeting loci L1/L2/L3, specified in Extended Data Fig. 1a. MALAT1: P1 and P2 represent two different qPCR primer pairs enriched regions in MALAT1 transcript as controls. e, RNA-binding specificity of TAF15 as determined using HTR-SELEX. The gkm-SVM scores were calculated by the gkm-SVM model trained with HTR-SELEX data to predict the potential of TAF15 binding to the corresponding locus in XIST transcript.The blue curve shows the predicted binding affinity of TAF15 along XIST, compared with the average value of 1,000 randomly selected genomic fragments (orange). f, X-linked GFP de-repression under depletion of TAF15 with short hairpin RNAs (shRNA; shTAF15-07 and shTAF15-44) and SNF2L (shSNF2L-29 and shSNF2L-31) in iMEF cells, using SMCHD1 (shSMCHD1) as a positive control (mean ± s.d., n = 3 independent experiments, two-sided unpaired Student’s t-test using NT + 5-aza as a control). NT, non-specific-targeting shRNA. 5-aza, 5-aza-2'-deoxycytidine, a DNA-demethylating agent to derepress gene expression in the inactive X chromosome.

Article Snippet: We generated gRNA sets composed of 2 gRNAs spaced by 30-nucleotide direct repeats to target 2 adjacent loci on the same lncRNA and cloned them into an empty gRNA expressing vector (Addgene no. 109054).

Techniques: Control, Labeling, Western Blot, Immunoprecipitation, Biomarker Discovery, RNA Binding Assay, Binding Assay, shRNA, Positive Control, Gene Expression

Journal: Cell host & microbe

Article Title: The Epstein-Barr Virus Regulome in Lymphoblastoid Cells

doi: 10.1016/j.chom.2017.09.001

Figure Lengend Snippet: (A) RNAPII ChIA-PET linkages at the MYC locus. ChIP-seq tracks were shown on the top. MYCSE1 (-525kb from TSS) and MYCSE2 (-428kb from TSS) were indicated by red box below the tracks. RNAPII ChIA-PET links were shown by magenta lines. Orange boxes indicated the regions targeted by CRISPR. (B) Validation of MYCSE1 Deletion. Cas9 stable LCLs or BJAB were transduced with paired gRNA targeting the edges of ESE. After puromycin selection, genomic DNA was prepared from LCLs and BJAB cells transduced with dual gRNA. The targeted region was PCR amplified. The presence or absence of the MYC SE1 was shown. (C) MYCSE1 deletion reduced MYC mRNA level. 2 independent pairs of gRNAs both decreased MYC mRNA by qRT-PCR normalized to b-actin. Control gRNA treated cells were set to 1. (D) Growth of MYCSE1 deleted LCLs and LCLs with deletion in a control region. (E) MYCSE2 deletion also reduced MYC mRNA level. 2 independent pairs of gRNAs both decreased MYC mRNA by qRT-PCR. All data are represented as mean+/−SE. See also Figure S3.

Article Snippet: Dual gRNAs were cloned into pLentiGuide-Puro (Addgene Plasmid #52963) using the Multiplex gRNA kit (System Biosciences) according to the manufacturer protocol.

Techniques: ChIA Pet Assay, ChIP-sequencing, CRISPR, Biomarker Discovery, Transduction, Selection, Amplification, Quantitative RT-PCR, Control

Journal: Nucleic Acids Research

Article Title: SIRT6 transcriptionally regulates global protein synthesis through transcription factor Sp1 independent of its deacetylase activity

doi: 10.1093/nar/gkz648

Figure Lengend Snippet: ( A ) qPCR analysis of relative mRNA expression levels of mTOR signalling genes in WT and SIRT6-KO mice heart tissues. n = 4–10 mice per group. Data are presented as mean ± s.d, * P < 0.05. AKT1 was used as a positive control. GAPDH was used as a negative control. ( B ) ChIP analysis to detect SIRT6 binding to the promoters of the indicated genes performed with a SIRT6-specific antibody or IgG control antibody in WT 293T cells. n = 3–4. Data are presented as mean ± s.d, * P < 0.05. ( C ) ChIP analysis to detect Sp1 binding to the promoters of the indicated genes performed with a Sp1-specific antibody or IgG control antibody in WT 293T cells. n = 3–4. Data are presented as mean ± s.d, * P < 0.05. ( D ) Representative images of western blotting analysis of mTOR signalling under Sp1 depleted (Sp1-KD) conditions in 293T cells. The Sp1 levels were depleted by transfecting the cells with specific shRNA and the knockdown was confirmed by western blotting. (E) Representative images of western blotting analysis of mTOR signalling in Sp1-KO HeLa cells. Sp1 was deleted using a specific guide RNA and the CRISPR-Cas9 system. ( F ) Representative images of western blotting analysis of mTOR signalling under Sp1 overexpression conditions in 293T cells. The cells were transfected with empty vector or Sp1 expressing plasmid for 48 h and Sp1 overexpression was confirmed by immunoblotting. ( G ) Sp1 was immunoprecipitated from 293T cells and the interaction with SIRT6 was tested by western blotting. IgG was used as control. Whole cell lysate (WCL) was probed for indicated proteins by western blotting. The results presented here are representative of three independent experiments. ( H ) Luciferase reporter assay to assess the transcriptional activity of Sp1 was performed in pcDNA, SIRT6-WT or SIRT6 H133Y expressing 293T cells. The results are expressed as the fold change relative to pcDNA expressing cells. n = 6. Data are presented as mean ± s.d, * P < 0.05. ( I ) Realtime qPCR analysis of relative mRNA levels of GLUT1 and GLUT3 in WT and SIRT6-KO mice heart tissues. n = 6 mice per group. Data are presented as mean ± s.d, * P < 0.05. ( J ) Sp1 was immunoprecipitated from the 293T cells transfected with pcDNA, SIRT6-WT or SIRT6-H133Y plasmids and western blotting analysis was performed to detect the levels of Sp1 acetylation (Ac-Lys) by anti-acetyl-lysine antibody. IgG was used as a negative control in this assay. Whole cell lysate (WCL) was probed for indicated proteins by western blotting. (K) Representative immunofluorescence images depicting the levels and localization of Sp1 transcription factor (green) under control and SIRT6-KD conditions in Hela cells. SIRT6 was depleted using specific siRNA and knockdown was confirmed by immunostaining for SIRT6 (green) in a parallel experiment . The nuclei were stained with Hoechst 33342 and are shown in blue. Scale bar = 20 μm.

Article Snippet: For generation of stable Sp1 knockout cell line, gRNA targeting Sp1 cloned in plentiCRISPR v2 vector was obtained from GenScript ® .

Techniques: Expressing, Positive Control, Negative Control, Binding Assay, Control, Western Blot, shRNA, Knockdown, CRISPR, Over Expression, Transfection, Plasmid Preparation, Immunoprecipitation, Luciferase, Reporter Assay, Activity Assay, Immunofluorescence, Immunostaining, Staining

Journal: Nucleic Acids Research

Article Title: SIRT6 transcriptionally regulates global protein synthesis through transcription factor Sp1 independent of its deacetylase activity

doi: 10.1093/nar/gkz648

Figure Lengend Snippet: ( A ) ChIP analysis to detect changes in Sp1 binding to the Rheb, mTOR and p70S6K promoter regions in control or SIRT6 stable knockdown 293T cells performed with Sp1 or IgG control antibody. n = 3. Data are presented as mean ± s.d, * P < 0.05. ( B ) ChIP analysis to detect changes in acetylation status at H3K9 residue at Rheb, mTOR and p70S6K promoter regions in control or SIRT6 stable knockdown 293T cells performed with Sp1 or IgG control antibody. n = 3. Data are presented as mean ± s.d, * P < 0.05. ( C ) ChIP analysis to detect changes in acetylation status at H3K56 residue at Rheb, mTOR and p70S6K promoter regions in control or SIRT6 stable knockdown 293T cells performed with Sp1 or IgG control antibody. n = 3. Data are presented as mean ± s.d, * P < 0.05. ( D ) Overlay of 2D 15 N- 1 H TROSY-HSQC NMR spectra of free Sp1 ZFDBD (black) with Sp1 ZFDBD in complex with SIRT6-WT (in red) (at 1:2 molar ratio). The disappeared cross peaks upon addition of SIRT6 are highlighted in box. ( E ) Overlay of 15 N- 1 H TROSY-HSQC NMR spectra of free Sp1 ZFDBD (black) with Sp1 ZFDBD in complex with mutant SIRT6-H133Y (in red) (at 1:2 molar ratios). The disappeared cross peaks upon addition of mutant SIRT6-H133Y are highlighted in box. ( F ) Representative images of western blotting SUnSET analysis in control or SIRT6-KD HeLa cells in the presence vehicle or 200 nM of Sp1 inhibitor Mithramycin A. DMSO was used as a vehicle control. SIRT6 was depleted using specific siRNA and the knockdown was confirmed by immunoblotting for SIRT6. Reduction in mTOR phosphorylation was used to confirm the action of Mithramycin A. ( G ) Quantitative representation of cap-dependent translation rates measured using the luciferase reporter pRL-CMV in control or SIRT6 depleted HeLa cells (siRNA mediated knockdown) in the presence or absence of 200 nM Mithramycin A. Renilla luciferase readings were normalized against the mRNA levels of Renilla luciferase. n = 4. Data are presented as mean ± s.d, * P < 0.05. ( H ) Quantitative representation of cap-dependent translation rates measured using the luciferase reporter pRL-CMV in control or SIRT6 depleted HeLa cells (siRNA mediated knockdown) under control or Sp1 depleted conditions (shRNA mediated knockdown). Renilla luciferase readings were normalized against the mRNA levels of Renilla luciferase. n = 4. Data are presented as mean ± s.d, * P < 0.05.

Article Snippet: For generation of stable Sp1 knockout cell line, gRNA targeting Sp1 cloned in plentiCRISPR v2 vector was obtained from GenScript ® .

Techniques: Binding Assay, Control, Knockdown, Residue, Mutagenesis, Western Blot, Phospho-proteomics, Luciferase, shRNA

Journal: Indian Journal of Hematology & Blood Transfusion

Article Title: CRISP Points on Establishing CRISPR - Cas9 In Vitro Culture Experiments in a Resource Constraint Haematology Oncology Research Lab

doi: 10.1007/s12288-018-1008-z

Figure Lengend Snippet: Companies providing CRISPR-Cas tools

Article Snippet: The overall approximate costs as visible from the table appear to be within reasonable range of a basic molecular laboratory doing research in a LMIC. table ft1 table-wrap mode="anchored" t5 Table 4 caption a7 Company Product description Applied StemCells (Menlo Park, California) Genome engineering Gene editing Knock-in cell lines GenScript (Piscataway, New Jersey) gRNA design Transfection and cell pool evaluation Single-cell clone generation and validation Horizon Discovery Gene-editing tools Validated gRNAs Cas9 vectors Cell line generation kit Delivery vectors Thermo Scientific CRISPR Nuclease Vector Reporter Kit Genome-CRISPR sgRNA design Cloning services Origene CRISPR cloning kits CRISPR-Cas9 custom services Addgene Cas9 plasmids Open in a separate window Companies providing CRISPR-Cas tools table ft1 table-wrap mode="anchored" t5 Table 5 caption a7 S. no.

Techniques: CRISPR, Knock-In, Transfection, Biomarker Discovery, Plasmid Preparation, Cloning

Journal: Indian Journal of Hematology & Blood Transfusion

Article Title: CRISP Points on Establishing CRISPR - Cas9 In Vitro Culture Experiments in a Resource Constraint Haematology Oncology Research Lab

doi: 10.1007/s12288-018-1008-z

Figure Lengend Snippet: Comparision of different Cas9 construct used in CRISPR experiment

Article Snippet: The overall approximate costs as visible from the table appear to be within reasonable range of a basic molecular laboratory doing research in a LMIC. table ft1 table-wrap mode="anchored" t5 Table 4 caption a7 Company Product description Applied StemCells (Menlo Park, California) Genome engineering Gene editing Knock-in cell lines GenScript (Piscataway, New Jersey) gRNA design Transfection and cell pool evaluation Single-cell clone generation and validation Horizon Discovery Gene-editing tools Validated gRNAs Cas9 vectors Cell line generation kit Delivery vectors Thermo Scientific CRISPR Nuclease Vector Reporter Kit Genome-CRISPR sgRNA design Cloning services Origene CRISPR cloning kits CRISPR-Cas9 custom services Addgene Cas9 plasmids Open in a separate window Companies providing CRISPR-Cas tools table ft1 table-wrap mode="anchored" t5 Table 5 caption a7 S. no.

Techniques: Construct, CRISPR, Plasmid Preparation