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: 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