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: Cell & bioscience

Article Title: CRISPR/Cas-mediated mRNA knockdown in the embryos of Xenopus tropicalis.

doi: 10.1186/s13578-025-01397-8

Figure Lengend Snippet: Fig. 3 Effects of dCas9-KM system on reporter gene expression in embryos of X. tropicalis. (A) Schematic of CRISPRi-related gRNAs targeting the regions of mCherry reporter gene in pmCherry-N1 plasmid. (B) Schematic illustration of the experimental setup used to analyze dCas9-KM capacity targeting exog enous reporter gene in X. tropicalis embryos. (C) The qPCR validation of mCherry expression in embryos co-injected with dCas9-KM system and indicated gRNAs at 48 dpi (n = 4 per group). (D and E) Representative images (D) and quantification (E) of mCherry expression in embryos co-injected with dCas9- KM system and indicated gRNA at 48 hpi (n = ~ 60 embryos from 3 independent experiments). (F) Schematic of gR-187 targeting mCherry reporter gene in pCMV-mCherry-EF1α-EGFP dual-reporter plasmid. (G) The qPCR validation of mCherry expression in embryos co-injected with dCas9-KM system and indicated gRNA at 48 dpi (n = 5 per group). EGFP was used as the internal control. (H and I) Representative images (H) and quantification (I) of mCherry fluorescence in embryos co-injected with dCas9-KM system and indicate gRNA at 48 hpi (n = 24 embryos from 3 independent experiments). All data are presented as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 versus control. Ns, no significant differences versus control

Article Snippet: The human codon-optimized PspCas13b (pC0046, #103862) and RfxCas13d (pXR001, #109049) expressing plasmids as well as their guide RNA (gRNA) expression plasmids (pC0043, #103854 for PspCas13b; pXR003, #109053 for RfxCas13d) were purchased from Addgene.

Techniques: Gene Expression, Plasmid Preparation, Biomarker Discovery, Expressing, Injection, Control, Fluorescence

Journal: Cell & bioscience

Article Title: CRISPR/Cas-mediated mRNA knockdown in the embryos of Xenopus tropicalis.

doi: 10.1186/s13578-025-01397-8

Figure Lengend Snippet: Fig. 4 Effects of dCas9-KM system on tyrosinase expression in embryos of X. tropicalis. (A) Schematic illustration of dCas9-KM system-related gRNAs targeting the DNA locus of endogenous tyrosinase (Tyr) gene. Fertilized eggs were co-injected with dCas9-KM mRNAs and indicated gRNA sets, followed by embryo evaluation at 48 hpi as follows. (B and C) Representative images (B) and quantification (C) of Tyr production in embryos co-injected with dCas9-KM mRNA and indicated gRNAs at 48 hpi (n = 26 ~ 30 embryos from 3 independent experiments). Scale bar = 250 μm. (D) The qPCR validation of Tyr expression in embryos co-injected with dCas9-KM mRNA and indicated gRNAs at 48 dpi (n = 5 per group). (E and F) Representative images (E, scale bar = 250 μm) and quantification (F) of embryos with different phenotypes were counted and compared with the total developed ones 48 h post co-injection of dCas9-KM system. (G and H) Representative images (G) and quantification (H) of embryos with different phenotypes were counted and compared with the total developed ones 72 h post co-injection of dCas9-KM system. Total embryos evaluated for each group (n) is shown above each column. All data are present as mean ± SEM. ****p < 0.0001 versus gNC group

Article Snippet: The human codon-optimized PspCas13b (pC0046, #103862) and RfxCas13d (pXR001, #109049) expressing plasmids as well as their guide RNA (gRNA) expression plasmids (pC0043, #103854 for PspCas13b; pXR003, #109053 for RfxCas13d) were purchased from Addgene.

Techniques: Expressing, Injection, Biomarker Discovery

Journal: Molecular Therapy. Nucleic Acids

Article Title: Protection of animals against devastating RNA viruses using CRISPR-Cas13s

doi: 10.1016/j.omtn.2024.102235

Figure Lengend Snippet: Schematic of FMDV genome and targeted regions by CRISPR-Cas and RNAi systems Scissors indicate target sites. Conserved regions of the 3D gene were tiled with nine crRNAs belonging to different subtypes of CRISPR-Cas type VI. Lwa Cas13a, Psp Cas13b, and Rfx Cas13d spacers are indicated in blue, green, and orange, respectively. The nucleotides in red highlight residues in the guide (antisense) strand of shRNA. Proper non-targeting controls as negative control “scramble” for nonspecific effects were designed. NTCs have the shuffled nucleotide sequence of the target-specific crRNA or shRNA and have passed characteristics of a functional crRNA or shRNA such as off-target analysis ( Table 1 ).

Article Snippet: Spacers were cloned into pC0040- Lwa Cas13a, pC0043- Psp Cas13b, or pXR003- Rfx Cas13d guide RNA expression backbone plasmids (Addgene #103851, #103854, and #109053, respectively) and shRNAs were cloned into pMKO.1 puro plasmid (Addgene #8452).

Techniques: CRISPR, shRNA, Negative Control, Sequencing, Functional Assay

Journal: Molecular Therapy. Nucleic Acids

Article Title: Protection of animals against devastating RNA viruses using CRISPR-Cas13s

doi: 10.1016/j.omtn.2024.102235

Figure Lengend Snippet: Effect of Cas13 and RNAi on FMDV inhibition, serotype O (A) Experimental procedure for evaluating Cas13s and RNAi antiviral activity in BHK-21 cells. BHK-21 cells were transfected with plasmids containing targeting and non-targeting shRNAs or Cas13 and crRNAs that target or do not target FMDV 24 h before infection. Then the supernatants were collected 24 hpi to evaluate the antiviral activity. Silencing efficiency of Lwa Cas13a (B), Psp Cas13b (C), Rfx Cas13d (D), and RNAi system (E) on FMDV virus according to RT-qPCR results. In (B) to (E), data points in the graph are five independent biological experiments performed in technical replicates, lines indicate means; error bars represent SD; t test was performed to compare each treatment to non-targeting crRNA or shRNA (green and red color of the boxes indicate p < 0.05 and p < 0.01, respectively).

Article Snippet: Spacers were cloned into pC0040- Lwa Cas13a, pC0043- Psp Cas13b, or pXR003- Rfx Cas13d guide RNA expression backbone plasmids (Addgene #103851, #103854, and #109053, respectively) and shRNAs were cloned into pMKO.1 puro plasmid (Addgene #8452).

Techniques: Inhibition, Activity Assay, Transfection, Infection, Virus, Quantitative RT-PCR, shRNA

Journal: Viruses

Article Title: Efficient Inhibition of HIV Using CRISPR/Cas13d Nuclease System

doi: 10.3390/v13091850

Figure Lengend Snippet: CRISPR/CasRx with HIV-specific single guide RNAs (gRNA) inhibits HIV-1 replication ( A ) schematic representation of HIV genome and gRNA binding sites (red stars; HIV sequence compendium 2018); ( B ) Lenti-XTM cells with stable CasRx-GFP expression (LRx) cells were co-transfected with plasmid encoding gRNA and HIV-encoding iRFP670 fluorescent marker (HIV-iRFP). Forty-eight hours after transfection, expression of CasRx-GFP and HIV-iRFP were measured by flow cytometry. Positions of gRNA sequences in HIV transcript are indicated on the X -axis; ( C ) Supernatants collected from the LRx cells transfected with HIV-iRFP and HIV-gRNA or NT plasmids were added to the reporter cell line TZM-bl containing luciferase gene under HIV-LTR. Relative luciferase expression was measured after 48 h and presented as a fold change in relative luciferase activity normalized to cells only control (CC). The means ± s.e.m. are depicted as horizontal and vertical bars for each group, respectively. ANOVA tests with Bonferroni correction were used for statistical comparisons, and two-tailed p values are indicated. **** p < 0.0001; *** p < 0.001, ** p < 0.01.

Article Snippet: The CasRx, (pXR001; Addgene #109049), dCasRx (PXR002; Addgene #109050) plasmids, gRNA expression vectors (pXR003, pXR004; Addgene #109053 and #109054), Vesicular Stomatitis Virus glycoprotein (VSV-G) envelope expression vector (pMD2.G; Addgene #12259), lentiviral packaging plasmid (psPax2; Addgene # 12260), iRFP670 fluorescent reporter vector (piRFP670-N1; Addgene#79987) and pKLV2-U6gRNA5(BbsI)-PGKpuro2ABFP-W (Addgene #67974) were obtained from the Addgene repository (Watertown, MA, USA).

Techniques: CRISPR, Binding Assay, Sequencing, Expressing, Transfection, Plasmid Preparation, Marker, Flow Cytometry, Luciferase, Activity Assay, Control, Two Tailed Test

Journal: Molecular cell

Article Title: Genome-scale exon perturbation screens uncover exons critical for cell fitness.

doi: 10.1016/j.molcel.2024.05.024

Figure Lengend Snippet: Figure 6. Combinatorial perturbation of neural-regulated exons using dCasRx-RBM25 (A) Splicing levels of Puf60 exon 5 and Ptbp1 exon 8 at different stages of in vitro differentiation of mESCs into mature neurons. EB, embryoid body; DIV, days in vitro. (B) Splicing levels of Puf60 exon 5 and Ptbp1 exon 8 in CGR8 mESCs stably expressing dCasRx-RBM25 and the indicated single gRNAs or two-spacer gRNA arrays. The order of the array is shown by the positions of gRNA1 (first spacer) and gRNA2 (second spacer). Wild-type (parental) mESCs are shown for com- parison. (C) RT-PCR assays monitoring splicing levels of Ptbp2 exon 10, Akap9 exon 18, and Snap91 exon 25, following expression of indicated gRNAs targeting Puf60 exon 5, and/or Ptbp1 exon 8, in mESCs stably expressing dCasRx-RBM25. (D) Bar plots quantifying change in PSI (dPSI) of Ptbp2 exon 10, Akap9 exon 18, and Snap91 exon 25, between indicated conditions of single Ptbp1 exon 8 perturbation (light gray bars), single Puf60 exon 5 perturbation (dark gray bars), and dual Ptbp1 exon 8 + Puf60 exon 5 perturbations (orange and blue bars). p values from two-sided Student’s t tests.

Article Snippet: Expression plasmids for RBFOX1N-dCasRx-C (Addgene #118635), and gRNAs targeting the SMN2 downstream intron (Addgene#118646-118648) are as previously described.22 ForMef2d reporter experiments, gRNAswere cloned into the pXR003 backbone (Addgene#109053) by annealed oligonucleotide ligation.

Techniques: In Vitro, Stable Transfection, Expressing, Reverse Transcription Polymerase Chain Reaction