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: Science advances

Article Title: HIV-1 Vpr induces ciTRAN to prevent transcriptional repression of the provirus.

doi: 10.1126/sciadv.adh9170

Figure Lengend Snippet: Fig. 2. Nanopore DRS reveals infection-induced circRNAs. (A) Schematics depicting processing of enriched circRNA pools for nanopore DRS (method detailed in the Supplementary Materials). (B) Table showing mapped reads obtained from three flow cells per sample after high-precision basecalling. (C) Schematics showing a com- putational pipeline developed for the detection of BSJs using circBase, circAtlas, and virtual exonic circRNA library. (D and E) Number of circRNAs obtained using the circBase-specific library in mock (D) and infection (E). (F) Numbers of circRNAs obtained after analysis of virtual exonic circRNA library using a pipeline in (C). (G) Infection- specific high-confidence circRNA candidates validated using RT-qPCR from HIV-1–infected Jurkat E6.1 cells. Data were normalized to GAPDH. (H) Luciferase activity measured as a function of HIV-1 Luc infection from JTAg cells depleted of indicated circRNAs. gRFP served as nonrelevant guide RNA (gRNA). n = 3; ±SD. M, million; GB, gigabyte.

Article Snippet: CRISPR-assisted RNA-protein interaction detection method We generated gRNA targeting the back-splicing junction of ciTRAN and cloned it into an empty gRNA-expressing vector (Addgene #109054).

Techniques: Infection, Quantitative RT-PCR, Luciferase, Activity Assay

Journal: Science advances

Article Title: HIV-1 Vpr induces ciTRAN to prevent transcriptional repression of the provirus.

doi: 10.1126/sciadv.adh9170

Figure Lengend Snippet: Fig. 3. A post-integration event is targeted by ciTRAN. Effects of ciTRAN knockdown during HIV-1–zsGreen infection on: (A) reverse transcription products as assessed by measuring cytoplasmic HIV-1 DNA (AZT served as a control), (B) proviral integration as assessed by Alu-Gag PCR for HIV-1 genomic DNA integration [raltegravir (RAL) served as a control], and (C) cytoplasmic HIV-1 RNA (gag) expressed from the provirus (α-amanitin served as a control). Hours post-infection (hpi). (D) Nuclear transcription (gag) from an integrated provirus in ciTRAN depleted condition assessed by nuclear run-on assay. GAPDH served as control. gRNA to luciferase served as a nonrelevant gRNA. (E) Immunoblot indicating intracellular viral Gag levels; actin served as a loading control. n = 3; ±SD.

Article Snippet: CRISPR-assisted RNA-protein interaction detection method We generated gRNA targeting the back-splicing junction of ciTRAN and cloned it into an empty gRNA-expressing vector (Addgene #109054).

Techniques: Knockdown, Infection, Reverse Transcription, Control, Nuclear Run-on Assay, Luciferase, Western Blot