Journal: PLOS One

Article Title: Down-regulation of circ0001361 induces apoptosis and suppresses the progression of glioma

doi: 10.1371/journal.pone.0343681

Figure Lengend Snippet: (A) MiRNAs targeted by circ0001361 were predicted. (B) Expression of hsa-miR-525-5p in glioma (Group A) and normal brain tissue (Group B) was shown. (C) Expression of hsa-miR-525-5p in glioma grade Ⅱ -Ⅳ was shown. (D) Prognostic significance of hsa-miR-525-5p was analyzed. (E) Potential binding sites between circ0001361 and hsa-miR-525-5p were predicted. (F) Circ0001361-hsa-miR-525-5p interaction was confirmed by dual-luciferase reporter assay.

Article Snippet: After 48 hours, the Firefly luciferase and Renilla Luciferase were detected using the Dual Luciferase Reporter Assay Kit (Vazyme, China).

Techniques: Expressing, Binding Assay, Luciferase, Reporter Assay

Journal: PLOS One

Article Title: Down-regulation of circ0001361 induces apoptosis and suppresses the progression of glioma

doi: 10.1371/journal.pone.0343681

Figure Lengend Snippet: (A) Genes targeted by hsa-miR-525-5p were predicted. (B) Correlation between hsa-miR-525-5p and MEIS1 was analyzed. (C) Prognostic significance of MEIS1 was analyzed. (D) Expression of MEIS1 in GBM, LGG and normal brain tissues was shown. (E) Expression of MEIS1 in glioma grade Ⅱ -Ⅳ was shown. (F) The relationship between MEIS1 and IDH mutation status was shown. (G) The relationship between MEIS1 and 1p/19q co−deletion status was shown. (H) Potential binding sites between hsa-miR-525-5p and MEIS1 were predicted. (I) Hsa-miR-525-5p and MEIS1 interaction was confirmed by dual-luciferase reporter assay.

Article Snippet: After 48 hours, the Firefly luciferase and Renilla Luciferase were detected using the Dual Luciferase Reporter Assay Kit (Vazyme, China).

Techniques: Expressing, Mutagenesis, Binding Assay, Luciferase, Reporter Assay

Journal: Science Advances

Article Title: Oligo-CALL: A next-generation barcoding platform for studying resistance to targeted therapy

doi: 10.1126/sciadv.adw9990

Figure Lengend Snippet: ( A ) Inducible gRNA barcode design: A spacer-blocking hairpin structure is engineered to include a 15-base semirandom genetic barcode within the hairpin loop. In addition, an 8A8G poly(A) tail is appended at the 3′ end of the barcoding gRNA. ( B ) Oligo-CALL system constructs: (i) Dual gRNA barcoding construct: two distinct barcoding gRNA, each driven by human or mouse U6 promoters, separately, with Thy1.1 as a constitutive selection marker. (ii) The green fluorescent protein (GFP) reporter construct: contains three tandem repeats of the barcoding gRNA target site upstream of a miniCMV (minimal cytomegalovirus) promoter controlling GFP expression; blue fluorescent protein (BFP) serves as a selection marker. (iii) CRISPRa construct: constitutively expresses dCas9-VPR [deactivated Cas9 fused to a transcriptional activator VPR (VP64, p65, and Rta)] and the red fluorescent protein (RFP; Cherry variant). All constructs use the lentiviral backbone to facilitate efficient cell transduction. hU6, human U6; mU6, mouse U6; EF1α, elongation factor-1 alpha promoter. ( C ) GFP activation with Oligo-CALL system: In the default state, a self-folding hairpin within the gRNA spacer prevents CRISPRa from activating GFP expression. Introduction of BC-ASOs disrupts this hairpin, exposing the gRNA spacer and enabling robust GFP expression. Representative fluorescence images show strong GFP induction following BC-ASO transfection, in contrast to minimal activation in cells receiving no ASO or mismatched ASOs (mis ASOs). ( D to F ) Flow cytometry analyses of GFP activation in Oligo-CALL–labeled cells: H358 cells bearing BC1 barcodes were transfected with ASOs against BC1. Flow cytometry shows the percentage of GFP + cells (D and E) and the mean fluorescent intensity (F) across the overall population (48 hours post-ASO transfection; n = 2 biological replicates). MFI, mean fluorescence intensity. Cont., control.

Article Snippet: Lentiviral backbone vectors—Dual-sgRNA (plasmid #154194), CaTCH_empty (plasmid #157746), and dCas9-VPR_P2A_mCherry (plasmid #154193), developed initially in A. Obenauf’s laboratory ( )—were obtained from Addgene.

Techniques: Blocking Assay, Construct, Selection, Marker, Expressing, Variant Assay, Transduction, Activation Assay, Fluorescence, Transfection, Flow Cytometry, Labeling, Control