alkaline phosphatase seap reporter assay raw blue cells  (InvivoGen)

Journal: Non-coding RNA Research

Article Title: CircSMAD4 shapes matrix-remodeling TAMs in lung adenocarcinoma

doi: 10.1016/j.ncrna.2026.03.003

Figure Lengend Snippet: circSMAD4 functions as a cytoplasmic ceRNA to sequester miR-562 and de-repress COL4A1. (A) Subcellular distribution of circSMAD4 in TC-hMDMs and patient-derived TAMs assessed by nuclear/cytoplasmic fractionation. (B) Representative immunofluorescence/ISH images showing circSMAD4 signals in macrophages (CD163) with nuclear counterstaining (DAPI). Scale bar, 50 μm. (C) Venn diagram of predicted circSMAD4-interacting miRNAs from circInteractome and circBank, yielding a shortlist including miR-562. (D) miR-562 levels following circSMAD4 knockdown in TC-hMDMs. (E–G) pri-miR-562, pre-miR-562, and miR-562 promoter reporter activity after circSMAD4 overexpression. (H) AGO2-RIP enrichment of circSMAD4 and miR-562 relative to IgG in TC-hMDMs. (I) AGO2 immunoblotting after circSMAD4 sense/antisense RNA pull-down in TC-hMDMs. (J) Predicted pairing between miR-562 and circSMAD4 (WT) and the corresponding mutant design. Mutations were introduced within the predicted miR-562 seed-matching region using transition substitutions (A↔G, C↔U) to disrupt miRNA–target pairing while minimizing changes in sequence composition and local RNA structure. (K) Dual-luciferase assays for circSMAD4-WT/MUT reporters in the presence of miR-562 mimics or inhibitor. (L) Intersection of miRNA target predictions (miRTarBase, miRmap, TargetScan, and miRDB) identifying candidate miR-562 targets. (M) COL4A1 mRNA levels after miR-562 mimics or inhibitor in TC-hMDMs. (N) Predicted miR-562 binding site within the COL4A1 3′UTR (WT) and mutant design. Mutations were introduced within the predicted miR-562 seed-matching region using transition substitutions (A↔G, C↔U) to disrupt miRNA–target pairing while minimizing changes in sequence composition and local RNA structure. (O) Dual-luciferase assays for COL4A1 3′UTR WT/MUT reporters with miR-562 mimics or inhibitor. ∗P < 0.05; ∗∗P < 0.01; ∗∗∗P < 0.001; ∗∗∗∗P < 0.0001; ns, not significant.

Article Snippet: At 48 h post-transfection, luciferase activities were measured using the Dual Luciferase Reporter Assay Kit (Vazyme, Cat# DL101-01), and relative luciferase activity was calculated by normalizing Firefly to Renilla signals.

Techniques: Derivative Assay, Fractionation, Immunofluorescence, Knockdown, Activity Assay, Over Expression, Western Blot, Mutagenesis, Sequencing, Luciferase, Binding Assay

Journal: Non-coding RNA Research

Article Title: CircSMAD4 shapes matrix-remodeling TAMs in lung adenocarcinoma

doi: 10.1016/j.ncrna.2026.03.003

Figure Lengend Snippet: circSMAD4 facilitates IGF2BP2-dependent stabilization of m6A-marked transcripts. (A) Venn diagram intersecting ENCORI-predicted IGF2BP2 targets with DEGs from shIGF2BP2 versus shNC and shcircSMAD4 versus shNC mRNA-seq, identifying shared candidates. (B) MeRIP–qPCR showing m6A enrichment on COL4A1, SPI1, and ACTA2 candidate regions (CRDs) in shNC and shIGF2BP2 cells. (C) IGF2BP2-RIP–qPCR showing IGF2BP2 binding to COL4A1, SPI1, and ACTA2 CRDs in shNC + Vector, shcircSMAD4 + Vector, shNC + IGF2BP2, and shcircSMAD4 + IGF2BP2 groups. (D) Biotin-circSMAD4 pull-down followed by qPCR showing enrichment of COL4A1, SPI1, and ACTA2 CRDs in Vector + shNC, circSMAD4 + shNC, Vector + shIGF2BP2, and circSMAD4 + shIGF2BP2 groups. (E–G) Schematics of m6A-site mutations introduced into COL4A1, SPI1, and ACTA2 reporters. (H–J) Dual-luciferase assays for CRD reporters (WT and m6A-mutant) in Vector, circSMAD4, and IGF2BP2 groups. (K–M) MeRIP–qPCR for WT and m6A-mutant CRD reporters in Vector, circSMAD4, and IGF2BP2 groups. (N–P) mRNA decay assays of endogenous COL4A1, SPI1, and ACTA2 following circSMAD4 knockdown with Vector or IGF2BP2 overexpression. Half-life estimated by one-phase decay (Y0 = 1, Plateau = 0). (Q–S) mRNA decay assays of endogenous COL4A1, SPI1, and ACTA2 following circSMAD4 overexpression with shNC or shIGF2BP2. Half-life estimated by one-phase decay (Y0 = 1, Plateau = 0). ∗P < 0.05; ∗∗P < 0.01; ∗∗∗P < 0.001; ∗∗∗∗P < 0.0001; ns, not significant.

Article Snippet: At 48 h post-transfection, luciferase activities were measured using the Dual Luciferase Reporter Assay Kit (Vazyme, Cat# DL101-01), and relative luciferase activity was calculated by normalizing Firefly to Renilla signals.

Techniques: Binding Assay, Plasmid Preparation, Luciferase, Mutagenesis, Knockdown, Over Expression

Journal: Development (Cambridge, England)

Article Title: Hemidesmosomes and Notch signaling regulate epidermal differentiation via delamination

doi: 10.1242/dev.205210

Figure Lengend Snippet: Notch signaling regulates integrin-β4 levels and delamination. (A-D) Confocal images of E17 Cre-negative control versus Rosa NICD epidermis (A) and E16 WT littermate versus Rbpj cKO epidermis (C), with single channel images of integrin-β4 on the right, and associated quantification of fluorescence intensity (B,D); n =3 except for WT control in Rbpj cohort ( n =2). (E-G) Images of Notch reporter (NR) transgenic (E) and LUGGIGE NR-transduced epidermis (F), and associated quantification (G). NR shown in green; RFP marks cells transduced with reporter. (H) Quantification of nuclear YAP in Itgb4 4124 epidermis. (I,K) Confocal images of E17 Cre-negative control versus Rosa NICD epidermis (I) and E16 WT littermate versus Rbpj cKO epidermis (K); asterisks indicate dual-positive cells. (J,L) Quantification of dual-positive cells in WT versus mutant. Each dot represents a biological replicate in G,H,J,L, or FOV in B,D, where shapes designate litters. Basement membrane is indicated with cyan dashed line. Scale bars: 25 μm (A,C,E,F,I,K); 10 µm (F, insets). ns, not significant; * P <0.05; **** P <0.0001 ( t -test).

Article Snippet: Transgenic Notch reporter animals were obtained from The Jackson Laboratory [ Tg(Cp-EGFP)25Gaia/J, stock #005854].

Techniques: Negative Control, Fluorescence, Control, Transgenic Assay, Transduction, Mutagenesis, Membrane