addgene 43802  (Addgene inc)

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 physically associates with IGF2BP2 in macrophages. (A) LC–MS/MS summary of proteins enriched by circSMAD4 RNA pull-down. (B) Western blot validation of IGF2BP2 in circSMAD4 sense (vs antisense) pull-down from TC-hMDMs. (C) IGF2BP2 RIP–qPCR showing circSMAD4 enrichment over IgG in TC-hMDMs. (D–E) catRAPID prediction and ViennaRNA RNAfold secondary-structure modeling indicating multiple candidate IGF2BP2-binding regions on circSMAD4. (F) Western blot of IGF2BP2 after pull-down with circSMAD4 fragments (1#–3#). (G) Schematic of IGF2BP2 domain architecture and the Flag-tagged truncation/deletion constructs used for mapping circSMAD4 interaction (designed based on catRAPID prediction and annotated RRM/KH domain boundaries). (H) Anti-Flag RIP–qPCR showing circSMAD4 enrichment precipitated by the indicated Flag-tagged IGF2BP2 truncation/deletion constructs (presented as % input and normalized to IgG). (I) Nuclear–cytoplasmic fractionation followed by RT–qPCR showing circSMAD4 distribution and the effect of IGF2BP2 knockdown on the nuclear-to-cytoplasmic ratio of circSMAD4 in TC-hMDMs. Fractionation quality was validated using nuclear/cytoplasmic marker transcripts/proteins. (J) Representative immunofluorescence/ISH images showing circSMAD4 signals and IGF2BP2 staining in macrophages (CD163) with nuclear counterstaining (DAPI). Scale bar, 50 μm. (K–N) qPCR and Western blot showing no reciprocal change in expression between circSMAD4 and IGF2BP2 upon knockdown/overexpression. ∗P < 0.05; ∗∗P < 0.01; ∗∗∗P < 0.001; ∗∗∗∗P < 0.0001; ns, not significant.

Article Snippet: Gene silencing was performed using lentiviral shRNAs. shRNAs targeting the human circSMAD4 back-splice junction (and the murine circSmad4 ortholog, avoiding linear Smad4) as well as IGF2BP2 were cloned into the pLKO.1-puro vector (Addgene, #8453).

Techniques: Liquid Chromatography with Mass Spectroscopy, Western Blot, Biomarker Discovery, Binding Assay, Construct, Fractionation, Quantitative RT-PCR, Knockdown, Marker, Immunofluorescence, Staining, Expressing, Over Expression

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: Gene silencing was performed using lentiviral shRNAs. shRNAs targeting the human circSMAD4 back-splice junction (and the murine circSmad4 ortholog, avoiding linear Smad4) as well as IGF2BP2 were cloned into the pLKO.1-puro vector (Addgene, #8453).

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

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: Proposed model: circSMAD4 drives matrix-remodeling TAM programs in LUAD. Schematic summary illustrating that circSMAD4 in tumor-associated macrophages promotes a matrix-remodeling, M2-like state through two post-transcriptional routes: (i) circSMAD4 sequesters miR-562 in an AGO2-dependent manner to relieve repression of COL4A1 mRNA; (ii) circSMAD4 associates with IGF2BP2 to enhance the stability of m6A-marked transcripts, including COL4A1, SPI1, and ACTA2 (α-SMA). These combined outputs reinforce extracellular matrix remodeling within the LUAD tumor microenvironment.

Article Snippet: Gene silencing was performed using lentiviral shRNAs. shRNAs targeting the human circSMAD4 back-splice junction (and the murine circSmad4 ortholog, avoiding linear Smad4) as well as IGF2BP2 were cloned into the pLKO.1-puro vector (Addgene, #8453).

Techniques: