Journal: iScience

Article Title: CFIm25-dependent alternative polyadenylation in AKT2 mRNA programs macrophage polarization

doi: 10.1016/j.isci.2026.115791

Figure Lengend Snippet: CFIm25 regulates macrophage biochemical properties in THP-1 cells (A) Analysis of nitric oxide synthase (NOS) and arginase (Arg) activity in CFIm25-overexpressing (OE) THP-1 cells. Left: NOS activity measured by DAF-FM DA fluorescence in M0, M1, and M2 polarized cells with or without CFIm25 OE. Right: Arginase activity assay in the same conditions. (B) Analysis of NOS and arginase in CFIm25-knockdown (KD) HL-60 cells. Left: NOS activity measured by DAF-FM DA fluorescence in M0, M1, and M2 polarized cells with or without CFIm25 KD. Right: Arginase activity assay in the same conditions. For A-B, data are normalized to respective controls (M0 state). (C) ELISA measurement of M1 cytokines in CFIm25 OE cells. Left: TNF-α levels in M0, M1, and M2 conditions. Right: IL-12 levels in the same condition. (D) ELISA measurement of M1 cytokines in CFIm25 KD cells. Left: TNF-α levels in M0, M1, and M2 conditions. Right: IL-12 levels in the same condition. (E) ELISA measurement of M2 cytokines in CFIm25 OE cells. Left: TGF-β levels in M0, M1, and M2 conditions. Right: IL-10 levels in the same conditions. (F) ELISA measurement of M2 cytokines in CFIm25 KD cells. Left: TGF-β levels in M0, M1, and M2 conditions. Right: IL-10 levels in the same conditions. For C-F, data are represented as relative fluorescence units. Data for all panels are shown as mean ± SEM from three independent experiments ∗∗ p < 0.01 and ∗ p < 0.05.

Article Snippet: DAF-FM DA (fluorescent NO indicator) , Fisher Scientific , Cat# 25-152-01MG.

Techniques: Activity Assay, Fluorescence, Arginase Activity Assay, Knockdown, Enzyme-linked Immunosorbent Assay

Journal: iScience

Article Title: CFIm25-dependent alternative polyadenylation in AKT2 mRNA programs macrophage polarization

doi: 10.1016/j.isci.2026.115791

Figure Lengend Snippet: Effects of blocking AKT2 proximal polyadenylation with antisense morpholino oligonucleotides (AMOs) on M1 properties (A) Schematic representation of AKT2 mRNA shows the coding sequence (CDS), polyadenylation sites (red bolts), and AMO position (purple bar) blocking the proximal polyadenylation site. (B) Effect of AKT2 proximal polyadenylation site blockade using AMO compared to control AMO. Left: APA analysis shows the log2 ratio of long/total AKT2 mRNA. Right: Log2 fold change in total AKT2 mRNA levels. (C) Western blot shows Akt2 protein levels in M1 polarized cells with or without AKT2 AMO treatment, with GAPDH as a loading control. (D) Effect of control or AKT2 AMO on macrophage surface markers by dual staining shows the percentage of cells expressing CD80 and CD206 surface markers in M1 polarized cells. (E) NOS activity measured by DAF-FM DA fluorescence in M1 polarized cells with or without AKT2 AMO. (F) Arginase activity assay in M1 polarized cells with control or AKT2 AMO. (G) Arginase (ARG1) gene expression by RT-qPCR in M1 polarized cells with control or AKT2 AMO. (H) Impact of AKT2 AMO on cytokine production. Left: M1 cytokine (TNF-α and IL-12) levels. Right: M2 cytokine (TGF-β and IL-10) levels measured by ELISA. (I) Western blot analysis of NF-κB pathway components in M1 polarized cells treated with control or AKT2 polyadenylation site AMO for phospho-p65, total NF-κB p65, and IκB-α, where GAPDH serves as a loading control. In relevant panels, data are shown as mean ± SEM from three independent experiments ∗∗∗ p < 0.001, ∗∗ p < 0.01 and ∗ p < 0.05.

Article Snippet: DAF-FM DA (fluorescent NO indicator) , Fisher Scientific , Cat# 25-152-01MG.

Techniques: Blocking Assay, Sequencing, Control, Western Blot, Staining, Expressing, Activity Assay, Fluorescence, Arginase Activity Assay, Gene Expression, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay