Journal: Inflammation

Article Title: FTO-dependent m 6 A Demethylation Activates Mxd1 To Enhance Vitamin D-induced Suppression of Neuroinflammation Via PTEN/AKT/PGC-1α Signaling Pathways in Microglia

doi: 10.1007/s10753-026-02450-5

Figure Lengend Snippet: VitD promotes M2 microglial polarization in LPS-stimulated BV-2 cells and mice. A-B CCK-8 assays of BV-2 cell viability following treatment with various concentrations of VitD, with or without LPS stimulation. Data are representative of three independent experiments and shown as mean ± SD. C Western blot analysis of CD16, CD18 (M1 markers), CD206 and ARG1 (M2 markers) in LPS- or LPS + VitD (VitD)-treated BV-2 cells and primary mouse microglia. Data are representative of three independent experiments and shown as mean ± SD. n = 3 ** P < 0.01. One-way ANOVA and Tukey’s test. D Immunofluorescence analysis of the levels of CD16 and CD206 in LPS- or LPS + VitD (VitD)-treated BV-2 cells. Scale bar: 50 μm. Right panel: Quantification of CD16 and CD206 IF intensity. Six regions were randomly selected for each group, and the IF intensities of CD16 and CD206 were quantified using the ImageJ software. Data are shown as mean ± SD. ** P < 0.01. One-way ANOVA and Tukey’s test. Red arrows indicate CD16-positive microglia and green arrows indicate CD206-positive microglia. E ELISA analysis of supernatant levels of TNF-α, IL1β, IL-6, IL-4 and IL-10 in LPS- or LPS + VitD (VitD)-treated BV-2 cells. n = 6. Data are shown as mean ± SD. ** P < 0.01. One-way ANOVA and Tukey’s test. F Immunofluorescence analysis of the levels of CD16 and CD206 in cerebral cortex and hippocampal CA1 area of LPS- or LPS + VitD(VitD)-treated mice. Scale bar: 20 μm. n = 6 mice per group. Data are shown as mean ± SD. ** P < 0.01. One-way ANOVA and Tukey’s test. Red arrows indicate CD16-positive microglia and green arrows indicate CD206-positive microglia.

Article Snippet: BV-2 cells were pretreated with various doses of active VitD (calcitriol, MedChemExpress, USA), and 1 h later LPS (100 ng/mL, Sigma-Aldrich Co., USA) was added to the medium for 24 h. To investigate the role of PI3K/AKT and PGC-1α in the regulation of BV-2 microglial polarization, BV-2 cells were pretreated with LY294002 (10μM, MedChemExpress) or SR-18,292 (10μM, MedChemExpress) for 1 h, both followed by stimulation with calcitriol for 1 h, and then stimulated with LPS (100 ng/mL) for 24 h.

Techniques: CCK-8 Assay, Western Blot, Immunofluorescence, Software, Enzyme-linked Immunosorbent Assay

Journal: Inflammation

Article Title: FTO-dependent m 6 A Demethylation Activates Mxd1 To Enhance Vitamin D-induced Suppression of Neuroinflammation Via PTEN/AKT/PGC-1α Signaling Pathways in Microglia

doi: 10.1007/s10753-026-02450-5

Figure Lengend Snippet: Mxd1 is essential for VitD-induced microglial M2 polarization in LPS-stimulated conditions. A Immunohistochemistry analysis of Mxd1 expression in cerebral cortex and hippocampal CA1 area hippocampal CA1 area of mice receiving LPS or LPS + VitD (VitD) treatment. Scale bar: 20 μm. Quantification of Mxd1-positive area is shown in the right panel. n = 6 mice per group. Data are shown as mean ± SD. ** P < 0.01. One-way ANOVA and Tukey’s test. B Western blot analysis of the protein levels of Mxd1 and c-MYC in BV-2 cells treated with LPS or LPS + VitD (VitD). Data are representative of three independent experiments and shown as mean ± SD. n = 3. ** P < 0.01. One-way ANOVA and Tukey’s test. C Immunofluorescence analysis of the levels of Mxd1 in in BV-2 cells treated with LPS or LPS + VitD (VitD). Scale bar: 100 μm. Right panel: Quantification of Mxd1 IF intensity. Six regions were randomly selected for each group, and the IF intensity of Mxd1 was quantified using the ImageJ software. Data are shown as mean ± SD. ** P < 0.01. One-way ANOVA and Tukey’s test. D Western blot analysis of the protein levels of CD16, CD18, CD206, ARG1 and Mxd1 in LPS or LPS + VitD (VitD)-treated BV-2 cells with either negative control siRNA (siNC), or Mxd1 siRNA (siMxd1). Data are representative of three independent experiments and shown as mean ± SD. n = 3. ns: not significant; * P < 0.05, ** P < 0.01. Two-way ANOVA and Tukey’s test. E ELISA analysis of supernatant levels of TNF-α, IL1β, IL-6, IL-4 and IL-10 in LPS or VitD-treated BV-2 cells with siNC or siMxd1. Data are representative of three independent experiments and shown as mean ± SD. n = 3 ** P < 0.01. Two-way ANOVA and Tukey’s test.

Article Snippet: BV-2 cells were pretreated with various doses of active VitD (calcitriol, MedChemExpress, USA), and 1 h later LPS (100 ng/mL, Sigma-Aldrich Co., USA) was added to the medium for 24 h. To investigate the role of PI3K/AKT and PGC-1α in the regulation of BV-2 microglial polarization, BV-2 cells were pretreated with LY294002 (10μM, MedChemExpress) or SR-18,292 (10μM, MedChemExpress) for 1 h, both followed by stimulation with calcitriol for 1 h, and then stimulated with LPS (100 ng/mL) for 24 h.

Techniques: Immunohistochemistry, Expressing, Western Blot, Immunofluorescence, Software, Negative Control, Enzyme-linked Immunosorbent Assay

Journal: Inflammation

Article Title: FTO-dependent m 6 A Demethylation Activates Mxd1 To Enhance Vitamin D-induced Suppression of Neuroinflammation Via PTEN/AKT/PGC-1α Signaling Pathways in Microglia

doi: 10.1007/s10753-026-02450-5

Figure Lengend Snippet: Effect of FTO on VitD-induced microglial M2 polarization. A Global m 6 A RNA methylation analysis of total mRNAs in LPS- and LPS + VitD(VitD)-treated BV-2 cells. Data are representative of three independent experiments and shown as mean ± SD. * P < 0.05; ** P < 0.01. One-way ANOVA and Tukey’s test. B Western blot of m 6 A methylation regulator (METTL3, METTL14, WTAP, ALKBH5, and FTO) in the same treatment conditions. Data are representative of three independent experiments and shown as mean ± SD. ns: not significant; ** P < 0.01. One-way ANOVA and Tukey’s test. C m 6 A quantification in BV-2 cells transfected with negative control siRNA (siNC) or FTO siRNA (siFTO) and treated with LPS or LPS + VitD (VitD). Data are representative of three independent experiments and shown as mean ± SD. * P < 0.05; ** P < 0.01. Two-way ANOVA and Tukey’s test. D Western blot of CD16, CD18, CD206, ARG1 and Mxd1 in the same treatment conditions. Data are representative of three independent experiments and shown as mean ± SD. ns: not significant; * P < 0.05; ** P < 0.01. Two-way ANOVA and Tukey’s test. E ELISA of cytokine levels in corresponding supernatants. Data are representative of three independent experiments and shown as mean ± SD. ns: not significant; ** P < 0.01. Two-way ANOVA and Tukey’s test. F Immunohistochemistry for Iba-1 in the cerebral cortex and hippocampal CA1 area of mice treated with vehicle, LPS, LPS + VitD, and LPS + VitD + FB23-2. Scale bar: 20 μm. Quantification data are shown in the right panel. n = 6 mice per group. Data are shown as mean ± SD. ** P < 0.01. One-way ANOVA and Tukey’s test.

Article Snippet: BV-2 cells were pretreated with various doses of active VitD (calcitriol, MedChemExpress, USA), and 1 h later LPS (100 ng/mL, Sigma-Aldrich Co., USA) was added to the medium for 24 h. To investigate the role of PI3K/AKT and PGC-1α in the regulation of BV-2 microglial polarization, BV-2 cells were pretreated with LY294002 (10μM, MedChemExpress) or SR-18,292 (10μM, MedChemExpress) for 1 h, both followed by stimulation with calcitriol for 1 h, and then stimulated with LPS (100 ng/mL) for 24 h.

Techniques: Methylation, Western Blot, Transfection, Negative Control, Enzyme-linked Immunosorbent Assay, Immunohistochemistry

Journal: Inflammation

Article Title: FTO-dependent m 6 A Demethylation Activates Mxd1 To Enhance Vitamin D-induced Suppression of Neuroinflammation Via PTEN/AKT/PGC-1α Signaling Pathways in Microglia

doi: 10.1007/s10753-026-02450-5

Figure Lengend Snippet: FTO stabilizes Mxd1 mRNA by preventing m 6 A/YTHDF2-mediated mRNA degradation in VitD-treated microglia. A Prediction m 6 A modification sites on Mxd1 mRNA using SRAMP bioinformatics analysis. The primer sets for MeRIP-qPCR assay are indicated by the red arrows in the schematic diagram. The mutation strategy of the m 6 A sites is shown. B MeRIP-qPCR showing m 6 A enrichment on Mxd1 mRNA in BV-2 cells. C Assessment of RIP assays detecting Mxd1 mRNA + 3635 site by a FTO antibody or by IgG in LPS or LPS + VitD treated BV-2 cells. Data are representative of three independent experiments and shown as mean ± SD. ** P < 0.01. Two-way ANOVA and Tukey’s test. D MeRIP-qPCR showing m 6 A enrichment on Mxd1 mRNA in cells treated with LPS or LPS + VitD, transfected with negative control siRNA (siNC) or FTO siRNA (siFTO). Data are representative of three independent experiments and shown as mean ± SD. * P < 0.05; ** P < 0.01. Two-way ANOVA and Tukey’s test. E-F RT-qPCR and Western blot analysis of Mxd1 mRNA and protein levels, respectively, under the same conditions. Data are representative of three independent experiments and shown as mean ± SD. ns: not significant; ** P < 0.01. Two-way ANOVA and Tukey’s test. G-H RT-qPCR and Western blot of Mxd1 in siNC- or siFTO-silenced BV-2 cells transfected with with negative control siRNA (siNC) or YTHDF2 siRNA (siYTHDF2), respectively. Data are representative of three independent experiments and shown as mean ± SD. ns: not significant; * P < 0.05; ** P < 0.01. Two-way ANOVA and Tukey’s test. I RT-qPCR analysis of Mxd1 mRNA stability in cells co-transfected as above. Data are representative of three independent experiments and shown as mean ± SD. ** P < 0.01. One-way ANOVA and Tukey’s test. J In BV-2 cells with or without YTHDF2 overexpression or FTO overexpression, luciferase activity of wild-type or mutated Mxd1 reporters were determined. n = 3, Data are shown as mean ± SD. ** P < 0.01. Two-way ANOVA and Tukey’s test.

Article Snippet: BV-2 cells were pretreated with various doses of active VitD (calcitriol, MedChemExpress, USA), and 1 h later LPS (100 ng/mL, Sigma-Aldrich Co., USA) was added to the medium for 24 h. To investigate the role of PI3K/AKT and PGC-1α in the regulation of BV-2 microglial polarization, BV-2 cells were pretreated with LY294002 (10μM, MedChemExpress) or SR-18,292 (10μM, MedChemExpress) for 1 h, both followed by stimulation with calcitriol for 1 h, and then stimulated with LPS (100 ng/mL) for 24 h.

Techniques: Modification, Mutagenesis, Transfection, Negative Control, Quantitative RT-PCR, Western Blot, Over Expression, Luciferase, Activity Assay

Journal: Inflammation

Article Title: FTO-dependent m 6 A Demethylation Activates Mxd1 To Enhance Vitamin D-induced Suppression of Neuroinflammation Via PTEN/AKT/PGC-1α Signaling Pathways in Microglia

doi: 10.1007/s10753-026-02450-5

Figure Lengend Snippet: Mxd1 mediates VitD-induced activation of PTEN/AKT/PGC-1α signaling pathway. A RT-qPCR analysis of PTEN mRNA in BV-2 cells treated with LPS or LPS + VitD (VitD) and transfected with negative control siRNA (siNC) or Mxd1 siRNA (siMxd1). Data are representative of three independent experiments and shown as mean ± SD. * P < 0.05; ** P < 0.01. Two-way ANOVA and Tukey’s test. B Western blot analysis of PGC-1α, PTEN, AKT and p-AKT and Mxd1 under the same conditions. Data are representative of three independent experiments and shown as mean ± SD. ns: not significant; * P < 0.05; ** P < 0.01. Two-way ANOVA and Tukey’s test. C The predicted binding sites of Mxd1 in the promoter of PTEN. The primer sets for ChIP-qPCR are indicated by the red arrows in the schematic diagram. D ChIP-qPCR assessing Mxd1 binding to the PTEN promoter in LPS or LPS + VitD (VitD)-treated cells. Data are representative of three independent experiments and shown as mean ± SD. ** P < 0.01. Two-way ANOVA and Tukey’s test. E Dual-luciferase reporter assay was performed to analyze the effect of Mxd1 overexpression or knockdown on the luciferase activity of PTEN-wt reporter. n = 3. ** p < 0.01, unpaired Student’s two-tailed t-test. F Western blot analysis of PGC-1α, AKT and p-AKT in LPS- or LPS + VitD-treated BV-2 cells with vehicle or LY294002. Data are representative of three independent experiments and shown as mean ± SD. ns: not significant; * P < 0.05; ** P < 0.01. Two-way ANOVA and Tukey’s test. G Western blot of PGC-1α, AKT and p-AKT in cells overexpressing PTEN plasmid (PTEN) or empty plasmid (vector) with LPS or LPS + VitD (VitD) treatment. Data are shown as mean ± SD ( n = 3). ** P < 0.01. Data are representative of three independent experiments and shown as mean ± SD. ** P < 0.01. Two-way ANOVA and Tukey’s test.

Article Snippet: BV-2 cells were pretreated with various doses of active VitD (calcitriol, MedChemExpress, USA), and 1 h later LPS (100 ng/mL, Sigma-Aldrich Co., USA) was added to the medium for 24 h. To investigate the role of PI3K/AKT and PGC-1α in the regulation of BV-2 microglial polarization, BV-2 cells were pretreated with LY294002 (10μM, MedChemExpress) or SR-18,292 (10μM, MedChemExpress) for 1 h, both followed by stimulation with calcitriol for 1 h, and then stimulated with LPS (100 ng/mL) for 24 h.

Techniques: Activation Assay, Quantitative RT-PCR, Transfection, Negative Control, Western Blot, Binding Assay, ChIP-qPCR, Luciferase, Reporter Assay, Over Expression, Knockdown, Activity Assay, Two Tailed Test, Plasmid Preparation

Journal: Inflammation

Article Title: FTO-dependent m 6 A Demethylation Activates Mxd1 To Enhance Vitamin D-induced Suppression of Neuroinflammation Via PTEN/AKT/PGC-1α Signaling Pathways in Microglia

doi: 10.1007/s10753-026-02450-5

Figure Lengend Snippet: PGC-1α is required for VitD-induced M2 microglial polarization. A Immunofluorescence analysis of PGC-1α nuclear localization in LPS- or LPS + VitD-treated BV-2 cells. Scale bar: 100 μm. Right panel: Quantification of PGC-1α IF intensity. Six regions were randomly selected for each group, and the IF intensity of PGC-1α was quantified using the ImageJ software. Data are shown as mean ± SD. ** P < 0.01. One-way ANOVA and Tukey’s test. B Western blot analysis of CD16, CD18, CD206 and ARG1 in LPS- or LPS + VitD(VitD)-treated BV-2 cells with vehicle or SR-18,292. Data are representative of three independent experiments and shown as mean ± SD. ns: not significant; ** P < 0.01. Two-way ANOVA and Tukey’s test. C ELISA quantification of TNF-α, IL1β, IL-6, IL-4 and IL-10 in culture supernatants from the same experimental groups. Data are representative of three independent experiments and shown as mean ± SD. ns: not significant; ** P < 0.01. Two-way ANOVA and Tukey’s test. D Immunohistochemical staining and quantification of Iba-1-positive cells in cerebral cortex and hippocampal CA1 area of LPS- or VitD-treated mice with vehicle or SR-18,292, and the quantification of Iba-1 positive cells in each group was shown in the low panel. Scale bar: 20 μm. n = 6 mice per group. Data are shown as mean ± SD. ** P < 0.01. One-way ANOVA and Tukey’s test.

Article Snippet: BV-2 cells were pretreated with various doses of active VitD (calcitriol, MedChemExpress, USA), and 1 h later LPS (100 ng/mL, Sigma-Aldrich Co., USA) was added to the medium for 24 h. To investigate the role of PI3K/AKT and PGC-1α in the regulation of BV-2 microglial polarization, BV-2 cells were pretreated with LY294002 (10μM, MedChemExpress) or SR-18,292 (10μM, MedChemExpress) for 1 h, both followed by stimulation with calcitriol for 1 h, and then stimulated with LPS (100 ng/mL) for 24 h.

Techniques: Immunofluorescence, Software, Western Blot, Enzyme-linked Immunosorbent Assay, Immunohistochemical staining, Staining