Journal: Clinical and Translational Medicine

Article Title: Single‐cell transcriptome sequencing of B‐cell heterogeneity and tertiary lymphoid structure predicts breast cancer prognosis and neoadjuvant therapy efficacy

doi: 10.1002/ctm2.1346

Figure Lengend Snippet: Correlation analysis between TIL‐B and mature tertiary lymphoid structures (TLS). (A) Among the 200 patients included in the BRCA dataset from the TCGA research consortium, a subset of 18 patients who exhibited TLS positivity were identified. H&E images of characteristic TLS in these patients. (B) Co‐immunofluorescence subimages depicting the co‐localization of CD23 and CD21 within CD23‐positive TLS were obtained from a collection of 70 BC samples that we compiled. (C) In 9 TLS‐positive patients of 70 BC patients, representative images of H&E staining and IHC staining of CD4, CD8, CD20, CD23 and BCL6 expression in TLS‐positive BC patients. (D) Analysis of DEGs in 200 TCGA BRCA cases categorized as TLS‐negative and TLS‐positive. Source data are available at: https://portal.gdc.cancer.gov . (E and F) H&E (E) and CD23 IHC (F) images of the typical TLS‐positive case in 70 BC patients. (G) Different tumour types exhibit FCER2 expression, according to TIMER2. * p < .05;** p < .01;*** p < .001. (H) Utilizing the EPIC and TIDE algorithms within TIMER2, the relationship between FCER2 expression and tumour infiltrating immune cells in BC was computed. (I‐J) Kaplan–Meier Survival curve of FCER2(I), co‐acting with FCER2 and B cells(J) in BC by TIMER2. TIL‐B, tumor‐infiltrating B cells.

Article Snippet: 1% bovine serum albumin for 10 min at room temperature, followed by overnight incubation at 4°C with the following primary antibodies: monoclonal antibody against FCER2 (CD23; 1:400; 60208‐2‐Ig; Proteintech), anti‐CD21 (1:400; 24374‐1‐AP; Proteintech), anti‐CD20 (1:400; 60271‐1‐Ig; Proteintech), anti‐CD4 (1:400; 67786‐1‐Ig; Proteintech), anti‐CD8 (1:400; 66868‐1‐Ig; Proteintech) and anti‐BCL6 (1:400; 66340‐1‐Ig; Proteintech).

Techniques: Immunofluorescence, Staining, Immunohistochemistry, Expressing

Journal: Clinical and Translational Medicine

Article Title: Single‐cell transcriptome sequencing of B‐cell heterogeneity and tertiary lymphoid structure predicts breast cancer prognosis and neoadjuvant therapy efficacy

doi: 10.1002/ctm2.1346

Figure Lengend Snippet: Different tertiary lymphoid structures (TLS) states in breast cancer (BC) display metabolic variability and survival variations. (A) A bar graph depicting the distribution of immune cells that have infiltrated tumours in TLS‐positive and TLS‐negative groups among 9 BC patients. (B) Genetic analysis of immune cell differences in TLS‐positive and TLS‐negative groups among 9 BC patients. (C) Heatmap of metabolic pathway scores in TLS‐positive and TLS‐negative groups among 9 BC patients. (D) Heatmap showing average metabolic gene expression in TLS‐positive and TLS‐negative groups among 9 BC patients. (E) To ascertain the expression correlation between CD23 and the biomarkers citrate synthase (CS), HK2, lactate dehydrogenase A (LDHA), and IDH3G in BC, the cBioportal tool ( https://www.cbioportal.org ) was utilized, employing the TCGA dataset (Firehose Legacy dataset; encompassing 1108 BC samples) for this purpose. (F) Kaplan–Meier survival curves depicting the disparity in BC outcomes between cohorts with high and low APOD expression, as analyzed through GEPIA. (G) Imaging before and after neoadjuvant chemotherapy of TLS‐positive patients with significant effectiveness. (H) Imaging before and after neoadjuvant immunotherapy of TLS‐positive patients with significant effectiveness.

Article Snippet: 1% bovine serum albumin for 10 min at room temperature, followed by overnight incubation at 4°C with the following primary antibodies: monoclonal antibody against FCER2 (CD23; 1:400; 60208‐2‐Ig; Proteintech), anti‐CD21 (1:400; 24374‐1‐AP; Proteintech), anti‐CD20 (1:400; 60271‐1‐Ig; Proteintech), anti‐CD4 (1:400; 67786‐1‐Ig; Proteintech), anti‐CD8 (1:400; 66868‐1‐Ig; Proteintech) and anti‐BCL6 (1:400; 66340‐1‐Ig; Proteintech).

Techniques: Gene Expression, Expressing, Imaging

Journal: Clinical and Translational Medicine

Article Title: Single‐cell transcriptome sequencing of B‐cell heterogeneity and tertiary lymphoid structure predicts breast cancer prognosis and neoadjuvant therapy efficacy

doi: 10.1002/ctm2.1346

Figure Lengend Snippet: The potential significance of tertiary lymphoid structures (TLS) and TLS‐specific markers for breast cancer (BC) prognosis. After matching with Fitness score matching (PSM), survival analysis and prognostic factor analysis were performed. For survival analysis, the Kaplan–Meier method and the log‐rank test were employed. For prognostic factors, COX proportional hazard regression model was used. The two‐sided log rank test was used to determine the p values. (A) Kaplan–Meier survival curves for the disease‐free survival (DFS) (left) and OS (right) of 920 TCGA BRCA patients based on single gene expression (CD20, CD23 and CD8). (B) The impact of CD20, CD23, CD8, CD4 and BCL6 on 920 TCGA BRCA patients’ prognosis. The forest map shows HRs (center pink and blue squares) and 95% confidence interval (horizontal ranges), and PSM matching has been made for factors such as molecular typing, lymph node status, tumour size, diagnosis age and histological grading of BC. (C) Kaplan–Meier survival curves for the DFS of 70 BC patients based on TLS expression.

Article Snippet: 1% bovine serum albumin for 10 min at room temperature, followed by overnight incubation at 4°C with the following primary antibodies: monoclonal antibody against FCER2 (CD23; 1:400; 60208‐2‐Ig; Proteintech), anti‐CD21 (1:400; 24374‐1‐AP; Proteintech), anti‐CD20 (1:400; 60271‐1‐Ig; Proteintech), anti‐CD4 (1:400; 67786‐1‐Ig; Proteintech), anti‐CD8 (1:400; 66868‐1‐Ig; Proteintech) and anti‐BCL6 (1:400; 66340‐1‐Ig; Proteintech).

Techniques: Gene Expression, Biomarker Discovery, Expressing

Journal: Evidence-based Complementary and Alternative Medicine : eCAM

Article Title: Tylosema esculentum (Marama) Tuber and Bean Extracts Are Strong Antiviral Agents against Rotavirus Infection

doi: 10.1155/2011/284795

Figure Lengend Snippet: Dose-dependent cytopathic effect of rotavirus on bovine intestinal epithelial cells (CIEB), pig intestinal epithelial cells (CLAB), and human intestinal epithelial cells (H4). The graphs for cytopathic effect of rotavirus on the different cells are shown in terms of mean absorbance at each concentration ± standard deviation at 595 nm.

Article Snippet: NO 2 release was significant from human intestinal epithelial cells (H4) exposed to all extracts used {seed coat water and ethanolic extracts (MSCW and MSCE, resp.), cotyledon water and ethanolic extracts (MCW and MCE, resp.), and tuber water extract (MTW)}, while only MCE and MTW significantly increased NO in CLAB cells ( ).

Techniques: Concentration Assay, Standard Deviation

Journal: Evidence-based Complementary and Alternative Medicine : eCAM

Article Title: Tylosema esculentum (Marama) Tuber and Bean Extracts Are Strong Antiviral Agents against Rotavirus Infection

doi: 10.1155/2011/284795

Figure Lengend Snippet: Survival of bovine intestinal epithelial cells (CIEB), pig intestinal epithelial cells (CLAB), and human intestinal epithelial cells (H4) from cytopathic effects of rotavirus in the presence of T. esculentum extracts. Shown above are bars of % survival of epithelial cells {% change in cell viability after coincubation of rotavirus with marama extracts compared to rotavirus only wells at OD 595 nm ± % standard deviation}, respective levels of significance in terms of T values (*). T values were determined using Statsoft Statistica software ( P < .05). Tests were done in triplicate wells. * T value between 0 and 5 (low level significance), ** T value greater than 5, but less than 10 (middle level significance), and *** T value greater than 10 (high level significance). MCE: T. esculentum bean cotyledon ethanolic extract, MSCE: T. esculentum bean husk ethanolic extract, MTW: T. esculentum tuber water extract, MSCW: T. esculentum bean husk water extract, MCW: T. esculentum bean cotyledon water extract, and Combivir: (lamivudine and zidovudine).

Article Snippet: NO 2 release was significant from human intestinal epithelial cells (H4) exposed to all extracts used {seed coat water and ethanolic extracts (MSCW and MSCE, resp.), cotyledon water and ethanolic extracts (MCW and MCE, resp.), and tuber water extract (MTW)}, while only MCE and MTW significantly increased NO in CLAB cells ( ).

Techniques: Standard Deviation, Software

Journal: Evidence-based Complementary and Alternative Medicine : eCAM

Article Title: Tylosema esculentum (Marama) Tuber and Bean Extracts Are Strong Antiviral Agents against Rotavirus Infection

doi: 10.1155/2011/284795

Figure Lengend Snippet: T. esculentum extracts show direct inhibition of rotavirus infectivity on bovine intestinal epithelial cells (CIEB), pig intestinal epithelial cells (CLAB), and human intestinal epithelial cells (H4). Shown above are bars of % survival of epithelial cells {% change in cell viability after pre-exposure of rotavirus to T. esculentum extracts compared to rotavirus only wells at OD 595 nm ± % standard deviation}, respective levels of significance in terms of T values (*). T values were determined using Statsoft Statistica software ( P < .05). Tests were done in triplicate wells. * T value between 0 and 5 (low level significance), ** T value greater than 5, but less than 10 (middle level significance), and *** T value greater than 10 (high level significance). MCE: T. esculentum bean cotyledon ethanolic extract, MSCE: T. esculentum bean husk ethanolic extract, MTW: T. esculentum tuber water extract, MSCW: T. esculentum bean husk water extract, MCW: T. esculentum bean cotyledon water extract, and AZT: Combivir (lamivudine and zidovudine).

Article Snippet: NO 2 release was significant from human intestinal epithelial cells (H4) exposed to all extracts used {seed coat water and ethanolic extracts (MSCW and MSCE, resp.), cotyledon water and ethanolic extracts (MCW and MCE, resp.), and tuber water extract (MTW)}, while only MCE and MTW significantly increased NO in CLAB cells ( ).

Techniques: Inhibition, Infection, Standard Deviation, Software

Journal: Evidence-based Complementary and Alternative Medicine : eCAM

Article Title: Tylosema esculentum (Marama) Tuber and Bean Extracts Are Strong Antiviral Agents against Rotavirus Infection

doi: 10.1155/2011/284795

Figure Lengend Snippet: Effect of coincubation of rotavirus with T. esculentum extracts on monolayer polarity (TER) (Ω/cm 2 ) of H4 cells over time. Shown graphs of change in epithelial cell monolayer polarity exposed to extracts or AZT and rotavirus (treatment wells), extracts or AZT alone (control), or rotavirus alone (negative control) over time compared to respective controls. Tests were done in triplicate wells (12-well Transwell plates). MCE: T. esculentum bean cotyledon ethanolic extract (1 mg/mL), MSCE: T. esculentum bean husk ethanolic extract (0.1 mg/mL), MTW: T. esculentum tuber water extract (0.1 mg/mL), MSCW: T. esculentum bean husk water extract (0.01 mg/mL), MCW: T. esculentum bean cotyledon water extract (2 mg/mL), and AZT: Combivir (lamivudine and zidovudine) (0.75 mg/mL).

Article Snippet: NO 2 release was significant from human intestinal epithelial cells (H4) exposed to all extracts used {seed coat water and ethanolic extracts (MSCW and MSCE, resp.), cotyledon water and ethanolic extracts (MCW and MCE, resp.), and tuber water extract (MTW)}, while only MCE and MTW significantly increased NO in CLAB cells ( ).

Techniques: Control, Negative Control

Journal: Evidence-based Complementary and Alternative Medicine : eCAM

Article Title: Tylosema esculentum (Marama) Tuber and Bean Extracts Are Strong Antiviral Agents against Rotavirus Infection

doi: 10.1155/2011/284795

Figure Lengend Snippet: Effect of T. esculentum extracts on release of nitric oxide (NO) (% release compared to control) in human small intestine epithelial cells (H4) and pig small intestine epithelial cells (CLAB). MCE: T. esculentum bean cotyledon ethanolic extract (1 mg/mL), MSCE: T. esculentum bean husk ethanolic extract (0.1 mg/mL), MTW: T. esculentum tuber water extract (0.1 mg/mL), MSCW: T. esculentum bean husk water extract (0.01 mg/mL), and MCW: T. esculentum bean cotyledon water extract (2 mg/mL).

Article Snippet: NO 2 release was significant from human intestinal epithelial cells (H4) exposed to all extracts used {seed coat water and ethanolic extracts (MSCW and MSCE, resp.), cotyledon water and ethanolic extracts (MCW and MCE, resp.), and tuber water extract (MTW)}, while only MCE and MTW significantly increased NO in CLAB cells ( ).

Techniques: Control

Journal: Oncotarget

Article Title: Efficacy of a Mer and Flt3 tyrosine kinase small molecule inhibitor, UNC1666, in acute myeloid leukemia

doi:

Figure Lengend Snippet: (A) Chemical structure of UNC1666, with inhibition constant (K i ) of 0.16 nM for Mer (enzymatic IC 50 : 0.55 nM) and 0.67 nM for Flt3 (enzymatic IC 50 : 0.69 nM). (B) Chemical structure of UNC1653, which lacks significant activity against Mer (enzymatic IC 50 : 560 nM) and Flt3 (enzymatic IC 50 : 220 nM) and is used as a negative control in these studies. (C) Whole cell lysates from AML cell lines with known Flt3 mutation status were analyzed by immunoblot and demonstrate presence or absence of the Mer tyrosine kinase (above) and the Flt3 tyrosine kinase (middle). Actin is shown as an indicator of total protein (below).

Article Snippet: Additionally, UNC1666 inhibits Flt3 (MCE IC 50 0.69 nM; K i 0.67 nM) equipotently in enzymatic MCE assays.

Techniques: Inhibition, Activity Assay, Negative Control, Mutagenesis, Western Blot

Journal: Oncotarget

Article Title: Efficacy of a Mer and Flt3 tyrosine kinase small molecule inhibitor, UNC1666, in acute myeloid leukemia

doi:

Figure Lengend Snippet: (A) Mer was immunoprecipitated from AML cell lysates and phosphorylated ( p -Mer) and total Mer (~180 kDa) levels were assessed by immunoblot analysis. This representative blot of the Kasumi-1 cell line demonstrates decreased Mer phosphorylation after treatment with increasing doses of UNC1666. (B) Flt3 was immunoprecipitated from AML cell lysates and phosphorylated ( p -Flt3) and total Flt3 (130/160 kDa) levels were assessed by immunoblot analysis. This representative blot of the MV4;11 cell line demonstrates decreased Flt3 phosphorylation after treatment with increasing doses of UNC1666. (C) Inhibition of downstream signaling after administration of UNC1666 in a Mer expressing AML cell lines that does not express a Flt3-ITD mutation (Kasumi-1) compared with equivalent concentrations of vehicle (DMSO) or inactive control TKI UNC1653. Actin is shown as an indicator of total protein. (D) Downstream signaling after administration of UNC1666 in a Flt3-ITD AML cell line that does not express Mer (MV4;11). Representative blots from at least 3 independent experiments are shown. nM = nanomolar

Article Snippet: Additionally, UNC1666 inhibits Flt3 (MCE IC 50 0.69 nM; K i 0.67 nM) equipotently in enzymatic MCE assays.

Techniques: Immunoprecipitation, Western Blot, Phospho-proteomics, Inhibition, Expressing, Mutagenesis, Control

Journal: Oncotarget

Article Title: Efficacy of a Mer and Flt3 tyrosine kinase small molecule inhibitor, UNC1666, in acute myeloid leukemia

doi:

Figure Lengend Snippet: Mer pos or Flt3-ITD AML cell lines were treated with UNC1666, vehicle (DMSO), or inactive control TKI UNC1653 for 72 hours and then analyzed by flow cytometry after staining with YO-PRO-1 iodide and propidium iodide to identify apoptotic and dead cells. (A) Representative flow cytometry profiles of Kasumi-1 cells are shown. The percentages of live (lower left quadrant), early apoptotic (lower right quadrant), and late apoptotic/dead cells (upper quadrants) are shown. (B) Graphic representation of flow cytometric analyses of apoptotic/dead cells. Mean values and standard errors were derived from at least 3 independent experiments. * p < 0.05, *** p < 0.001, NS = not significant. (C) Cells were treated as indicated for 72 hours, whole cell lysates were prepared and the indicated apoptotic proteins were assessed by immunoblot analysis. Actin is shown as a loading control.

Article Snippet: Additionally, UNC1666 inhibits Flt3 (MCE IC 50 0.69 nM; K i 0.67 nM) equipotently in enzymatic MCE assays.

Techniques: Control, Flow Cytometry, Staining, Derivative Assay, Western Blot

Journal: Oncotarget

Article Title: Efficacy of a Mer and Flt3 tyrosine kinase small molecule inhibitor, UNC1666, in acute myeloid leukemia

doi:

Figure Lengend Snippet: Mer positive or Flt3-ITD AML cell lines were treated with UNC1666, vehicle (DMSO), or inactive control TKI UNC1653 for 72 hours, fixed with 100% ethanol and then analyzed by flow cytometry after staining with propidium iodide to identify stage of cell cycle. Graphic representation of cell cycle progress in Kasumi-1 and MV4;11 cells using ModFit analysis are shown. The percentages of cells in G2/M (light gray), S (white), and G1 (dark gray) phases are shown. Mean values and standard errors were derived from at least 3 independent experiments. * p < 0.05, ** p < 0.01.

Article Snippet: Additionally, UNC1666 inhibits Flt3 (MCE IC 50 0.69 nM; K i 0.67 nM) equipotently in enzymatic MCE assays.

Techniques: Control, Flow Cytometry, Staining, Derivative Assay

Journal: Oncotarget

Article Title: Efficacy of a Mer and Flt3 tyrosine kinase small molecule inhibitor, UNC1666, in acute myeloid leukemia

doi:

Figure Lengend Snippet: (A) Diagram of the replating assay. Cells were treated with UNC1666 or vehicle for 72 hours, then washed to remove any residual compound and equal numbers of viable cells (1.5 × 10 4 /ml) were replated in growth medium on Day 0. On Day 6 after replating, the number of viable cells was determined. (B) Graphic representation of the results of the replating assay in Mer or Flt3-ITD expressing cell lines demonstrating decreased rebound growth after treatment with UNC1666. Mean values and standard errors were derived from at least 3 independent experiments. (C) Colony-formation assays were performed using Mer or Flt3-ITD expressing AML cell lines. Cells were grown in soft agar with the indicated treatments. Graphic representation of reduced colony number after treatment with UNC1666, compared to vehicle or negative control TKI UNC1653. Mean values and standard errors were derived from at least 3 independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001, NS = not significant.

Article Snippet: Additionally, UNC1666 inhibits Flt3 (MCE IC 50 0.69 nM; K i 0.67 nM) equipotently in enzymatic MCE assays.

Techniques: Expressing, Derivative Assay, Negative Control

Journal: Oncotarget

Article Title: Efficacy of a Mer and Flt3 tyrosine kinase small molecule inhibitor, UNC1666, in acute myeloid leukemia

doi:

Figure Lengend Snippet: (A) Immunoblot analysis of Mer and Flt3 expression in lysates prepared from AML patient samples. (B) Flt3 mutation status of patient samples determined by molecular profiling. (C, D) Dose-dependent inhibition of Mer and Flt3 phosphorylation in response to treatment with UNC1666. AML blasts from patient sample #10510 (Mer positive, Flt3-ITD high allelic ratio) were treated with UNC1666 or vehicle for two hours. (C) Mer and Flt3 were immunoprecipitated from cell lysates and phosphorylated Mer ( p -Mer), total Mer (~180 kDa), phosphorylated Flt3 ( p -Flt3) and total Flt3 (130/160 kDa) were detected by immunoblot. (D) Phosphorylation of downstream signaling molecules was assessed by immunoblot after treatment with UNC1666 or vehicle.

Article Snippet: Additionally, UNC1666 inhibits Flt3 (MCE IC 50 0.69 nM; K i 0.67 nM) equipotently in enzymatic MCE assays.

Techniques: Western Blot, Expressing, Mutagenesis, Inhibition, Phospho-proteomics, Immunoprecipitation

Journal: Oncotarget

Article Title: Efficacy of a Mer and Flt3 tyrosine kinase small molecule inhibitor, UNC1666, in acute myeloid leukemia

doi:

Figure Lengend Snippet: (A) Graphic representation of apoptosis and cell death in AML patient samples after treatment with UNC1666 or vehicle for 72 hours. Apoptotic and dead cells were determined by flow cytometry after staining with YO-PRO-1 iodide and propidium iodide. Values derived from each sample are shown. (B) Colony-forming assays were performed in methylcellulose with the indicated treatments. Graphic representation of reduced colony number after treatment with UNC1666, compared to vehicle. Mean values and standard errors derived from triplicate samples are shown. (C) Graphic representation of the effect of UNC1666 on normal cord blood colony forming potential. Mean values and standard errors were derived from 3 independent experiments. ** p < 0.01, NS = not significant.

Article Snippet: Additionally, UNC1666 inhibits Flt3 (MCE IC 50 0.69 nM; K i 0.67 nM) equipotently in enzymatic MCE assays.

Techniques: Flow Cytometry, Staining, Derivative 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: 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: For immunofluorescence analysis, brain sections were cut at 7 μm, washed with 0.1 M PBS, permeabilized with 0.3% (v/v) Triton X-100-PBS, blocked with 10% (v/v) donkey serum for 1 h, and then incubated with primary antibodies against CD16 (1:200, ProteinTech, Wuhan, China), CD206 (1:200, RecordBio) at 4 °C overnight.

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: For immunofluorescence analysis, brain sections were cut at 7 μm, washed with 0.1 M PBS, permeabilized with 0.3% (v/v) Triton X-100-PBS, blocked with 10% (v/v) donkey serum for 1 h, and then incubated with primary antibodies against CD16 (1:200, ProteinTech, Wuhan, China), CD206 (1:200, RecordBio) at 4 °C overnight.

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: For immunofluorescence analysis, brain sections were cut at 7 μm, washed with 0.1 M PBS, permeabilized with 0.3% (v/v) Triton X-100-PBS, blocked with 10% (v/v) donkey serum for 1 h, and then incubated with primary antibodies against CD16 (1:200, ProteinTech, Wuhan, China), CD206 (1:200, RecordBio) at 4 °C overnight.

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: 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: For immunofluorescence analysis, brain sections were cut at 7 μm, washed with 0.1 M PBS, permeabilized with 0.3% (v/v) Triton X-100-PBS, blocked with 10% (v/v) donkey serum for 1 h, and then incubated with primary antibodies against CD16 (1:200, ProteinTech, Wuhan, China), CD206 (1:200, RecordBio) at 4 °C overnight.

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