Journal: The Journal of Neuroscience

Article Title: Regulatory B Cells Normalize CNS Myeloid Cell Content in a Mouse Model of Multiple Sclerosis and Promote Oligodendrogenesis and Remyelination

doi: 10.1523/JNEUROSCI.2840-19.2020

Figure Lengend Snippet: Oligodendroglial lineage after adoptive transfer with B cells or Bregs in EAE mice. Mice were killed starting 2 weeks after adoptive cell transfer, and spinal cords were collected for analysis by flow cytometry and Western blotting. Naive mice and untreated EAE mice were used as control. A, Gating strategy: single cells in side and forward scatter, cells, live cells followed by CD45–/low gate. B, Expression of oligodendroglial markers 2 (and 3.5 weeks for Bregs** treated animals) after adoptive transfer with Bregs or B cells compared with WT and EAE-untreated mice: A2B5, CD140, GalC, and O1 in WT mice and EAE C57BL/6 treated either with B cells or Bregs. C, Statistical analysis of the oligodendroglial marker expression in the spinal cords and brains 2 weeks after adoptive transfer. D, Oligodendroglial phenotyping from spinal cords in WT, Bregs, and B-cell-treated mice: A2B5+CD140+, early OPCs, and O1+GALC+ mature oligodendrocytes. E, Western blotting analysis on the expression of MOG, MBP, and paired-related homeobox protein 1 (PRXX1) by spinal cord oligodendrocytes from EAE C57BL/6 mice untreated, treated with Bregs or B cells, and WT mice 2 weeks after adoptive transfer, load control GADPH. Data are representative of three independent experiments in vivo with n ≥ 6. Data are mean ± SEM. *p < 0.05; **p ≤ 0.01; ***p ≤ 0.001; compared with the corresponding control (Student's t test).

Article Snippet: For oligodendrocyte analysis, the following markers were used as per the manufacturer's recommendation: R&D Systems anti-A2B5 AlexaFluor-647 (clone #105), R&D Systems anti-oligodendrocyte marker O1 AlexaFluor-405-conjugated antibody, Sigma Millipore anti-galactocerebroside FITC antibody (clone mGalC), and Invitrogen anti-CD140 (PDGFRA) FITC (clone APA5).

Techniques: Adoptive Transfer Assay, Flow Cytometry, Western Blot, Expressing, Marker, In Vivo

Journal: The Journal of Neuroscience

Article Title: Regulatory B Cells Normalize CNS Myeloid Cell Content in a Mouse Model of Multiple Sclerosis and Promote Oligodendrogenesis and Remyelination

doi: 10.1523/JNEUROSCI.2840-19.2020

Figure Lengend Snippet: Model of repair process induced by Bregs and effect in lymphoid organs and CNS. EAE manifested with an increase in autoreactive T cells and inflammation in lymphoid organs and the CNS causing demyelination and axon loss occur. In lymphoid organs, autoreactive T cells (Th1 and Th17) are induced after MOG immunization, and they then migrate in the CNS. The inflammatory microenvironment of the CNS induced demyelination and loss of oligodendrocytes. Myeloid-derived cells, both resident microglia and blood-derived monocytes/macrophages, showed a proinflammatory phenotype in B-cell-treated mice compared with WT or Bregs-treated animals (left). Adoptive transfer of Bregs can decrease the frequency of autoreactive T cell in the periphery and the CNS with concomitant increased of IL-10-producing T cells, both Tr-1 and canonical Tregs. Moreover, in mice that received Bregs, myeloid-derived cells, both monocytes/macrophages and microglia, displayed a more immunosuppressive phenotype, and their frequency is lowered respect to EAE mice (right). Concomitantly, a maturation/differentiation of OPCs was evident in the spinal cords of mice that received Bregs concurrent with remyelination.

Article Snippet: For oligodendrocyte analysis, the following markers were used as per the manufacturer's recommendation: R&D Systems anti-A2B5 AlexaFluor-647 (clone #105), R&D Systems anti-oligodendrocyte marker O1 AlexaFluor-405-conjugated antibody, Sigma Millipore anti-galactocerebroside FITC antibody (clone mGalC), and Invitrogen anti-CD140 (PDGFRA) FITC (clone APA5).

Techniques: Derivative Assay, Adoptive Transfer Assay

Journal: Aging Cell

Article Title: Lipoteichoic acid restrains macrophage senescence via β‐catenin/ FOXO1 / REDD1 pathway in age‐related osteoporosis

doi: 10.1111/acel.14072

Figure Lengend Snippet: LTA prevents senescence‐induced FOXO1 downregulation and deactivation. Representative images (a) of double‐immunofluorescence staining of F4/80 (red) and FOXO1 (green) and quantification (b) of the percentage of FOXO1 expression area on F4/80 + cells in femoral bone. n = 6/group. Scale bars, 50 μm. BMDMs were treated with H 2 O 2 together with LTA. Representative images (c) and quantification of Western blot of the relative intensity of total FOXO1 (d) and phosphorylated FOXO1 (f). n = 3/group. Quantitative real‐time PCR analysis of FOXO1 (e) for three groups of BMDMs. n = 3/group. Representative images (g) and quantification (h) of immunofluorescence staining of nuclear FOXO1 in three groups of BMDMs. n = 3/group. Scale bars, 50 μm. Representative images (i) and quantification of Western blot of the relative intensity of cytoplasmic FOXO1 (j) and nuclear FOXO1 (k) in three groups of BMDMs. n = 3/group. * p < 0.05, ** p < 0.01, *** p < 0.001. Data are presented as mean ± SD. One‐way ANOVA with Tukey's test.

Article Snippet: For immunofluorescence staining, we incubated the cells with primary antibodies to FOXO1 (18592‐1‐AP; Proteintech, China) overnight at 4 °C, followed by incubation with Fluorescein (FITC)–conjugated Affinipure Goat Anti‐Rabbit IgG (H+L; SA00003‐2; Proteintech ) secondary antibodies.

Techniques: Double Immunofluorescence Staining, Expressing, Western Blot, Real-time Polymerase Chain Reaction, Immunofluorescence, Staining

Journal: Aging Cell

Article Title: Lipoteichoic acid restrains macrophage senescence via β‐catenin/ FOXO1 / REDD1 pathway in age‐related osteoporosis

doi: 10.1111/acel.14072

Figure Lengend Snippet: LTA inhibits mTOR phosphorylation through FOXO1/REDD1 signaling pathway. Representative images (a) of double‐immunofluorescence staining of F4/80 (red) and p‐mTOR (green) and quantification (b) of the percentage of p‐mTOR expression area on F4/80 + cells in femoral bone of vehicle‐treated 2‐month‐old mice, vehicle‐treated 12‐month‐old mice and LTA‐treated 12‐month‐old mice in the presence or absence of AS. n = 6/group. Scale bars, 50 μm. Representative images (c) of double‐immunofluorescence staining of F4/80 (red) and REDD1 (green) and quantification (d) of the percentage of REDD1 expression area on F4/80 + cells in the femoral bone of four groups of mice. n = 6/group. Scale bars, 50 μm. BMDMs were treated with H 2 O 2 together with LTA and AS. Representative images (e) and quantification of Western blot of the relative intensity of p‐mTOR (f) and REDD1 (g) in four groups of BMDMs. n = 3/group. AS: AS1842856. * p < 0.05, ** p < 0.01, *** p < 0.001. Data are presented as mean ± SD. One‐way ANOVA with Tukey's test.

Article Snippet: For immunofluorescence staining, we incubated the cells with primary antibodies to FOXO1 (18592‐1‐AP; Proteintech, China) overnight at 4 °C, followed by incubation with Fluorescein (FITC)–conjugated Affinipure Goat Anti‐Rabbit IgG (H+L; SA00003‐2; Proteintech ) secondary antibodies.

Techniques: Phospho-proteomics, Double Immunofluorescence Staining, Expressing, Western Blot

Journal: Aging Cell

Article Title: Lipoteichoic acid restrains macrophage senescence via β‐catenin/ FOXO1 / REDD1 pathway in age‐related osteoporosis

doi: 10.1111/acel.14072

Figure Lengend Snippet: LTA stimulates FOXO1 expression and promotes FOXO1 nuclear translocation through β‐catenin. Representative images (a) of double‐immunofluorescence staining of F4/80 (red) and FOXO1 (green) and quantification (b) of the percentage of FOXO1 expression area on F4/80 + cells in the femoral bone of three groups of 12‐month‐old mice. n = 6/group. Scale bars, 50 μm. BMDMs were treated with H 2 O 2 together with LTA and IWR‐1. Representative images (c) and quantification of Western blot of the relative intensity of total FOXO1 (d) and phosphorylated FOXO1 (e) in four groups of BMDMs. n = 3/group. Representative images (f) and quantification (g) of immunofluorescence staining of nuclear FOXO1 in four groups of BMDMs. n = 3/group. Scale bars, 50 μm. Representative images (h) and quantification of Western blot of the relative intensity of cytoplasmic FOXO1 (i) and nuclear FOXO1 (j) in four groups of BMDMs. n = 3/group. * p < 0.05, ** p < 0.01, *** p < 0.001. Data are presented as mean ± SD. One‐way ANOVA with Tukey's test.

Article Snippet: For immunofluorescence staining, we incubated the cells with primary antibodies to FOXO1 (18592‐1‐AP; Proteintech, China) overnight at 4 °C, followed by incubation with Fluorescein (FITC)–conjugated Affinipure Goat Anti‐Rabbit IgG (H+L; SA00003‐2; Proteintech ) secondary antibodies.

Techniques: Expressing, Translocation Assay, Double Immunofluorescence Staining, Western Blot, Immunofluorescence, Staining

Journal: Bioengineered

Article Title: Long noncoding RNA matrilineal expression gene 3 inhibits hepatocellular carcinoma progression by targeting microRNA-5195-3p and regulating the expression of forkhead box O1.

doi: 10.1080/21655979.2021.2005986

Figure Lengend Snippet: Figure 4. FOXO1 is a target gene of miR-5195-3p. (a) The binding site between miR-5195-3p and FOXO1 was predicted by bioinformatics analysis. (b) Luciferase reporter assay was used to confirm the interaction between FOXO1 and miR-5195-3p. (c) RNA pull-down assay was performed to determine whether miR-5195-3p targets FOXO1. (d, e) FOXO1 expression level was detected in miR-5195-3p overexpression or knockdown group using qPCR and Western blotting. (f) qPCR was performed to evaluate the expression of FOXO1 in HCC tissues. **P < 0.01.

Article Snippet: The The membrane was blocked with 5% nonfat milk in Tris-buffered saline with Tween 20 at room temperature for 2 h and then probed with FOXO1 and GAPDH primary antibody (1:2000 dilution; Boster, China) at 4°C overnight.

Techniques: Binding Assay, Luciferase, Reporter Assay, Pull Down Assay, Expressing, Over Expression, Knockdown, Western Blot

Journal: Bioengineered

Article Title: Long noncoding RNA matrilineal expression gene 3 inhibits hepatocellular carcinoma progression by targeting microRNA-5195-3p and regulating the expression of forkhead box O1.

doi: 10.1080/21655979.2021.2005986

Figure Lengend Snippet: Figure 5. Co-expression of MEG3 and FOXO1 inhibits HCC progression in liver cancer cell lines. (a) qPCR was performed to detect FOXO1 expression in HepG2 and Huh7 cell lines. (b) CCK-8 was used to detect HCC cell proliferation after transfection. (c, d) Transwell assay was used to assess HCC cell migration and invasion. **P < 0.01.

Article Snippet: The The membrane was blocked with 5% nonfat milk in Tris-buffered saline with Tween 20 at room temperature for 2 h and then probed with FOXO1 and GAPDH primary antibody (1:2000 dilution; Boster, China) at 4°C overnight.

Techniques: Expressing, CCK-8 Assay, Transfection, Transwell Assay, Migration

Journal: Bioengineered

Article Title: Long noncoding RNA matrilineal expression gene 3 inhibits hepatocellular carcinoma progression by targeting microRNA-5195-3p and regulating the expression of forkhead box O1.

doi: 10.1080/21655979.2021.2005986

Figure Lengend Snippet: Figure 6. Xenograft tumor model demonstrates that overexpression of MEG3 inhibites the HCC progression. (a) The images of tumor in two groups. (b) The growth curve was established. (c) Tumors weight in each group was detected. (d-f) MEG3, miR-5195-3p and FOXO1 expression level was detected in the tumor tissues using qPCR. **P < 0.01, ***P < 0.001.

Article Snippet: The The membrane was blocked with 5% nonfat milk in Tris-buffered saline with Tween 20 at room temperature for 2 h and then probed with FOXO1 and GAPDH primary antibody (1:2000 dilution; Boster, China) at 4°C overnight.

Techniques: Over Expression, Expressing

Journal: Journal of psychiatric research

Article Title: TCF7L2: A potential key regulator of antidepressant effects on hippocampal astrocytes in depression model mice.

doi: 10.1016/j.jpsychires.2024.01.007

Figure Lengend Snippet: Fig. 3. Successful isolation of hippocampal astrocytes by enzymatic dispersion and FACS (a) Overview of the experimental manipulations to disperse and isolate hippocampal astrocytes. The image shows the cell lysate after dispersion under an optical microscope. For this series of experiments, the number of mice that proceeded to RNA-seq and sequence data analysis was 5–6 per group. (b) Gating strategy used for flow cytometry. Cells were initially differentiated from debris using FSC-A and SSC-A thresholds, with doublets subsequently excluded via FSC-A and FSC-H gating. Thereafter, DAPI was used for dead-live exclusion. This was followed by ACSA-2-PE and O1-APC detection, enabling the segregation of live cells into four distinct populations. Three of these regions were used for subsequent analysis. (c) The separation of cells was confirmed using RT-RamDA and PCR. For the positive control, RNA was extracted from the whole hippocampus and RT-RamDA was performed. Atp1b2 and Aqp4 are known markers of astrocytes, Mog for oligodendrocytes, Cx3cr1 for microglia, Snap25 for neurons, and Tek for vascular endothelial cells. The expected bands for astrocytes, oligodendrocytes, and microglia could be detected.

Article Snippet: The dissociated single-cell suspension was resuspended and incubated in FcR Blocking Reagent (130–097-678; Miltenyi Biotec) at 4 ◦C for 10 min, and then costained for 15 min at 4 ◦C with PE-conjugated anti-astrocyte cell surface antigen-2 (ACSA-2) antibody (130-116-244; Miltenyi Biotec) and Alexa Fluor 647-conjugated anti-O1 antibody (FAB1327R; R&D Systems, Minneapolis, USA).

Techniques: Isolation, Dispersion, Microscopy, RNA Sequencing, Sequencing, Flow Cytometry, Positive Control