Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: MiR-155 -targeted IcosL controls tumor rejection.

doi: 10.1073/pnas.2408649121

Figure Lengend Snippet: Fig. 1. MiR-155 impairs T cell antitumor response at least in part by targeting ICOSL transcripts. (A) Detection of miR-155 (dark blue) in mice with miR-155 ON and OFF using ISH. Left, miR-155 detection (dark blue) when this microRNA is turned ON in Nestin-Cre x miR-155LSLtTAoffspring; Right, Lack of miR-155 expression when this microRNA has been turned OFF in the same mice. (B) Tumor infiltrates in mice with miR-155 ON for 3 mo (Top panels) then back OFF for 1 wk (Bottom panels). Top panels: Left: H&E staining of the kidney showing infiltrates (dark blue color); Middle: B220 (red signal) and CD4 (brown signal) staining of the infiltrate from the kidney indicated by the square); Right: B220 (red signal) and CD4 (brown signal) staining of a skeletal muscle. Bottom panels, tumor infiltrates in kidneys. From left to right: H&E staining at low (first panel) and high magnification (second panel); B220 staining (red, third panel, notice lack of B220-positive cells); CD4 staining (brown, fourth panel). (C) Top panels: Left: IHC for CD4 (brown) showing T cell infiltration surrounding a lymph node. Right: Magnification of a part of the image on the Left. Bottom panels: IHC for Icos (brown) on lymphomas with miR-155 ON (Left) and then turned back OFF (Right). (D) Dual-Luciferase reporter assays performed in Raw264.7 macrophages cotransfected with a Renilla luciferase reporter vector containing the human ICOSL-3′-UTR with either the WT miR-155 binding site or its mutated version, along with either a miR-Control RNA, a human miR-155 RNA, or a human miR-155-Inhibitor RNA, as indicated. n = 6 replicates/each experimental setting; P = 5.09182E-07. (E) Raw264.7 cells transfected with either a miR-Control RNA, a mouse miR-155 RNA, or a mouse miR-155- Inhibitor RNA, as indicated, were treated with either LPS (100 ng/mL) or the vehicle 24 h after transfection. Two days posttransfection, cell lysates were analyzed by western blot for IcosL expression. GAPDH was used as a loading control. (F) Two days after transfection with either a miR-Control RNA or human-miR-155 RNA, BL cell lines BJAB and NAMALWA were analyzed for ICOSL expression by western blotting. α-tubulin and GAPDH were used as loading control. (G) Inverse correlation between miR-155 and ICOSL transcripts as measured by qRT-PCR in CLL (MEC1 and MEC2) and BL (NAMALWA, BJAB, RAJI, PH3R1, DAUDI) cell lines.

Article Snippet: The specific antibodies used (source and catalog numbers) were as follows: ICOS (ProSci; 8685), ICOSL (ProSci; 8687), T cell (CD3) (Abcam; ab16669), CD163 (Abcam; ab182422), CD31 (Abcam; ab28364); and CD20 B cells (Biogenex; AM537GP).

Techniques: Expressing, Staining, Luciferase, Plasmid Preparation, Binding Assay, Control, Transfection, Western Blot, Quantitative RT-PCR

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: MiR-155 -targeted IcosL controls tumor rejection.

doi: 10.1073/pnas.2408649121

Figure Lengend Snippet: Fig. 2. Turning ON the expression of miR-155 in lymphocytes of Nestin-Cre x miR-155LSLtTA mice leads to development of B cell lymphomas lacking IcosL expression, while turning miR-155 back OFF allows the formation of B (IcosL) - T (Icos) cells synapses mostly located around blood vessels. (A) Flow cytometry analyses for B220 and IcosL expression in lymph nodes of littermates with miR-155 OFF (Left) or ON (Right) for 2 mo. Representative images. (B) First row: IcosL (green; Left) and CD20 (red; Middle) staining in a WT spleen. Colocalization (yellow; Right). Second row: Lack of IcosL (green; Left) expression by CD20-positive cells (red; Middle) in lymphomas with miR-155 ON (no yellow signal, Right). IcosL-positive cells in the left are distinct from malignant B cells. Third row: IcosL-positive cells (green; Left) in lymphomas with miR-155 ON are CD31-positive (red, Middle) with colocalization (yellow; Right). Fourth row: A few benign B cells present in the mice with miR-155 ON then OFF for 1 wk had started to re-express IcosL. CD20 (green; Left); IcosL (red; Middle); colocalization (yellow; Right). Panels are representative of staining of 3 mice/group. (C) IHC for B220 (red; Top Left), CD3 (green; Top Right) in infiltrates from mice with miR-155 ON for 10 wk then back OFF for 4 d. Scattered B220-CD3 synapses are seen as yellow fluorescence in the pictures at the Bottom Left after merging B220-CD3 signals; Bottom Right is a magnification of the square in the left picture. (D) IHC for Icos (red; Top) and IcosL (green; Middle) and coexpression (yellow; Bottom) in infiltrates from mice with miR-155 ON for 2 to 3 mo then OFF for 4 d. The immunological synapses between B cells (IcosL) and T cells (Icos) are located mostly around blood vessels (ovals). (Scale bar, 100 µm.)

Article Snippet: The specific antibodies used (source and catalog numbers) were as follows: ICOS (ProSci; 8685), ICOSL (ProSci; 8687), T cell (CD3) (Abcam; ab16669), CD163 (Abcam; ab182422), CD31 (Abcam; ab28364); and CD20 B cells (Biogenex; AM537GP).

Techniques: Expressing, Flow Cytometry, Staining, Fluorescence

Journal: Nutrients

Article Title: Evaluation of the Relationship Between Orexin A, Peptide YY, AgRP, and POMC Levels and Sleep Disorders in Children with Malnutrition

doi: 10.3390/nu18030377

Figure Lengend Snippet: Box plots showing the distribution of the orexin A, proopiomelanocortin (POMC), agouti-related protein (AgRP), and peptide yy (PYY) levels in the malnourished children and typically developing (TD) healthy controls. The Mann–Whitney U test was used to compare the peptide levels between the two groups.

Article Snippet: Human POMC, orexin A, AgRP, and PYY enzyme-linked immunosorbent assay (ELISA) kits (Shanghai Korain Biotech Co., Ltd., Shanghai, China) were used to measure the plasma peptide levels.

Techniques: MANN-WHITNEY

Journal: Inflammation

Article Title: Methylprednisolone Modulates the Tfr/Tfh ratio in EAE-Induced Neuroinflammation through the PI3K/AKT/FoxO1 and PI3K/AKT/mTOR Signalling Pathways

doi: 10.1007/s10753-024-02099-y

Figure Lengend Snippet: Flow cytometry analysis of different subsets of splenic Tfr and Tfh cells in the control, Pre-EAE, P-EAE, and EAE + MP groups. A The percentages of CD4 + CXCR5 + Foxp3 + Tfr and CD4 + CXCR5 + Foxp3-Tfh cells in the spleens of EAE mice in these four groups. B The percentages of CD4 + CXCR5 + Foxp3-ICOS + and CD4 + CXCR5 + Foxp3-PD-1 + Tfh cells in the spleens of EAE mice in these four groups. n = 5 per group. The data are presented as the mean ± SEM. * P < 0.05, ** P < 0.01*** P < 0.001.

Article Snippet: The membranes were blocked in a solution containing TBST with 5% skim milk for one hour and then incubated respectively with anti-PI3K (Cell Signaling Technology, 34,050), anti-phospho-AKT (Affinity, AF0016), anti-AKT (Affinity, AF6261), anti-phospho-FoxO1 (Cell Signaling Technology, 9464), anti-FoxO1(Affinity, AF3417), anti-phospho-mTOR (Cell Signaling Technology, 5536), anti-mTOR (Cell Signaling Technology, 2983), CXCR5 (ABclonal, A8950), Foxp3 (Affinity, AF6544), ICOS (ABclonal, A1811), PD-1 (Cell Signaling Technology, 84,651), TGF-β1 (Proteintech, 21,898–1-AP), IL-21 (Affinity, DF4818), GFAP (Cell Signaling Technology, 3670), Iba-1 (Cell Signaling Technology, 17,198), Arg-1 (Proteintech,16,001–1-AP), iNOS (Proteintech, 18985–1-AP) and GAPDH (BOSTER, BM3874) antibodies overnight at 4 °C.

Techniques: Flow Cytometry, Control

Journal: Inflammation

Article Title: Methylprednisolone Modulates the Tfr/Tfh ratio in EAE-Induced Neuroinflammation through the PI3K/AKT/FoxO1 and PI3K/AKT/mTOR Signalling Pathways

doi: 10.1007/s10753-024-02099-y

Figure Lengend Snippet: EAE mice were classified into the control group, the Pre-EAE group, the P-EAE group and the EAE + MP group. Representative blots bands of CXCR5, PD-1, iCOS, TGF-β1, FoxP3, and IL-21 in the spinal cords of EAE mice were analysed. n = 3 per group. The data are presented as the mean ± SEM. * P < 0.05, ** P < 0.01.*** P < 0.001, and **** P < 0.0001.

Article Snippet: The membranes were blocked in a solution containing TBST with 5% skim milk for one hour and then incubated respectively with anti-PI3K (Cell Signaling Technology, 34,050), anti-phospho-AKT (Affinity, AF0016), anti-AKT (Affinity, AF6261), anti-phospho-FoxO1 (Cell Signaling Technology, 9464), anti-FoxO1(Affinity, AF3417), anti-phospho-mTOR (Cell Signaling Technology, 5536), anti-mTOR (Cell Signaling Technology, 2983), CXCR5 (ABclonal, A8950), Foxp3 (Affinity, AF6544), ICOS (ABclonal, A1811), PD-1 (Cell Signaling Technology, 84,651), TGF-β1 (Proteintech, 21,898–1-AP), IL-21 (Affinity, DF4818), GFAP (Cell Signaling Technology, 3670), Iba-1 (Cell Signaling Technology, 17,198), Arg-1 (Proteintech,16,001–1-AP), iNOS (Proteintech, 18985–1-AP) and GAPDH (BOSTER, BM3874) antibodies overnight at 4 °C.

Techniques: Control