Journal: Autophagy

Article Title: RNF186 regulates EFNB1 (ephrin B1)-EPHB2-induced autophagy in the colonic epithelial cells for the maintenance of intestinal homeostasis

doi: 10.1080/15548627.2020.1851496

Figure Lengend Snippet: EFNB1-induced autophagy is independent of MTOR inhibition. (A) Ls174t cells were treated as indicated, followed by western blot analysis of the indicated proteins. Densitometry quantification from three independent experiments was shown in the right panel. (B-D) Ls174t cells were pretreated with SBI-0206965 (10 μM) (B), 3-MA (5 mM) (C) or wortmannin (100 nM) (D) for 1 h, followed by the treatment of plate-coated Fc or ephrin-B1-Fc (10 ug/ml) in presence of the inhibitors for the indicated time. Cell lysates were analyzed by western blot for the indicated proteins. Densitometry quantification from three independent experiments was shown in the right panel. (E) Ls174t cells were pretreated with DMSO, 3-MA (5 mM) or wortmannin (100 nM) for 1 h, followed by plate-coated Fc or ephrin-B1-Fc (10 μg/ml) for 2 h in presence of the inhibitors and followed by confocal microscopy analysis of MAP1LC3B puncta. Quantification of cells with puncta of MAP1LC3B-II was shown in the bottom panel. Scale bar: 5 μm. (F) Ls174t cells were pretreated with VPS34-IN1 (2 μM) for 1 h, followed by the treatment of plate-coated Fc or ephrin-B1-Fc (10 ug/ml) in presence of the inhibitor for the indicated time. Cell lysates were analyzed by western blot for the indicated proteins. Densitometry quantification was shown in the right panel. (G) Control-gRNA or ATG5-gRNA infected Ls174t cells were treated with 10 μg/ml plate-coated Fc (0 time point) or ephrin-B1-Fc, followed by western blot analysis of the indicated proteins. Densitometry quantification from three independent experiments was shown in the right panel. WT: wild type; KO: knockout; 3-MA: 3-methyladenine. All error bars represent SEM of technical replicates. *: P < 0.05, **: P < 0.01, ***: P < 0.001, ****: P < 0.0001 based on two-sided unpaired T test (A-G). Densitometry quantification of A-D and F-G were based on three independent experiments. Data are representative of three independent experiments

Article Snippet: ULK inhibitor SBI-0206965 (T2128), PtdIns3K inhibitor 3-MA (T1879), wortmannin (T6283) and Vps34-IN-1 (T7015) were bought from TargetMol.

Techniques: Inhibition, Western Blot, Confocal Microscopy, Infection, Knock-Out

Journal: Advanced Science

Article Title: Dual Activity of Type III PI3K Kinase Vps34 is Critical for NK Cell Development and Senescence

doi: 10.1002/advs.202309315

Figure Lengend Snippet: Deletion of Vps34 at an early stage impairs NK‐cell development. A) Flow cytometric analysis of NK cells in the spleen (SP) and bone marrow (BM) of the indicated mice. Representative plots and quantifications of NK cells (CD3 − NKp46 + ) are shown. B) Flow cytometric analysis of NK‐cell subsets (gated on live CD3 − CD122 + ), including NKp (NK1.1 − CD11b − ), iNK (NK1.1 + CD11b − ), and mNK (NK1.1 + CD11b + ). Representative plots and quantifications are shown. C,D) Bone marrow chimera experiment. A mixture of bone marrow cells from wild‐type (WT) mice expressing CD45.1 and Vps34‐KO CD122 mice expressing CD45.2 was injected into Rag1 −/− γc − mice. Representative flow cytometry plots show the percentages of CD3 − NK1.1 + NK cells (gated on CD45.1 and CD45.2, respectively), as well as three NK subsets (gated on CD3 − CD122 + ) from recipient SP and BM. E) RMA‐S tumor rejection assay. A mixture of RMA and RMA‐S cells was co‐injected into the indicated mice. Left: representative plots showing the relative ratio of GFP + RMA‐S and DsRed + RMA cells. Right: quantification. F) β2m −/− splenocyte rejection assay. A mixture of CFSE‐labeled splenocytes was co‐injected into the indicated mice. Left: representative plots showing residual CFSE+ splenocytes in the SP and lymph node (LN): R1, WT splenocyte; R2, β2m −/− splenocyte. Right: quantification. G) B16 melanoma metastasis. Left: representative lung photos; quantification of lung weight; and B16 colony (right). Each symbol represents an individual mouse. Data in panels A, B, and F are pooled from two or three independent experiments. The data in panels E and G are representative of three independent experiments (n = 3–5 per group). The graph represents the mean ± SD.

Article Snippet: Vps34‐PK‐III (MedChem Express, Cat#HY‐12794), a chemical inhibitor for Vps34, was applied at a concentration of 40 n m . For the in vivo assay, mice were intraperitoneally injected with LPS (10 mg LPS per kg body weight).

Techniques: Expressing, Injection, Flow Cytometry, Tumor Rejection Assay, Labeling

Journal: Advanced Science

Article Title: Dual Activity of Type III PI3K Kinase Vps34 is Critical for NK Cell Development and Senescence

doi: 10.1002/advs.202309315

Figure Lengend Snippet: Vps34 is necessary to maintain the responsiveness of NK cells to IL‐15. A,B) Representative histograms display the expression of NK cell development‐associated markers on splenic NK cells (gated on CD3 − NK1.1 + ) from the indicated mice. The percentages of NK cells expressing these markers are quantified. C,D) Flow cytometric analysis of CD122 on total NK cells (gated on CD3 − NK1.1 + ) and four NK‐cell subsets (refer to Figure , Supporting Information) in the spleen (SP) and bone marrow (BM) of the indicated mice. Representative histograms are shown, and the mean fluorescence intensity (MFI) of CD122 is quantified. Isotype control was used as a negative staining control. E–H) Splenocytes were stimulated with IL‐15/IL15Rα complex (IL‐15) or left unstimulated (Ctr), followed by intracellular staining of p‐AKT T308 , p‐AKT S473 , p‐S6 (E‐F), or E4BP4, p‐STAT5 (G‐H) in NK cells (gated on CD3 − NK1.1 + ). Representative histograms (E, G) and quantifications (F, H) are shown. Isotype control was used as a negative staining control, respectively. Each symbol represents an individual mouse. Data in (A) and (C) are representative of at least 3 independent experiments. The graphs represent the mean ±SD.

Article Snippet: Vps34‐PK‐III (MedChem Express, Cat#HY‐12794), a chemical inhibitor for Vps34, was applied at a concentration of 40 n m . For the in vivo assay, mice were intraperitoneally injected with LPS (10 mg LPS per kg body weight).

Techniques: Expressing, Fluorescence, Control, Negative Staining, Staining

Journal: Advanced Science

Article Title: Dual Activity of Type III PI3K Kinase Vps34 is Critical for NK Cell Development and Senescence

doi: 10.1002/advs.202309315

Figure Lengend Snippet: Vps34 regulates CD122 membrane trafficking. A) Flow cytometric analysis was performed to examine the expression of CD122 on NK cells (gated on CD3 − NK1.1 + ) in terms of membrane, intracellular, and total (membrane plus intracellular) forms. Representative histograms (left) and quantification of MFI (right) are shown. Blue represents Vps34 fl/fl , red represents Vps34‐KO CD122 . B,C) Splenocytes from wild‐type mice or the specified mice were stimulated with or without the IL‐15/IL‐15Rα complex for 4 h. After washing out IL‐15, the cells were further cultured for 4 h or treated with Vps34‐PK‐III (B, DMSO used as a control). Flow cytometry was employed to detect CD122 expression on the surface of NK cells at the indicated time points. Representative histograms (left) and quantification of CD122 MFI (right) are presented. Black represents 0 h; Blue represents 4 h; Red represents 8 h. Isotype (filled grey) was used as a negative staining control. “NS” denotes not significant. D) Vps34 fl/fl and Vps34‐KO CD122 mice were stimulated in vivo with LPS, and the expression of CD122 on the membrane of NK cells was examined by flow cytometry at the specified time points. Representative histograms (left) and quantification of CD122 MFI (right) are displayed. Black represents 0 h; Blue represents 8 h; Red represents 24 h. “NS” denotes not significant. E) Similar to (B), CD3 − CD49b + NK cells were sorted for imaging analysis of CD122. NK cells were collected at the indicated time points, fixed, permeabilized, and stained for the specific proteins of interest. Images were acquired using confocal microscopy. Scale bar: 2 µm. The images presented are representative of two experiments (3 mice were analyzed). Each symbol represents an individual mouse. The data in (A–D) are representative of three independent experiments (n = 3–4 per group). The graph represents the mean ± SD.

Article Snippet: Vps34‐PK‐III (MedChem Express, Cat#HY‐12794), a chemical inhibitor for Vps34, was applied at a concentration of 40 n m . For the in vivo assay, mice were intraperitoneally injected with LPS (10 mg LPS per kg body weight).

Techniques: Membrane, Expressing, Cell Culture, Control, Flow Cytometry, Negative Staining, In Vivo, Imaging, Staining, Confocal Microscopy

Journal: Advanced Science

Article Title: Dual Activity of Type III PI3K Kinase Vps34 is Critical for NK Cell Development and Senescence

doi: 10.1002/advs.202309315

Figure Lengend Snippet: Vps34 deficiency at terminal stage leads to gradual loss of NK cells with aging. A,B) The frequency and absolute number of NK cells (gated on CD3 − NK1.1 + ) in the spleen and bone marrow of Vps34 fl/fl and Vps34‐KO Ncr1 mice at various weeks of age were analyzed using flow cytometry. C,D) Flow cytometric analysis of four subsets of NK cells (gated on CD3 − NK1.1 + ) in the spleen of six‐week‐old or sixty‐week‐old Vps34 fl/fl and Vps34‐KO Ncr1 mice. E,F) Flow cytometric analysis of the expression of specific receptors on splenic NK cells from sixty‐week‐old mice. Representative histograms and quantitative data are presented. Each symbol represents an individual mouse. The data in panels (A–F) are representative of three independent experiments (n = 3–5 per group). The graph shows the mean ± SD.

Article Snippet: Vps34‐PK‐III (MedChem Express, Cat#HY‐12794), a chemical inhibitor for Vps34, was applied at a concentration of 40 n m . For the in vivo assay, mice were intraperitoneally injected with LPS (10 mg LPS per kg body weight).

Techniques: Flow Cytometry, Expressing

Journal: Advanced Science

Article Title: Dual Activity of Type III PI3K Kinase Vps34 is Critical for NK Cell Development and Senescence

doi: 10.1002/advs.202309315

Figure Lengend Snippet: Loss of Vps34 accelerates NK‐cell senescence. A) Flow cytometric analysis of the expression of KLRG1 on NK cells (gated on CD3 − NK1.1 + ). Representative histograms (left) and quantification of KLRG1 MFI (right) on NK cells from six‐week‐old (young) or sixty‐week‐old (old) mice are shown. B,C) Flow cytometric analysis of the expression of KLRG1 in Vps34 fl/fl and Vps34‐KO Ncr1 mice. Representative plots (gated on CD3 − NK1.1 + ) are displayed in, and the frequency of KLRG1 + NK cells and KLRG1 MFI on total NK cells were quantified in. D–F) Intracellular staining of p21 was performed on NK cells (CD3 − NK1.1 + ) from the indicated mice: six‐week‐old (young), sixty‐week‐old wild‐type mice (old). Isotype control was used for negative staining (filled grey). SA‐βgal staining of NK cells (CD3 − CD49b + ) from the indicated mice: six‐week‐old (young), sixty‐week‐old (old) wild‐type mice, or old Vps34 fl/fl and Vps34‐KO Ncr1 G). Representative images are shown(left). The percentage of SA‐βgal + NK cells was quantified (right). H) Intracellular staining of T‐bet in NK cells (gated on CD3 − NK1.1 + ). Representative histograms and the MFI of T‐bet are presented. Vps34 fl/fl , blue, Vps34‐KO Ncr1 , red (I‐J) Evaluation of B16 melanoma metastasis. Representative lung photos are shown in I), and the weight of the lungs and the number of B16 colonies in Vps34 fl/fl and Vps34‐KO Ncr1 mice were quantified in J). K) Analysis of NK cell function ex vivo. Splenocytes were stimulated as indicated (medium alone as a negative control; PMA plus ionomycin (P+I) as a positive control), and then CD107a + (left) or IFN‐γ + (right) NK cells (gated on CD3 − NKp46 + CD27 + CD11b + ) were analyzed by flow cytometry. Each symbol represents an individual mouse. Data in panels A‐C are pooled from 2 or 3 independent experiments (n = 3–4 per group). Data in panels D‐K are representative of 2 or 3 independent experiments (n = 3–5 per group). The graph represents the mean ± SD.

Article Snippet: Vps34‐PK‐III (MedChem Express, Cat#HY‐12794), a chemical inhibitor for Vps34, was applied at a concentration of 40 n m . For the in vivo assay, mice were intraperitoneally injected with LPS (10 mg LPS per kg body weight).

Techniques: Expressing, Staining, Control, Negative Staining, Cell Function Assay, Ex Vivo, Negative Control, Positive Control, Flow Cytometry

Journal: Advanced Science

Article Title: Dual Activity of Type III PI3K Kinase Vps34 is Critical for NK Cell Development and Senescence

doi: 10.1002/advs.202309315

Figure Lengend Snippet: Clearance of ROS by Vps34‐mediated autophagy protects old NK cells from senescence. A) Gene ontology (GO) analysis of RNA‐sequencing data: The top 19 hallmark pathways were enriched in Vps34‐KO Ncr1 NK cells (CD3 − CD49b + ) compared to Vps34 fl/fl NK cells. B) Flow cytometric detection of reactive oxygen species (ROS) in NK cells by staining with DCFH‐DA: Representative histograms and MFI of DCFH‐DH on NK cells from sixty‐week‐old mice. Blue, Vps34 fl/fl ; Red, Vps34‐KO Ncr1 ; filled grey, unstained negative control. C) Flow cytometric detection of mitochondrial aggregates in NK cells by MitoTracker staining: Representative histograms and MFI of MitoTracker on NK cells from six‐week‐old (young) or sixty‐week‐old (old) mice. Blue, Vps34fl/fl; Red, Vps34‐KO Ncr1 . D) Flow cytometric detection of mitochondrial function in NK cells by MitoSOX staining: Representative histograms (left) and MFI of MitoSOX (right) on NK cells from sixty‐week‐old mice. Blue,Vps34 fl/fl ; Red, Vps34‐KO Ncr1 ; filled grey, unstained negative control. E) Vps34 fl/fl and Vps34 fl/f l/UBC‐Cre/ERT2 mice were intraperitoneally injected with tamoxifen for five continuous days. Flow cytometric detection of mitochondrial aggregates in NK cells: Representative histograms and MFI of MitoTracker on NK cells from sixty‐week‐old (old) mice. Blue, Vps34fl/fl; Red, Vps34 fl/fl /UBC‐Cre/ERT2; filled grey, unstained negative control. F) Splenocytes were treated with rapamycin (Rap) or not (Ctr) for 4 h in vitro, and then autophagosomes were detected by flow cytometry‐based staining of Cyto‐ID. Blue, Vps34 fl/fl ; Red, Vps34‐KO Ncr1 ; filled grey, unstained negative control. G) Intracellular staining of p62 in NK cells from sixty‐week‐old mice: Representative histograms (upper panel) and MFI of p62 (lower panel) on NK cells. Blue, Vps34 fl/fl ; Red, Vps34‐KO Ncr1 ; filled grey, unstained negative control. H) Splenocytes were treated with rotenone for 4 h, with or without rapamycin. DMSO was used as the untreated control (Ctr), and then ROS was monitored by DCFH‐DA staining. Representative histograms (left) and DCFH‐DH MFI (right) on NK cells from the indicated mice are shown. Black, Ctl; Blue,rotenone; Red, rotenone+Rap; filled grey, unstained negative control. I) Detection of ROS in NK cells: Representative histograms and MFI of DCFH‐DH (lower panel) in NK cells from six‐week‐old (young) or sixty‐week‐old (old) wild‐type mice. Blue, young; Red, old; filled grey, unstained. J) Detection of ROS in NK cells treated with N ‐acetyl‐ L ‐cysteine (NAC): Representative histograms and MFI of DCFH‐DH (lower) in NK cells treated with NAC (red), DMSO (Ctr, black), or unstained (filled grey). K) For SA‐βgal staining, splenic NK cells (CD3 − CD49b + ) were sorted from the indicated mice and treated with NAC or not (Ctr) for 96 h in vitro. Representative images were acquired (left), and the percentage of SA‐βgal + NK cells was calculated (right). Scale bar, 10 µm. Each symbol represents an individual mouse. The data in panels (A–G) and (I–K) are representative of 3 independent experiments (n = 3–5 mice per group). The data in H are pooled from two independent experiments (n = 3 per group). The graph represents the mean ± SD

Article Snippet: Vps34‐PK‐III (MedChem Express, Cat#HY‐12794), a chemical inhibitor for Vps34, was applied at a concentration of 40 n m . For the in vivo assay, mice were intraperitoneally injected with LPS (10 mg LPS per kg body weight).

Techniques: RNA Sequencing Assay, Staining, Negative Control, Injection, In Vitro, Flow Cytometry, Control