Journal: iScience

Article Title: A purinergic mechanism underlying metformin regulation of hyperglycemia

doi: 10.1016/j.isci.2023.106898

Figure Lengend Snippet:

Article Snippet: Rabbit anti-mouse lgG (light chain specific) secondary antibody, HRP , Cell Signaling , #58802.

Techniques: Virus, Recombinant, SYBR Green Assay, Calcium Mobilization Assay, ATP Assay, Glucose Assay, Detection Assay, Control, Software, Plasmid Preparation, Inverted Microscopy

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: Targeting CD73 increases therapeutic response to immunogenic chemotherapy by promoting dendritic cell maturation

doi: 10.1007/s00262-023-03416-4

Figure Lengend Snippet: The expression of tumor CD73 was significantly associated with the risk of distant metastasis after surgical operation and poor survival outcome. A CD73 proteins were mainly expressed on the membrane of cancer cells. CD39 proteins were majorly expressed in the stromal cells and partially expressed in the inflammatory cells. B The H-score of tumor CD73 was statistically associated with the risk of distant metastasis (n = 421 and unpaired T-test p = 0.0353). C Stromal CD39 expression was not associated with the present of LVI, PNI and distant metastasis (n = 421) by unpaired T-test. D Tumor CD73 expression was significantly associated with pathological T stage (n = 421, p = 0.0008). Stromal CD39 expression was not associated with pathological T stage (n = 421 and p = 0.70). One-way ANOVA test. E Patients with high tumor CD73 expression were clinically associated with worsen disease-free survival (DFS) in CRC patients (n = 421, p = 0.0076). High stromal CD39 expression was associated with favorable DFS in CRC patients (n = 421 p = 0.0034). F Tumor CD73 expression was significantly associated with distant metastasis-free survival (DMFS) and DFS in stage II-III COAD patients who received adjuvant chemotherapy (n = 177, log-rank p = 0.0077). G Tumor CD73 expression was conversely associated with the density of CD8+ TILs and CD45.+ TILs within tumor microenvironment (TME). Unpaired t test (n = 421)

Article Snippet: After incubation with the primary rabbit monoclonal antibody against CD73 (#13,160, 1:100, Cell Signaling Tech.) or CD39 (#14,481, 1:100, Cell Signaling Tech.) at room temperature for 2 h, the sections were stained according to the manufacture’s manual (VECTASTAIN Elite ABC Kit, Vector Laboratories) and incubated with the substrate DAB chromogen (Vector Laboratories), and then were counterstained with hematoxylin [ 21 , 22 ].

Techniques: Expressing, Membrane, Adjuvant

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: Targeting CD73 increases therapeutic response to immunogenic chemotherapy by promoting dendritic cell maturation

doi: 10.1007/s00262-023-03416-4

Figure Lengend Snippet: Correlation between clinicopathologic parameters, DFS and OS

Article Snippet: After incubation with the primary rabbit monoclonal antibody against CD73 (#13,160, 1:100, Cell Signaling Tech.) or CD39 (#14,481, 1:100, Cell Signaling Tech.) at room temperature for 2 h, the sections were stained according to the manufacture’s manual (VECTASTAIN Elite ABC Kit, Vector Laboratories) and incubated with the substrate DAB chromogen (Vector Laboratories), and then were counterstained with hematoxylin [ 21 , 22 ].

Techniques:

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: Targeting CD73 increases therapeutic response to immunogenic chemotherapy by promoting dendritic cell maturation

doi: 10.1007/s00262-023-03416-4

Figure Lengend Snippet: Univariate and multivariate analysis of DFS and known prognostic factors in stage I-IV colon adenocarcinoma patients

Article Snippet: After incubation with the primary rabbit monoclonal antibody against CD73 (#13,160, 1:100, Cell Signaling Tech.) or CD39 (#14,481, 1:100, Cell Signaling Tech.) at room temperature for 2 h, the sections were stained according to the manufacture’s manual (VECTASTAIN Elite ABC Kit, Vector Laboratories) and incubated with the substrate DAB chromogen (Vector Laboratories), and then were counterstained with hematoxylin [ 21 , 22 ].

Techniques:

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: Targeting CD73 increases therapeutic response to immunogenic chemotherapy by promoting dendritic cell maturation

doi: 10.1007/s00262-023-03416-4

Figure Lengend Snippet: Genomic correlates of NT5E and CD8 signatures in the TCGA dataset A CD73/NT5E was highly expressed on tumor tissues compared to normal tissues. B High CD73 mRNA expression was remarkably associated with poor survival outcome in CRC patients that retrieved from TCGA database (cut-off mRNA = 9.0, n = 368, p = 0.0453). Moreover, CD39 mRNA expression was not associated with poor survival outcome in CRC patients that retrieved from TCGA database (cut-off mRNA = 3.37, n = 515, p = 0.7079). C High CD73 mRNA expression was conversely associated with less expression of T-cell mediated signatures (TCGA database, n = 368, unpaired T test). ***p < 0.001

Article Snippet: After incubation with the primary rabbit monoclonal antibody against CD73 (#13,160, 1:100, Cell Signaling Tech.) or CD39 (#14,481, 1:100, Cell Signaling Tech.) at room temperature for 2 h, the sections were stained according to the manufacture’s manual (VECTASTAIN Elite ABC Kit, Vector Laboratories) and incubated with the substrate DAB chromogen (Vector Laboratories), and then were counterstained with hematoxylin [ 21 , 22 ].

Techniques: Expressing

Journal: The Journal of Biological Chemistry

Article Title: CORO7 functions as a scaffold protein for the core kinase complex assembly of the Hippo pathway

doi: 10.1074/jbc.RA120.013297

Figure Lengend Snippet: CORO7 forms a complex with the components of the Hippo pathway. A–C, schematic representation of domains and truncated constructs of CORO7 ( A ), LATS1 ( B ), and SAV1 ( C ) used in coimmunoprecipitation assays. D – F, HA-LATS1 ( D ), HA-MST2 ( E ), or HA-SAV1 ( F ) was expressed together with wild-type (WT), N-terminal (N), or C-terminal (C) Flag-CORO7 in HEK293T cells. The lysates were immunoprecipitated by anti-Flag antibody and immunoblotted with anti-HA and anti-Flag antibodies. The whole cell lysate (WCL) samples were loaded for indicating the expression levels. G, HA-CORO7 was transfected with the Flag-tagged truncated forms of LATS1 in HEK293T cells, and the lysates were immunoprecipitated by anti-Flag antibody. Subsequently, they were immunoblotted with the same antibodies as in ( D – F ). The WCL samples were loaded for indicating the expression levels. H, Flag-CORO7 was transfected with the HA-tagged truncated forms of SAV1 in HEK293T cells. The lysates were immunoprecipitated by anti-HA antibody and immunoblotted with the same antibodies as in ( D – F ). The WCL samples were loaded for indicating the expression levels. I, HEK293T cells were transfected with HA-tagged truncated forms of SAV1, HA-MST2, and Flag-CORO7. The lysates were immunoprecipitated by anti-Flag antibody and immunoblotted with the same antibodies as in ( D – F ). The WCL samples were loaded for indicating the expression levels. Data information: Flag-tagged vector and HA-tagged vector were transfected as negative controls (−). All immunoblots are representative of at least three independent experiments. CORO7, coronin 7; FBM, FERM-binding motif; HA, hemagglutinin; LATS1, large tumor suppressor kinase 1/2; MOB1, MOB kinase activator 1; MST2, mammalian sterile 20-like kinase 2; SARAH, Sav/Rassf/Hpo; SAV1, salvador family WW domain–containing protein 1; WCL, whole cell lysate.

Article Snippet: The following antibodies were used for immunoblotting and immunoprecipitation: antibodies against HA (MBL Life Science; M132-3), Flag (MBL Life Science; M185-3L), Flag (CST; 2368), Myc (MBL Life Science; M192-3), CORO7 (Abcam; ab117446), YAP (Santa-Cruz; sc-101199), p-YAP (CST; 4911), LATS1 (CST; 3477), tubulin (DSHB; E7), SAV1 (CST; 13301), p-Tyr (CST; 9411), and IgG (Millipore; 2896738).

Techniques: Construct, Immunoprecipitation, Expressing, Transfection, Plasmid Preparation, Western Blot, Binding Assay, Sterility

Journal: The Journal of Biological Chemistry

Article Title: CORO7 functions as a scaffold protein for the core kinase complex assembly of the Hippo pathway

doi: 10.1074/jbc.RA120.013297

Figure Lengend Snippet: CORO7 is necessary for the formation of the core Hippo kinase complex. A, Flag-MST2, HA-LATS1, HA-SAV1, and HA-MOB1 were transfected in HEK293T cells to observe the coimmunoprecipitation between Flag-MST2 and the rest. siRNA targeting CORO7 was treated in the indicated lane (si- CORO7 ). The lysates were immunoprecipitated by anti-Flag antibody and immunoblotted with anti-HA and anti-Flag antibodies. The whole cell lysate (WCL) samples were loaded for indicating the expression levels, and CORO7 was immunoblotted to verify the efficiency of knockdown. B, flag-LATS1, HA-MST2, HA-SAV1, and HA-MOB1 were transfected in HEK293T cells to observe the coimmunoprecipitation between Flag-LATS1 and the rest. siRNA targeting CORO7 was treated in the indicated lane (si- CORO7 ). The lysates were immunoprecipitated by anti-Flag antibody and immunoblotted with anti-HA and anti-Flag antibodies. The WCL samples were loaded for indicating the expression levels, and CORO7 was immunoblotted to verify the efficiency of knockdown. C, flag-MST2 was expressed in HEK293T cells, and CORO7 was knocked down in the indicated lane (si- CORO7 ). Cells were deprived of FBS for 24 h, and the lysates were immunoprecipitated by anti-Flag antibody and immunoblotted with anti-LATS1, anti-SAV1, and anti-Flag antibodies. The WCL samples were loaded for indicating the expression levels, and CORO7 was immunoblotted to verify the efficiency of knockdown. All immunoblots are representative of at least three independent experiments. CORO7, coronin 7; HA, hemagglutinin; LATS1, large tumor suppressor kinase 1/2; MOB1, MOB kinase activator 1; MST2, mammalian sterile 20-like kinase 2; SAV1, salvador family WW domain–containing protein 1; WCL, whole cell lysate.

Article Snippet: The following antibodies were used for immunoblotting and immunoprecipitation: antibodies against HA (MBL Life Science; M132-3), Flag (MBL Life Science; M185-3L), Flag (CST; 2368), Myc (MBL Life Science; M192-3), CORO7 (Abcam; ab117446), YAP (Santa-Cruz; sc-101199), p-YAP (CST; 4911), LATS1 (CST; 3477), tubulin (DSHB; E7), SAV1 (CST; 13301), p-Tyr (CST; 9411), and IgG (Millipore; 2896738).

Techniques: Transfection, Immunoprecipitation, Expressing, Knockdown, Western Blot, Sterility

Journal: The Journal of Biological Chemistry

Article Title: CORO7 functions as a scaffold protein for the core kinase complex assembly of the Hippo pathway

doi: 10.1074/jbc.RA120.013297

Figure Lengend Snippet: Signals activating the Hippo pathway regulate complex formation between CORO7 and the components of the pathway. A, 24 h after seeded to 15% confluency, HEK293T cells transfected with HA-MST2, HA-SAV1, and Flag-CORO7 were deprived of serum (FBS) or treated with 0.25 μg/ml LatB for 1 h. The cell lysate samples were immunoprecipitated with anti-Flag antibody and immunoblotted with anti-HA and anti-Flag antibodies. The WCL samples were loaded for indicating the expression levels. The WCL samples were also immunoblotted with anti-pYAP antibody. B, HEK293T cells were transfected with Flag-CORO7, Myc-SAV1, and HA-NF2. Three different levels of HA-NF2 were expressed. The cell lysate samples were immunoprecipitated with anti-Myc antibody and immunoblotted with anti-HA, anti-Flag, and anti-Myc antibodies. The WCL samples were loaded for indicating the expression levels. C, HEK293T cells expressing Myc-CORO7, HA-NF2, and Flag-SAV1 were deprived of serum (FBS). The cell lysate samples were immunoprecipitated with anti-Flag antibody and immunoblotted with the same antibodies as in ( B ). The WCL samples were loaded for indicating the expression levels. All immunoblots are representative of at least three independent experiments. CORO7, coronin 7; FBS, fetal bovine serum; HA, hemagglutinin; LatB, latrunculin B; MST2, mammalian sterile 20-like kinase 2; NF2, neurofibromatosis type II; SAV1, salvador family WW domain–containing protein 1; WCL, whole cell lysate; YAP, yes-associated protein.

Article Snippet: The following antibodies were used for immunoblotting and immunoprecipitation: antibodies against HA (MBL Life Science; M132-3), Flag (MBL Life Science; M185-3L), Flag (CST; 2368), Myc (MBL Life Science; M192-3), CORO7 (Abcam; ab117446), YAP (Santa-Cruz; sc-101199), p-YAP (CST; 4911), LATS1 (CST; 3477), tubulin (DSHB; E7), SAV1 (CST; 13301), p-Tyr (CST; 9411), and IgG (Millipore; 2896738).

Techniques: Transfection, Immunoprecipitation, Expressing, Western Blot, Sterility