Journal: American journal of physiology. Renal physiology

Article Title: Array profiling reveals contribution of Cthrc1 to growth of the denervated rat urinary bladder.

doi: 10.1152/ajprenal.00499.2017

Figure Lengend Snippet: Fig. 4. A set of 15 genes change in opposite directions at the mRNA level in denervation and obstruction. The Venny analysis tool was used to identify genes that were increased in denervation (at Q 0.05) and reduced in obstruction (at Q 0.05) or vice versa. Fifteen mRNAs satisfied these criteria. Changes of these mRNAs are plotted in A–C. Italicized gene products met the Q 0 criterion, and the remainder met the Q 0.05 criterion. D: 4 examples of immunohistochemical stainings from the Human Protein Atlas for gene products in this set. Brown indicates positive staining. Antibodies for all of these targets stained the urothelial cell layer of the bladder. E: Western blots for Wdr77 and Flot2 in denervation and obstruction.

Article Snippet: Following one-dimensional (1D) separation and transfer to nitrocellulose, proteins were detected using the following antibodies: CTHRC1 (mmcri, vli55), PRC1 (no. 3639; Cell Signaling), PLOD2 (21214–1-AP; Proteintech), DKK3 (ab186409; Abcam), PERK (no. 3192; Cell Signaling), CAV1 (no. 3267; Cell Signaling), myosin phosphatase targeting subunit (MYPT/MYPT1, no. 2634; Cell Signaling), myosin light chain kinase (MYLK, M 7905; Sigma), pleiotrophin (PTN, ab79411; Abcam), gelsolin (GSN, no. 12953; Cell Signaling), osteopontin (SPP1, ab91665; Abcam), CD68 (MCA5709; Bio-Rad), WDR77 (D56B8, no. 2018S; Cell Signaling), FLOT2 (no. 3436S; Cell Signaling), -actin (A5441; Sigma Aldrich), and HSP90 (610418; BD Transduction Laboratories).

Techniques: Immunohistochemical staining, Staining, Western Blot

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: Disease models & mechanisms

Article Title: The ALK inhibitor PF-06463922 is effective as a single agent in neuroblastoma driven by expression of ALK and MYCN.

doi: 10.1242/dmm.024448

Figure Lengend Snippet: Fig. 1. Inhibition of neuroblastoma cell lines employing crizotinib and PF-06463922. Treatment of neuroblastoma cells with the ALK TKIs crizotinib and PF-06463922. (A,B) Proliferation was assessed over 5 days using the resazurin cell proliferation assay in the following neuroblastoma cell lines: CLB-BAR, CLB-GE, CLB-PE, SK-N-AS and IMR32. Neuroblastoma cell line characteristics: CLB-GE – MYCN amplified, 1p deletion, 17q gain, amplified ALK amplicon containing an ALKF1174V mutation; CLB-BAR – amplified MYCN/ALK, Δexon 4-11, 1p deletion, 17q gain; IMR32 – MYCN, wt sequence but exons 2-4 are amplified, 1p deletion, 17q gain; CLB-PE – 1p gain, amplified MYCN, 17q gain; SK-N-AS – IGF-1 overexpressing, 1p deletion, 1q gain, 17q gain, 17 deletion. Cells lines were treated with increasing concentrations of either PF-06463922 (A) or crizotinib (B) as indicated. Data are mean±s.d. of the fold-relative fluorescence from treated cells relative to untreated cells from three independent experiments. (C) CLB-BAR, CLB-GE neuroblastoma cells were treated for 6 h with either crizotinib or PF-04643922 as indicated. Cellswere harvested andpre-clearedcelllysates were analyzed onSDS PAGE followedbywesternblotting for ALK, phospho-ALK-Y1278,phospho-ERK5, pan-ERK5 phospho-ERK1/2 and pan-ERK. Actin was employed as a loading control. Protein band intensities were quantified by Image Studio Lite 3.1 and normalized to untreated samples.

Article Snippet: Primary antibodies were: phospho-ALK (Y1604 (1:2000; 3341) and Y1278 (1:2000; 12127), phospho-ERK1/2, actin (1:2000; 4967), phospho-Akt (1:2000; 92711), MYCN (1:2000; 9405), Ki67 (1:100; 9129), GADPH and phospho-ERK5 (1:1000; 3371) from Cell Signaling Technology (Danvers, MA).

Techniques: Inhibition, Proliferation Assay, Amplification, Mutagenesis, Sequencing, Fluorescence, Control

Journal: Disease models & mechanisms

Article Title: The ALK inhibitor PF-06463922 is effective as a single agent in neuroblastoma driven by expression of ALK and MYCN.

doi: 10.1242/dmm.024448

Figure Lengend Snippet: Fig. 3. Comparison of inhibition effects of crizotinib and PF-06463922 on WT and neuroblastoma gain-of-function mutant TKDs by in vitro kinase assay. (A,B) Different ALK TKD proteins were incubated with either PF-06463922 (A) or crizotinib (B) in the presence of ATP (0.1 mM) and substrate peptides (0.2 mM). The incorporation of labelled γ-32P was detected under different conditions. Background counts from no-enzyme controls were subtracted, and the data were normalized to the 0 nM inhibitor reactions. (C) IC50 values from A,B were calculated by fitting data to a log (inhibitor) versus normalized response (variable slope) equation in GraphPad Prism 6.0. All data are shown as mean±s.d. from at least two independent experiments. (D) Crystal structures of ALK kinase domain in complex with PF-06463922 (top) or crizotinib (bottom). Compounds indicated in black. Gain-of-function ALK mutations F1174, R1192P, F1245, G1269 and Y1278 are shown as red spheres. The ribbon diagram displays αC helix (1157-1173; orange), catalytic loop (1246-125; magenta), activation loop (1271-1288; cyan) with DFG motif marked in blue. Figures were generated with PyMol using published coordinates (Protein data bank code: 4CLI and 2XP2).

Article Snippet: Primary antibodies were: phospho-ALK (Y1604 (1:2000; 3341) and Y1278 (1:2000; 12127), phospho-ERK1/2, actin (1:2000; 4967), phospho-Akt (1:2000; 92711), MYCN (1:2000; 9405), Ki67 (1:100; 9129), GADPH and phospho-ERK5 (1:1000; 3371) from Cell Signaling Technology (Danvers, MA).

Techniques: Comparison, Inhibition, Mutagenesis, In Vitro, Kinase Assay, Incubation, Activation Assay, Generated

Journal: Disease models & mechanisms

Article Title: The ALK inhibitor PF-06463922 is effective as a single agent in neuroblastoma driven by expression of ALK and MYCN.

doi: 10.1242/dmm.024448

Figure Lengend Snippet: Fig. 5. Efficacy of PF-06463922 in an ALKF1174 CLB-GE xenograft neuroblastoma model. 4.5×106 CLB-GE cells were injected into left shoulder subcutaneously. (A) Tumor growth curves represent the average volume of vehicle group and PF-06463922-treated group, P≤0.05 (n=5 for each group). (B) Average tumor weights in vehicle group and PF-06463922 group are displayed, P=0.02. (C) Average body weight on the day of sacrifice is shown, P>0.05. (D) Immunoblotting analysis of indicated proteins from tumors collected after 8 days of treatment with vehicle or PF-0646399 and relapsed tumors. Tumor lysates were harvested, pre-cleared and analyzed by western blotting for ALK, phospho-ALK-Y1278, MYCN, phospho-ERK1/2 and phospho-AKT. Pan-ERK and pan-Akt were employed as loading controls. (E) Immunohistochemical staining of tumors for Ki-76 as a measure of proliferation rate as indicated. Ki67-positive cells were counted manually per field of vision and quantitative results (n=15). Statistical analysis shows significant difference between vehicle and PF-06463922- treated group, P<0.002 using unpaired t-test. Data in all graphs presented as mean±s.d.

Article Snippet: Primary antibodies were: phospho-ALK (Y1604 (1:2000; 3341) and Y1278 (1:2000; 12127), phospho-ERK1/2, actin (1:2000; 4967), phospho-Akt (1:2000; 92711), MYCN (1:2000; 9405), Ki67 (1:100; 9129), GADPH and phospho-ERK5 (1:1000; 3371) from Cell Signaling Technology (Danvers, MA).

Techniques: Injection, Western Blot, Immunohistochemical staining, Staining

Journal: Disease models & mechanisms

Article Title: The ALK inhibitor PF-06463922 is effective as a single agent in neuroblastoma driven by expression of ALK and MYCN.

doi: 10.1242/dmm.024448

Figure Lengend Snippet: Fig. 6. Preclinical efficacy of PF-06463922 in a murine model of Th-ALKF1174L/MYCN-driven neuroblastoma. (A) Waterfall plots of tumoral response in Th-ALKF1174L/MYCN mice treated with vehicle, crizotinib (100 mg/kg, once daily) or PF-06463922 (10 mg/kg, twice daily). Each bar indicates percent change in the volume of an individual tumor, as assessed by T2-weighted MRI, on day 7 compared with day 0 of treatment. (B) Representative MRI of each treatment group on day 0 and day 7 after treatment of indicated vehicle or drug. Hashed white line indicates tumor border. (C) Immunoblot analysis of phosphorylated ALK (phospho-ALK-Y1278), total ALK, and MYCN of tumors treated for 2 days with vehicle or PF-06463922. GAPDH was employed as loading control. Arrow indicates pALK-Y1278. (D) Quantification of band intensity of MYCN relative to GAPDH (left) and ratio of band intensity of pALK over total ALK (right) comparing vehicle versus treated samples. (E) Meso Scale Discovery assay depicted as electrochemiluminescence signal of treated samples relative to the respective vehicle controls for total ALK, phospho-ALK-Y1568, and the ratio of pALK to ALK. (F) Representative H&E (top) and immunohistochemical images for Ki67 (bottom) of vehicle and treated samples. Quantification of overall percentage of area positive for Ki67 (right). Error bars represent s.d. between individual animals (per group: n=4 in A, n=5 in C-F). P-values equal unpaired t-test comparison between vehicle and treatment groups.

Article Snippet: Primary antibodies were: phospho-ALK (Y1604 (1:2000; 3341) and Y1278 (1:2000; 12127), phospho-ERK1/2, actin (1:2000; 4967), phospho-Akt (1:2000; 92711), MYCN (1:2000; 9405), Ki67 (1:100; 9129), GADPH and phospho-ERK5 (1:1000; 3371) from Cell Signaling Technology (Danvers, MA).

Techniques: Western Blot, Control, Electrochemiluminescence, Immunohistochemical staining, Comparison

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