Journal: Cellular and molecular gastroenterology and hepatology

Article Title: Additive Effect of CD73 Inhibitor in Colorectal Cancer Treatment With CDK4/6 Inhibitor Through Regulation of PD-L1.

doi: 10.1016/j.jcmgh.2022.07.005

Figure Lengend Snippet: Figure 8. Negative corre- lation between ecto- enzymes and CCND1 expression in CRC. (A) IHC staining of human normal colon (n ¼ 40) and colon adenocarcinoma (n ¼ 64) specimens in a tissue microarray for CCND1 and CD163 (a macrophage marker). (B) Relative intensities of CCND1 staining in the lamina propria of a normal and cancerous colon. **P < .01 (unpaired t test). (C) Correlation analysis of NT5E and CCND1 expres- sion in the TCGA colorectal adenocarcinoma dataset. ***P < .001 (Student t test).

Article Snippet: For the fluorescence-activated cell sorting analysis, cells were stained with APC,-anti human CD163 (clone: GHI/61), PD-anti human CTLA4 (clone: L3D10), Alexa 647-anti human IDO1 (clone: 2E2/IDO1), PE-anti human DR4 (clone: DJR1), FITC-anti human CD47 (clone: REA220, FITC-anti human MICA&B (clone: 6D4), PE-anti human PD-L1 (clone: MIH2), PD-anti human CD69 (clone: FNM50), FITCanti human CD2 (clone: RPA-2.10), FITC-anti human CD20 (clone: 2H7), APC-anti mouse F4/80 (clone: BM8), Brilliant Violet 421TM-anti mouse CD11b (clone: M1/70), Brilliant Violet 570TM-anti mouse CD45 (clone: 104), PE-anti-human CD45 (clone: HI30), monoclonal antibodies (Biolegend; San Diego, CA, USA), FITC-anti-human CD40 (clone: REA733), PE-anti human CD80 (clone: 2D10), PE/vio770-anti human CD206 (clone: DCN228), PE-anti human CD62E (clone: REA280), PE-anti human CD192 (clone: REA264), APC-anti human I-CAM (clone: REA266), APC-anti human HLA-DR, DP, DQ (clone: REA332), APC-anti human CCL2 (clone: REA485), APC-anti human CD14 (clone: HI30), Vioblue-anti human CD31 (clone: TUK4), FTIC-anti-human CD86 (clone: FM95), PE-anti human CD73 (clone: AD2), PE-anti mouse PD-L1 (clone: 60533), PE-anti mouse CD39, and PE-anti human CD39 (clone: REA739) monoclonal antibodies (Miltenyi Biotec; Bergisch Gladbach, Germany), and PE/CF594anti human CD3 (clone: UCHT1), PE-anti human CD44 (clone: G44-26), PE-anti human CD183 (clone: 150503), and PD-1 (clone: REA739) monoclonal antibodies (BD Biosciences; San Jose, CA, USA) following the manufacturer’s protocol.

Techniques: Expressing, Immunohistochemistry, Microarray, Marker, Staining

Journal: Cancer cell

Article Title: Tinagl1 Suppresses Triple-Negative Breast Cancer Progression and Metastasis by Simultaneously Inhibiting Integrin/FAK and EGFR Signaling.

doi: 10.1016/j.ccell.2018.11.016

Figure Lengend Snippet: Figure 4. Identification of Tinagl1-Interacting Proteins (A–C) LM2 cells expressing the C-terminal HA-tagged Tinagl1 (Tinagl1-HA) were lysed and immunoprecipitated (IP) with immunoglobulin G (IgG) (control) or anti- HA antibody. The IP samples were subjected to silver staining and WB (A) before mass spectrometry analysis. Tinagl1-interacting partners were clustered with KEGG pathway analysis, and the three top pathways are shown in (B). The members of the top three pathways have overlaps. The EGFR and integrin b1 subunits are the core members of each pathways (C). (D and E) LM2 cells stably expressing Tinagl1-HA were lysed and IP with IgG or anti-HA antibodies. The IP samples were subjected to WB analysis with the indicated antibodies to detect the interaction with EGFR and the integrin b1 subunit (D), and with integrins av, or a5 subunits (E). See also Figure S4.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Tinagl1, in 1:1000 (WB), 2 mg (IP), Rabbit ProteinTech Cat#12077-1-AP; RRID: AB_2058942 Tinagl1, in 1:100 (IHC), Rabbit Sigma-Aldrich Cat#HPA048695; RRID: AB_2680497 b-actin, in 1:10,000 (WB), mouse Abcam Cat#ab6276; RRID: AB_2223210 EGFR, in 1:1000 (WB), Rabbit Cell Signaling Technology Cat#4267; RRID: AB_2246311 p-EGFR (Try1068), in 1:1000 (WB), 1:100 (IHC), Rabbit Cell Signaling Technology Cat#3777; RRID: AB_2096270 FAK, in 1:1000 (WB), Rabbit Cell Signaling Technology Cat#3285; RRID: AB_10694068 p-FAK (Try397), in 1:1000 (WB), 1:100 (IHC), Rabbit Cell Signaling Technology Cat#8556; RRID: AB_10891442 p-FAK (Try925), in 1:1000 (WB), Rabbit Cell Signaling Technology Cat#3284; RRID: AB_2253227 AKT, in 1:1000 (WB), Rabbit Cell Signaling Technology Cat#4691; RRID: AB_915783 p-ATK (S473), in 1:1000 (WB), Rabbit Cell Signaling Technology Cat#4060; RRID: AB_2315049 ERK1/2, in 1:1000 (WB), Rabbit Cell Signaling Technology Cat#4695; RRID: AB_390779 p-ERK1/2 (Thr202/Tyr204), in 1:1000 (WB), Rabbit Cell Signaling Technology Cat#4370; RRID: AB_2315112 Integrin b1 subunit, in 1:1000 (WB), 2 mg (IP), Rabbit Cell Signaling Technology Cat#34971 Integrin a5 subunit, in 1:1000 (WB), 2 mg (IP), Rabbit Cell Signaling Technology Cat#4705; RRID: AB_10827978 Integrin av subunit, in 1:1000 (WB), 2 mg (IP), Rabbit Abcam Cat#ab179475; RRID: AB_2716738 Integrin a3 subunit, in 1:1000 (WB), Rabbit Abcam Cat#ab190731 Integrin a4 subunit, in 1:1000 (WB), Rabbit Cell Signaling Technology Cat#8440P Integrin aM subunit, in 1:1000 (WB), Rabbit Abcam Cat#ab8878; RRID: AB_306831 HA, in 2 mg (IP), Mouse Santa Cruz Biotechnology Cat#sc-7392; RRID: AB_627809 HA, in 1:1000 (WB), Rat Roche, 11867423001 Cat#11867423001; RRID: AB_10094468 MYC, in 1:1000 (WB), 2 mg (IP), Mouse Santa Cruz Biotechnology Cat#sc-40; RRID: AB_627268 FLAG, in 1:1000 (WB), 2 mg (IP), Mouse Sigma-Aldrich Cat#F7425; RRID: AB_439687 His, in 1:1000 (WB), Mouse Sigma-Aldrich Cat#H1029; RRID: AB_260015 GFP, in 1:1000 (WB), Mouse Santa Cruz Biotechnology Cat#sc-9996; RRID: AB_627695 Fibronectin, in 1:1000 (WB), Rabbit ProteinTech Cat#15613-1-AP; RRID: AB_2105691 EGF, in 1:1000 (WB), Mouse Santa Cruz Biotechnology Cat#sc-275; RRID: AB_631417 (Continued on next page) e1 Cancer Cell 35, 1–17.e1–e7, January 14, 2019

Techniques: Expressing, Immunoprecipitation, Control, Silver Staining, Mass Spectrometry, Stable Transfection

Journal: Cancer cell

Article Title: Tinagl1 Suppresses Triple-Negative Breast Cancer Progression and Metastasis by Simultaneously Inhibiting Integrin/FAK and EGFR Signaling.

doi: 10.1016/j.ccell.2018.11.016

Figure Lengend Snippet: Figure 5. Tinagl1 Inhibits integrin/FAK and EGFR Signaling Pathways (A) Gene set enrichment analysis of lung metastatic nodules formed by LM2 cells stably expressing the vector control or Tinagl1 were dissected and digested. n = 3 per group. (B) Heatmap representation of microarray data displaying the expression of EGFR or integrin/FAK regulated genes in the control versus Tinagl1-expressing LM2 cells. (C) Heatmap representation of microarray data displaying the expression of genes compensated by integrin/FAK (left) or EGFR (right) in control versus Tinagl1- expressing LM2 cells.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Tinagl1, in 1:1000 (WB), 2 mg (IP), Rabbit ProteinTech Cat#12077-1-AP; RRID: AB_2058942 Tinagl1, in 1:100 (IHC), Rabbit Sigma-Aldrich Cat#HPA048695; RRID: AB_2680497 b-actin, in 1:10,000 (WB), mouse Abcam Cat#ab6276; RRID: AB_2223210 EGFR, in 1:1000 (WB), Rabbit Cell Signaling Technology Cat#4267; RRID: AB_2246311 p-EGFR (Try1068), in 1:1000 (WB), 1:100 (IHC), Rabbit Cell Signaling Technology Cat#3777; RRID: AB_2096270 FAK, in 1:1000 (WB), Rabbit Cell Signaling Technology Cat#3285; RRID: AB_10694068 p-FAK (Try397), in 1:1000 (WB), 1:100 (IHC), Rabbit Cell Signaling Technology Cat#8556; RRID: AB_10891442 p-FAK (Try925), in 1:1000 (WB), Rabbit Cell Signaling Technology Cat#3284; RRID: AB_2253227 AKT, in 1:1000 (WB), Rabbit Cell Signaling Technology Cat#4691; RRID: AB_915783 p-ATK (S473), in 1:1000 (WB), Rabbit Cell Signaling Technology Cat#4060; RRID: AB_2315049 ERK1/2, in 1:1000 (WB), Rabbit Cell Signaling Technology Cat#4695; RRID: AB_390779 p-ERK1/2 (Thr202/Tyr204), in 1:1000 (WB), Rabbit Cell Signaling Technology Cat#4370; RRID: AB_2315112 Integrin b1 subunit, in 1:1000 (WB), 2 mg (IP), Rabbit Cell Signaling Technology Cat#34971 Integrin a5 subunit, in 1:1000 (WB), 2 mg (IP), Rabbit Cell Signaling Technology Cat#4705; RRID: AB_10827978 Integrin av subunit, in 1:1000 (WB), 2 mg (IP), Rabbit Abcam Cat#ab179475; RRID: AB_2716738 Integrin a3 subunit, in 1:1000 (WB), Rabbit Abcam Cat#ab190731 Integrin a4 subunit, in 1:1000 (WB), Rabbit Cell Signaling Technology Cat#8440P Integrin aM subunit, in 1:1000 (WB), Rabbit Abcam Cat#ab8878; RRID: AB_306831 HA, in 2 mg (IP), Mouse Santa Cruz Biotechnology Cat#sc-7392; RRID: AB_627809 HA, in 1:1000 (WB), Rat Roche, 11867423001 Cat#11867423001; RRID: AB_10094468 MYC, in 1:1000 (WB), 2 mg (IP), Mouse Santa Cruz Biotechnology Cat#sc-40; RRID: AB_627268 FLAG, in 1:1000 (WB), 2 mg (IP), Mouse Sigma-Aldrich Cat#F7425; RRID: AB_439687 His, in 1:1000 (WB), Mouse Sigma-Aldrich Cat#H1029; RRID: AB_260015 GFP, in 1:1000 (WB), Mouse Santa Cruz Biotechnology Cat#sc-9996; RRID: AB_627695 Fibronectin, in 1:1000 (WB), Rabbit ProteinTech Cat#15613-1-AP; RRID: AB_2105691 EGF, in 1:1000 (WB), Mouse Santa Cruz Biotechnology Cat#sc-275; RRID: AB_631417 (Continued on next page) e1 Cancer Cell 35, 1–17.e1–e7, January 14, 2019

Techniques: Protein-Protein interactions, Stable Transfection, Expressing, Plasmid Preparation, Control, Microarray

Journal: Cancer cell

Article Title: Tinagl1 Suppresses Triple-Negative Breast Cancer Progression and Metastasis by Simultaneously Inhibiting Integrin/FAK and EGFR Signaling.

doi: 10.1016/j.ccell.2018.11.016

Figure Lengend Snippet: Figure 6. Tinagl1 Inhibits EGFR Dimerization and Blocks the Interaction between the Integrin b1 Subunit and FN (A) LM2 cells were transfected with plasmids to overexpress GFP-EGFR and EGFR-Myc. 48 hr after transfection, the cells were treated with or without 1 mg/mL of r-Tinagl1 for 1 hr, followed by 10 min of 1 ng/mL EGF treatment. The cells were then collected and immunoprecipitated with either IgG or anti-Myc antibody. IP samples were subjected to WB analysis (top), and the amount of EGFR-GFP that interacts with EGFR-Myc was quantified and normalized to the PBS treatment group (bottom). (B) LM2 cells stably expressing EGFR-Myc were pre-treated with PBS or 1 mg/mL of r-Tinagl1 for 1 hr and then treated with 1 ng/mL of EGF for another 10 min. Next, the cells were collected and the dimers were crosslinked with disuccinimidyl suberate (DSS) treatment, followed by WB analysis (top) and quantification of the ratio of dimerized EGFR in each treatment group (bottom). (C) HEK293T cells overexpressing the integrin b1 subunit were lysed. 20 mg of FN was added into the lysate, and the lysate was divided into eight groups. PBS or the indicated amount of proteins were added into each group followed by IP with IgG or anti-b1 antibody. The IP samples were then subjected to WB analysis. (D) HEK293T cells overexpressing both integrin b1 subunit and Tinagl1-HA were lysed and divided into eight groups. PBS or the indicated amount of proteins were added into the lysate, followed by IP and WB analysis.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Tinagl1, in 1:1000 (WB), 2 mg (IP), Rabbit ProteinTech Cat#12077-1-AP; RRID: AB_2058942 Tinagl1, in 1:100 (IHC), Rabbit Sigma-Aldrich Cat#HPA048695; RRID: AB_2680497 b-actin, in 1:10,000 (WB), mouse Abcam Cat#ab6276; RRID: AB_2223210 EGFR, in 1:1000 (WB), Rabbit Cell Signaling Technology Cat#4267; RRID: AB_2246311 p-EGFR (Try1068), in 1:1000 (WB), 1:100 (IHC), Rabbit Cell Signaling Technology Cat#3777; RRID: AB_2096270 FAK, in 1:1000 (WB), Rabbit Cell Signaling Technology Cat#3285; RRID: AB_10694068 p-FAK (Try397), in 1:1000 (WB), 1:100 (IHC), Rabbit Cell Signaling Technology Cat#8556; RRID: AB_10891442 p-FAK (Try925), in 1:1000 (WB), Rabbit Cell Signaling Technology Cat#3284; RRID: AB_2253227 AKT, in 1:1000 (WB), Rabbit Cell Signaling Technology Cat#4691; RRID: AB_915783 p-ATK (S473), in 1:1000 (WB), Rabbit Cell Signaling Technology Cat#4060; RRID: AB_2315049 ERK1/2, in 1:1000 (WB), Rabbit Cell Signaling Technology Cat#4695; RRID: AB_390779 p-ERK1/2 (Thr202/Tyr204), in 1:1000 (WB), Rabbit Cell Signaling Technology Cat#4370; RRID: AB_2315112 Integrin b1 subunit, in 1:1000 (WB), 2 mg (IP), Rabbit Cell Signaling Technology Cat#34971 Integrin a5 subunit, in 1:1000 (WB), 2 mg (IP), Rabbit Cell Signaling Technology Cat#4705; RRID: AB_10827978 Integrin av subunit, in 1:1000 (WB), 2 mg (IP), Rabbit Abcam Cat#ab179475; RRID: AB_2716738 Integrin a3 subunit, in 1:1000 (WB), Rabbit Abcam Cat#ab190731 Integrin a4 subunit, in 1:1000 (WB), Rabbit Cell Signaling Technology Cat#8440P Integrin aM subunit, in 1:1000 (WB), Rabbit Abcam Cat#ab8878; RRID: AB_306831 HA, in 2 mg (IP), Mouse Santa Cruz Biotechnology Cat#sc-7392; RRID: AB_627809 HA, in 1:1000 (WB), Rat Roche, 11867423001 Cat#11867423001; RRID: AB_10094468 MYC, in 1:1000 (WB), 2 mg (IP), Mouse Santa Cruz Biotechnology Cat#sc-40; RRID: AB_627268 FLAG, in 1:1000 (WB), 2 mg (IP), Mouse Sigma-Aldrich Cat#F7425; RRID: AB_439687 His, in 1:1000 (WB), Mouse Sigma-Aldrich Cat#H1029; RRID: AB_260015 GFP, in 1:1000 (WB), Mouse Santa Cruz Biotechnology Cat#sc-9996; RRID: AB_627695 Fibronectin, in 1:1000 (WB), Rabbit ProteinTech Cat#15613-1-AP; RRID: AB_2105691 EGF, in 1:1000 (WB), Mouse Santa Cruz Biotechnology Cat#sc-275; RRID: AB_631417 (Continued on next page) e1 Cancer Cell 35, 1–17.e1–e7, January 14, 2019

Techniques: Transfection, Immunoprecipitation, Stable Transfection, Expressing

Journal: Cancer cell

Article Title: Tinagl1 Suppresses Triple-Negative Breast Cancer Progression and Metastasis by Simultaneously Inhibiting Integrin/FAK and EGFR Signaling.

doi: 10.1016/j.ccell.2018.11.016

Figure Lengend Snippet: Figure 7. Tinagl1 Inhibits TNBC Progression by Simultaneously Targeting the Integrin/FAK and EGFR Signaling Pathways (A) 104 LM2 cells was injected into the MFP of NSG mice. Mice were intravenously treated with the indicated reagents twice per week when tumors reached 2 mm in diameter. n = 6 mice per group. (B) WB analysis for the activation of EGFR and FAK in primary tumor of each group after 5 weeks of the treatments as in (A). (C) Quantification of tumor volumes of each treatment group of (A). n = 12 tumors per group. (D and E) Lungs were collected and spontaneous metastasis was examined by ex vivo BLI at the endpoint. n = 6 lungs per group. Representative lungs (D) and quantitative data (E) is shown. Data represent means ± SEM. n.s., not significant; p > 0.05, **p < 0.001, ***p < 0.0001. Significance determined by one-way ANOVA analysis with Dunnett’s test for multiple comparisons. See also Figure S7.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Tinagl1, in 1:1000 (WB), 2 mg (IP), Rabbit ProteinTech Cat#12077-1-AP; RRID: AB_2058942 Tinagl1, in 1:100 (IHC), Rabbit Sigma-Aldrich Cat#HPA048695; RRID: AB_2680497 b-actin, in 1:10,000 (WB), mouse Abcam Cat#ab6276; RRID: AB_2223210 EGFR, in 1:1000 (WB), Rabbit Cell Signaling Technology Cat#4267; RRID: AB_2246311 p-EGFR (Try1068), in 1:1000 (WB), 1:100 (IHC), Rabbit Cell Signaling Technology Cat#3777; RRID: AB_2096270 FAK, in 1:1000 (WB), Rabbit Cell Signaling Technology Cat#3285; RRID: AB_10694068 p-FAK (Try397), in 1:1000 (WB), 1:100 (IHC), Rabbit Cell Signaling Technology Cat#8556; RRID: AB_10891442 p-FAK (Try925), in 1:1000 (WB), Rabbit Cell Signaling Technology Cat#3284; RRID: AB_2253227 AKT, in 1:1000 (WB), Rabbit Cell Signaling Technology Cat#4691; RRID: AB_915783 p-ATK (S473), in 1:1000 (WB), Rabbit Cell Signaling Technology Cat#4060; RRID: AB_2315049 ERK1/2, in 1:1000 (WB), Rabbit Cell Signaling Technology Cat#4695; RRID: AB_390779 p-ERK1/2 (Thr202/Tyr204), in 1:1000 (WB), Rabbit Cell Signaling Technology Cat#4370; RRID: AB_2315112 Integrin b1 subunit, in 1:1000 (WB), 2 mg (IP), Rabbit Cell Signaling Technology Cat#34971 Integrin a5 subunit, in 1:1000 (WB), 2 mg (IP), Rabbit Cell Signaling Technology Cat#4705; RRID: AB_10827978 Integrin av subunit, in 1:1000 (WB), 2 mg (IP), Rabbit Abcam Cat#ab179475; RRID: AB_2716738 Integrin a3 subunit, in 1:1000 (WB), Rabbit Abcam Cat#ab190731 Integrin a4 subunit, in 1:1000 (WB), Rabbit Cell Signaling Technology Cat#8440P Integrin aM subunit, in 1:1000 (WB), Rabbit Abcam Cat#ab8878; RRID: AB_306831 HA, in 2 mg (IP), Mouse Santa Cruz Biotechnology Cat#sc-7392; RRID: AB_627809 HA, in 1:1000 (WB), Rat Roche, 11867423001 Cat#11867423001; RRID: AB_10094468 MYC, in 1:1000 (WB), 2 mg (IP), Mouse Santa Cruz Biotechnology Cat#sc-40; RRID: AB_627268 FLAG, in 1:1000 (WB), 2 mg (IP), Mouse Sigma-Aldrich Cat#F7425; RRID: AB_439687 His, in 1:1000 (WB), Mouse Sigma-Aldrich Cat#H1029; RRID: AB_260015 GFP, in 1:1000 (WB), Mouse Santa Cruz Biotechnology Cat#sc-9996; RRID: AB_627695 Fibronectin, in 1:1000 (WB), Rabbit ProteinTech Cat#15613-1-AP; RRID: AB_2105691 EGF, in 1:1000 (WB), Mouse Santa Cruz Biotechnology Cat#sc-275; RRID: AB_631417 (Continued on next page) e1 Cancer Cell 35, 1–17.e1–e7, January 14, 2019

Techniques: Protein-Protein interactions, Injection, Activation Assay, Ex Vivo

Journal: The International journal of biological markers

Article Title: Cytoplasmic levels of high mobility group A2 determine survival prognoses in breast cancer patients.

doi: 10.1177/1724600820917990

Figure Lengend Snippet: Figure 2. Expression patterns of HMGA2 divided by subcellular localization and correlation with breast cancer pathological features and intrinsic subtypes. Correlation coefficients r for positive and negative correlations (0–0.16). HMGA: high mobility group A. *P < 0.05, **P < 0.01, ***P < 0.001.

Article Snippet: Immunohistochemistry was performed utilizing a monoclonal rabbit antibody directed against HMGA2 (dilution 1:200, D1A7, Cell Signaling, Leiden, Netherlands, [#8179]) using the autostainer BondTM Max System (Leica Microsystems GmbH, Wetzlar, Germany).

Techniques: Expressing

Journal: The International journal of biological markers

Article Title: Cytoplasmic levels of high mobility group A2 determine survival prognoses in breast cancer patients.

doi: 10.1177/1724600820917990

Figure Lengend Snippet: Figure 1. Representative staining for nuclear and cytoplasmic HMGA2 levels (score 0–3) in primary tumor specimens from the breast cancer tissue micro array. The addition of nuclear and cytoplasmic HMGA2 scores resulted in the overall score for each tumor specimen (score 0–6). The black scale bars indicate 100 µm. The dashed tumor area in each picture is presented in 7.5-fold magnification (400x). HMGA: high mobility group A.

Article Snippet: Immunohistochemistry was performed utilizing a monoclonal rabbit antibody directed against HMGA2 (dilution 1:200, D1A7, Cell Signaling, Leiden, Netherlands, [#8179]) using the autostainer BondTM Max System (Leica Microsystems GmbH, Wetzlar, Germany).

Techniques: Staining, Microarray

Journal: The International journal of biological markers

Article Title: Cytoplasmic levels of high mobility group A2 determine survival prognoses in breast cancer patients.

doi: 10.1177/1724600820917990

Figure Lengend Snippet: Figure 3. (a)Combined survival analyses (OS) in 342 breast cancer patients in our collective using Kaplan–Meier estimates. (b) OS in the breast cancer collective divided by subcellular levels of HMGA2: Overall and cytoplasmic, but not nuclear, HMGA2 correlated with better survival. Green curves: high HMGA2 scores. Blue curves: low HMGA2 scores. Vertical bars indicate censored patients. (c) Kaplan–Meier curves for all five intrinsic subtypes in our collective of 342 breast cancer patients. Patients of the HER2-positive subgroups did not receive HER2-targeted therapies routinely. (d) Cox regression models showing hazard ratios (HR) of subcellular HMGA2 distribution divided by intrinsic subtypes. Only in Luminal A and TNBC subtypes, high cytoplasmic HMGA2 scores led to better survival prognoses. HMGA: high mobility group A; OS: overall survival; TNBC: triple negative breast cancer.

Article Snippet: Immunohistochemistry was performed utilizing a monoclonal rabbit antibody directed against HMGA2 (dilution 1:200, D1A7, Cell Signaling, Leiden, Netherlands, [#8179]) using the autostainer BondTM Max System (Leica Microsystems GmbH, Wetzlar, Germany).

Techniques:

Journal: The International journal of biological markers

Article Title: Cytoplasmic levels of high mobility group A2 determine survival prognoses in breast cancer patients.

doi: 10.1177/1724600820917990

Figure Lengend Snippet: Figure 4. HMGA2 scores and OS: two-step hierarchical Cox regression model for conventional breast cancer risk factors and surrogate markers incorporating subcellular HMGA2 levels. Incorporating overall and cytoplasmic—but not nuclear—HMGA2- based models allowed more accurate risk stratification in our collective (P < 0.001). HMGA: high mobility group A; OS: overall survival.

Article Snippet: Immunohistochemistry was performed utilizing a monoclonal rabbit antibody directed against HMGA2 (dilution 1:200, D1A7, Cell Signaling, Leiden, Netherlands, [#8179]) using the autostainer BondTM Max System (Leica Microsystems GmbH, Wetzlar, Germany).

Techniques:

Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: Vitamin E relieves chronic obstructive pulmonary disease by inhibiting COX2-mediated p-STAT3 nuclear translocation through the EGFR/MAPK signaling pathway.

doi: 10.1038/s41374-021-00652-z

Figure Lengend Snippet: Fig. 2 Vitamin E downregulates EGFR and inactivates MAPK signaling pathway. A Venn diagram of the intersection of drug and disease targets. B Network diagram of gene-drug-disease interaction relationship. C KEGG enrichment analysis results of 30 candidate targets. D Box diagram of EGFR expression in COPD attained through microarray dataset GSE3320, wherein blue box on the left indicates normal tissue samples and red box on the right indicates COPD samples. E EGFR expression in lung tissues of rats after CS or vitamin E treatment was detected by immunohistochemistry. F EGFR, p38, p-p38, JNK, p-JNK, ERK1/2, and p-ERK1/2 protein expression in lung tissues of rats after CS or vitamin E treatment was detected by Western blot. G Statistical diagram of Panel F. The data in the figure are measurement data, which are expressed by the mean ± standard deviation. The data of each group are analyzed by unpaired t-test, and the data among multiple groups are compared by one-way ANOVA and Tukey’s post hoc test. *p < 0.05 vs. the control group, #p < 0.05 vs. the CS group, n = 6.

Article Snippet: Then the membrane was probed with primary antibodies against COX2 (1: 1000, ab179800, Abcam), EGFR (1:2000, ab52894, Abcam), STAT3 (1:1000, ab68153, Abcam), p-STAT3 (1:1000, ab76315, Abcam), p38 (1:1000, 9212, Cell Signaling Technologies [CST], Beverly, MA,USA), p-p38 (1:1000, 9211, CST), JNK (1:2000, 9252, CST), p-JNK (1:2000, 9255, CST), ERK1/2 (1:1000, 4695, CST), p-ERK1/2 (1:1000, 4370, CST), and GAPDH (1:5000, ab8254, Abcam) overnight at 4 °C.

Techniques: Expressing, Microarray, Immunohistochemistry, Western Blot, Standard Deviation, Control

Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: Vitamin E relieves chronic obstructive pulmonary disease by inhibiting COX2-mediated p-STAT3 nuclear translocation through the EGFR/MAPK signaling pathway.

doi: 10.1038/s41374-021-00652-z

Figure Lengend Snippet: Fig. 3 Vitamin E relieves CS-induced HBE cell damage. A RT-qPCR was used to assess the expression of EGFR in HBE cells of each group. B Western blot was used to assess the expression of EGFR, p38, p-p38, JNK, p-JNK, ERK1/2, and p-ERK1/2 in HBE cells of each group. C Statistical map of Panel B. D CCK8 assay was used to detect the viability of HBE cells in each group. E Annexin V/PI staining was used to detect the apoptosis of HBE cells in each group. F ELISA was used to detect the expression of inflammatory factors in the supernatant of HBE cells of each group. G ROS level in HBE cells of each group. H The SOD activity and MDA content in HBE cells of each group. The data in the figure were all measurement data, expressed as the mean ± standard deviation. The data of each group were analyzed by unpaired t-test, and the comparison of data among multiple groups was performed by one-way ANOVA and Tukey’s post hoc test. *p < 0.05 vs. the Vector group, #p < 0.05 vs. the vector + CS group, and $p < 0.05 vs. the vector + CS + vitamin E group.

Article Snippet: Then the membrane was probed with primary antibodies against COX2 (1: 1000, ab179800, Abcam), EGFR (1:2000, ab52894, Abcam), STAT3 (1:1000, ab68153, Abcam), p-STAT3 (1:1000, ab76315, Abcam), p38 (1:1000, 9212, Cell Signaling Technologies [CST], Beverly, MA,USA), p-p38 (1:1000, 9211, CST), JNK (1:2000, 9252, CST), p-JNK (1:2000, 9255, CST), ERK1/2 (1:1000, 4695, CST), p-ERK1/2 (1:1000, 4370, CST), and GAPDH (1:5000, ab8254, Abcam) overnight at 4 °C.

Techniques: Quantitative RT-PCR, Expressing, Western Blot, CCK-8 Assay, Staining, Enzyme-linked Immunosorbent Assay, Activity Assay, Standard Deviation, Comparison, Plasmid Preparation

Journal: Integrative zoology

Article Title: cba-miR-222-3p involved in photoperiod-induced apoptosis in testes of striped hamsters by targeting TRAF7.

doi: 10.1111/1749-4877.12918

Figure Lengend Snippet: Figure 3 Validation of cba-miR-222-3p targeting TRAF7 and TRAF7 expression in the testes of striped hamsters. (a) Sequences and peak maps of cba-miR-222-3p, TRAF7-WT, and TRAF7-MT. (b) Relative luciferase activity detected by Dual-Luciferase Reporter Assay. (c) Immunohistochemistry (IHC, tissue microarray [TMA]) of TRAF7 in testes. (d) Integrated density of TRAF7 detected by IHC (TMA; n = 4). (e) Protein expression levels of TRAF7 in the testes detected by western blot (n = 4). (f) Pearson correlation analysis of cba-miR-222-3p and TRAF7. LD, long daylength; MD, moderate daylength; SD, short daylength; ∗, P < 0.05; ∗∗, P < 0.01.

Article Snippet: After performing electrophoresis, the proteins were transferred to PVDF membranes, which were then incubated with TRAF7 antibodies (11780-1-AP, Proteintech, China, RRID:AB_2877793) at a 1:1000 dilution and β-actin antibodies (20536-1-AP, Proteintech, China, RRID:AB_10700003) at a 1:1000 dilution, and subsequently incubated with IRDye 800 CW goat anti-rabbit secondary antibodies (31 460, Thermo Fisher, USA).

Techniques: Biomarker Discovery, Expressing, Luciferase, Activity Assay, Reporter Assay, Immunohistochemistry, Microarray, Western Blot

Journal: Integrative zoology

Article Title: cba-miR-222-3p involved in photoperiod-induced apoptosis in testes of striped hamsters by targeting TRAF7.

doi: 10.1111/1749-4877.12918

Figure Lengend Snippet: Figure 4 Expression of cba-miR-222-3p and TRAF7 and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) analysis after in vivo injection in the testes of striped hamsters. (a) Schematic diagram of in vivo injection experiment of miRNA mimics. (b) Fluorescence in situ hybridization (FISH, tissue microarray [TMA]) of cba-miR-222-3p in testes after in vivo injection. (c) Fluorescence intensity of cba-miR-222-3p detected by FISH (TMA; n = 4). (d) Immunohistochemistry (IHC, TMA) of TRAF7 in testes after in vivo injection. (e) Integrated density of TRAF7 detected by IHC (TMA; n = 4). (f) TUNEL (TMA) staining of the testes after in vivo injection. (g) The proportion of TUNEL (TMA) staining with apoptotic activity in the testes (n = 4). (h) Relative expression of MEKK3 (n = 6). (i) Relative expression of p38 (n = 6). (j) Relative expression of p53 (n = 6). AG, agomir injection; NC, agomir negative control injection; DAPI, 4′6′-diamidino-2-phenylindole. ∗, P < 0.05; ∗∗, P < 0.01; ∗∗∗, P < 0.001.

Article Snippet: After performing electrophoresis, the proteins were transferred to PVDF membranes, which were then incubated with TRAF7 antibodies (11780-1-AP, Proteintech, China, RRID:AB_2877793) at a 1:1000 dilution and β-actin antibodies (20536-1-AP, Proteintech, China, RRID:AB_10700003) at a 1:1000 dilution, and subsequently incubated with IRDye 800 CW goat anti-rabbit secondary antibodies (31 460, Thermo Fisher, USA).

Techniques: Expressing, TUNEL Assay, In Vivo, Injection, Fluorescence, In Situ Hybridization, Microarray, Immunohistochemistry, Staining, Activity Assay, Negative Control

Journal: Cancer cell

Article Title: TGF-β Receptor Inhibitors Target the CD44(high)/Id1(high) Glioma-Initiating Cell Population in Human Glioblastoma.

doi: 10.1016/j.ccr.2010.10.023

Figure Lengend Snippet: Figure 1. Id1 Is a Target of the TbRI Inhibitor in PCTCs and in a Patient-Derived Mouse Model for Glioma (A) The genes were regulated by the treatment with 2 mM TbRI inhibitor for 3 hr in 11 human glioma PCTCs with a fold change over 1.4 or below 0.6 and a p < 0.001. (B) ID3, ID1, Smad7, and RhoB transcript levels were determined by qRT-PCR analysis. GAPDH RNA levels were used as an internal normalization control. *p < 0.05; **p < 0.001. Data are presented as means ± SD. (C and D) Cells from GBM1 and GBM2 neuro- spheres were inoculated in the brain of immuno- compromised mice. Twenty days after surgery, mice were orally treated twice a day with 100 mg/kg of TbRI inhibitor for 30 days. (C) Tumor area was quantified. (D) Images from the entire mouse brains were obtained by MRI (arrowheads indicate tumors). (E) Immunohistochemistry for Id1 (upper panel) and double immunofluorescence for Id1 and CD31 (middle panel) were performed in tumors generated by GBM neurospheres. For immunoflu- orescence, nuclei were counterstained with Hoechst. Scale bar, 100 mm. Representative images are shown and percentage of Id1-positive cells was calculated (lower panel). Data are pre- sented as means ± SD. (F) Cells from GBM1 neurospheres were inocu- lated in the brain of immunocompromised mice. Forty days after surgery, mice were orally treated twice a day with 100 mg/kg of TbRI inhibitor for 10 days. Subsequently, 10 mm slides from mouse brains were stained with H&E and tumors were localized and microdissected. ID1 tran- script levels were determined by qRT-PCR (n = 4 for vehicle; n = 3 for TbRI inhibitor). Data are presented as means ± SD. See also Fig- ure S1.

Article Snippet: Specific antibodies against p-Smad2, Smad2 (Cell Signaling), a-Tubulin (Sigma), Id1 (C20; Santa Cruz Biotechnology), Id3 (Biocheck), and lamin A/C and ATF3 (Santa Cruz Biotechnology) were used for immunoblotting.

Techniques: Derivative Assay, Quantitative RT-PCR, Control, Immunohistochemistry, Generated, Staining

Journal: Cancer cell

Article Title: TGF-β Receptor Inhibitors Target the CD44(high)/Id1(high) Glioma-Initiating Cell Population in Human Glioblastoma.

doi: 10.1016/j.ccr.2010.10.023

Figure Lengend Snippet: Figure 4. Patient-Derived Neurospheres Have a CD44high/Id1high Cell Population (A) Cells from GBM1 neurospheres were dissociated and seeded on coverslips. Immunocytochemistry for Id1and CD44 was performed and nuclei were counter- stained with Hoechst. Scale bar, 10 mm. Quantification of Id1 and CD44 expression levels per cell is shown. (B) FACS analysis of CD44 levels was performed in GBM neurospheres (upper panel). Isotype control is shown. Cells with high or low levels of CD44 from the indicated GBM neurospheres were sorted by FACS and CD44, ID1, ID2, and ID3 transcript levels were determined by qRT-PCR (lower panels). *p < 0.01; **p < 0.001 compared to CD44low. Data are presented as means ± SD. (C) Cells from GBM1 neurospheres were sorted by FACS according to CD44 levels, and the levels of Id1, Id3, and tubulin were determined by immunoblotting. See also Figure S4.

Article Snippet: Specific antibodies against p-Smad2, Smad2 (Cell Signaling), a-Tubulin (Sigma), Id1 (C20; Santa Cruz Biotechnology), Id3 (Biocheck), and lamin A/C and ATF3 (Santa Cruz Biotechnology) were used for immunoblotting.

Techniques: Derivative Assay, Immunocytochemistry, Staining, Expressing, Control, Quantitative RT-PCR, Western Blot

Journal: Cancer cell

Article Title: TGF-β Receptor Inhibitors Target the CD44(high)/Id1(high) Glioma-Initiating Cell Population in Human Glioblastoma.

doi: 10.1016/j.ccr.2010.10.023

Figure Lengend Snippet: Figure 6. Id Proteins Mediate the Effect of TGF-b on the CD44high Population and the Oncogenic Potential of GBM Neurospheres (A) Cells from GBM1 were infected with the indicated lentivirus. Neurospheres were treated with 100 pM TGF-b1 for 3 hr, 2 mM TbRI inhibitor for 8 hr, or left untreated, and the levels of Id1, Id3, p-Smad2, Smad2, and tubulin were determined by immunoblotting. (B) Cells from GBM1 neurospheres previously infected with the indicated lentivirus were treated with 100 pM TGF-b or 2 mM TbRI inhibitor for 7 days or left untreated. FACS analysis of CD44 levels (left panel) and quantification of the frequency of CD44high cells are shown (right panel). Bars represent the percent of CD44high cells in untreated, TGF-b-treated, and TbRI inhibitor-treated lentiviral-infected neurospheres, as indicated. Data are presented as means. (C–E) Cells from GBM1 neurospheres previously infected with the indicated lentivirus were treated for 7 days with 2 mM TbRI inhibitor, or left untreated. Subse- quently, cells were dissociated, counted, and equal numbers of cells were inoculated in the brain of NOD-SCID mice. (C) Images from the entire mouse brains were obtained by MRI. Arrowheads indicate tumors. (D) Tumor area was quantified (p = 0.004 comparing mice inoculated with untreated neurospheres with mice inoculated with neurospheres treated with the TbRI inhibitor; p = 0.002 comparing mice inoculated with neurospheres infected with the lentivirus control with mice inoculated with neurospheres infected with the sh2 ID1/ID3). (E) Tumor incidence was determined. Data are presented as means ± SD. (F–H) Cells from GBM1 neurospheres previously infected with the indicated lentivirus were treated during 7 days with 2 mM TbRI inhibitor or left untreated. Subse- quently, cells were dissociated, counted, and equal numbers of cells were inoculated in the brain of immunocompromised mice. (F) Images from the entire mouse brains were obtained by MRI. Arrowheads indicate tumors. (G) Tumor area was quantified (p = 0.04 comparing mice inoculated with control neurospheres with mice inoculated with Id1 overexpressing neurospheres), and (H) tumor incidence was determined. Data are presented as means ± SD. See also Figure S6.

Article Snippet: Specific antibodies against p-Smad2, Smad2 (Cell Signaling), a-Tubulin (Sigma), Id1 (C20; Santa Cruz Biotechnology), Id3 (Biocheck), and lamin A/C and ATF3 (Santa Cruz Biotechnology) were used for immunoblotting.

Techniques: Infection, Western Blot, Control

Journal: Cancer cell

Article Title: TGF-β Receptor Inhibitors Target the CD44(high)/Id1(high) Glioma-Initiating Cell Population in Human Glioblastoma.

doi: 10.1016/j.ccr.2010.10.023

Figure Lengend Snippet: Figure 7. TbRI Inhibitor Decreases Id1, Id3, and CD44 Levels in Tumors and Prevents Tumor Recurrence in Vivo (A) Scheme showing the experimental procedure. (B and C) Cells from GBM1 neurospheres were inoculated in the brain of immunocompromised mice. Forty days after surgery, mice were orally treated twice a day with 100 mg/kg of TbRI inhibitor for 10 days. Subsequently, human tumor cells were isolated from the brain of mice treated or untreated with TbRI inhibitor through sorting of human MHC-I-positive cells. (B) ID1, ID3, and CD44 transcripts levels were determined by qRT-PCR, and (C) CD44 levels were assessed by FACS. Isotype control is shown. Data are presented as means ± SD. (D and E) Human tumor cells obtained from mice treated or untreated with the TbRI inhibitor were inoculated in the brain of secondary mice. (D) Thirty days after surgery, images from the entire mouse brains were obtained by MRI, tumor area was quantified (n = 4 mice inoculated with cells from untreated mice; n = 8 mice inoculated with cells from TbRI inhibitor treated mice), and (E) tumor incidence was determined. Data are presented as means ± SD.

Article Snippet: Specific antibodies against p-Smad2, Smad2 (Cell Signaling), a-Tubulin (Sigma), Id1 (C20; Santa Cruz Biotechnology), Id3 (Biocheck), and lamin A/C and ATF3 (Santa Cruz Biotechnology) were used for immunoblotting.

Techniques: In Vivo, Isolation, Quantitative RT-PCR, Control

Journal: Cancer cell

Article Title: TGF-β Receptor Inhibitors Target the CD44(high)/Id1(high) Glioma-Initiating Cell Population in Human Glioblastoma.

doi: 10.1016/j.ccr.2010.10.023

Figure Lengend Snippet: Figure 8. Id1 and CD44 Coexpression Correlates with Overall Survival in GBM Patients (A) A tissue microarray of 43 different GBM patients was stained with an antibody against Id1 and the frequency of Id1-positive nuclei was calculated. (B) CD44/Id1 and CD31/Id3 double immunofluorescence of samples from GBM patients. Magnification of the indicated areas stained with Id1/CD44 and Id3/ CD31 are shown in the right panels. Nuclei were counterstained with Hoechst. Scale bar, 50 mm. (C) CD44, Id1, and CD31 immunohistochemistry of samples from GBM patients. Scale bar, 100 mm. (D) Kaplan-Meier curves showing that the overall survival of patients with both ID1 mRNA levels upregulated R3-fold and CD44 mRNA levels upregulated R10-fold is significantly lower than the rest of the patients (p = 0.03) by log-rank test. Data obtained from the Repository for Molecular Brain Neoplasia Data (REMBRANDT) program form the National Cancer Institute. See also Figure S7.

Article Snippet: Specific antibodies against p-Smad2, Smad2 (Cell Signaling), a-Tubulin (Sigma), Id1 (C20; Santa Cruz Biotechnology), Id3 (Biocheck), and lamin A/C and ATF3 (Santa Cruz Biotechnology) were used for immunoblotting.

Techniques: Microarray, Staining, Immunohistochemistry

Journal: Nature immunology

Article Title: Chronic signaling via the metabolic checkpoint kinase mTORC1 induces macrophage granuloma formation and marks sarcoidosis progression

doi: 10.1038/ni.3655

Figure Lengend Snippet: ( a , c ) Lung sections evaluated by H&E staining of Tsc2 fl/fl and Tsc2 fl/fl Lyz2 -Cre mice at the age of ( a ) 3 and ( c ) 6 months. ( b ) Immunofluorescence for Mac-2 and p-S6 in lung sections of 3 month-old mice. Boxed region shows p-S6 staining only. ( d ) Image of a Tsc2 fl/fl Lyz2 -Cre mouse at the age of 6 months (left) and immunohistochemistry of Mac-2 of a paw (right). ( e ) Immunofluorescence for Mac-2 and F4/80 in paw sections of 6 month-old Tsc2 fl/fl Lyz2 -Cre mice. ( f ) Flow cytometric scatterplot of the G1 population of Tsc2 fl/fl and Tsc2 fl/fl Lyz2 -Cre peritoneal macrophages. ( g ) Tsc2 fl/fl and Tsc2 fl/fl Lyz2 -Cre bone marrow was injected into irradiated wild-type recipient mice and evaluated after 3 months by histology. Left panel: H&E staining of lung sections. Right panel: IHC of Mac-2 in liver. ( h ) Flow cytometry analysis of hypertrophic macrophages in the lungs of Tsc2 fl/fl (n=5) and Tsc2 fl/fl Lyz2 -Cre (n= 4) mice. Shown are means. ( i ) Expression of the indicated mRNAs in the lungs of 6 month-old mice. Shown are means ± SE (n= 5). *p < 0.05, **p < 0.01, ***p < 0.001; n.s., not significant (Student's t test). Representative histological images or plots are from one mouse; each analysis was performed on three ( b , d , e , g ) or five ( a , c,f ) mice per genotype. Numerical data are representative of two independent ( h ) or cumulative of two independent ( i ) experiments. Scale bars, 100 μm ( a , c , e , g ), 10 μm ( b ), 1 mm ( d ).

Article Snippet: Primary antibodies were TSC2 (D93F12) 1:1000, p-Akt S473 (D9E) 1:1000, 4E-BP1 (53H11) 1:1000, p-S6 S240/244 (rabbit polyclonal) 1:3000, S6 (54D2) 1:1000, p-Akt T308 (D25E6) 1:1000, pan-Akt (40D4) 1:1000, β-tubulin (9F3) 1:1000 , p-RB Ser807/811 (D20B12) 1:1000, PDCD4 (D29C6) 1:1000, cleaved caspase 3 Asp175 (5A1E or rabbit polyclonal) 1:500, survivin (71G4B7) 1:1000 and p-S6K1 T389 (108D2) 1:1000, p-TSC2 S939 (rabbit polyclonal) 1:1000 obtained from Cell Signaling Technology.

Techniques: Staining, Immunofluorescence, Immunohistochemistry, Injection, Irradiation, Flow Cytometry, Expressing

Journal: Nature immunology

Article Title: Chronic signaling via the metabolic checkpoint kinase mTORC1 induces macrophage granuloma formation and marks sarcoidosis progression

doi: 10.1038/ni.3655

Figure Lengend Snippet: ( a ) Immunoblots of CSF1-deprived Tsc2 fl/fl and Tsc2 fl/fl Lyz2 -Cre bone marrow-derived macrophages (BMDM), treated for 18 hours with 100 nM rapamycin or solvent control, hybridized with the indicated antibodies. ( b ) Surface expression of the indicated proteins on Tsc2 fl/fl (n=6) and Tsc2 fl/fl Lyz2 -Cre (n=4) BMDM after differentiation. ( c ) Left: Images of IL-4 stimulated (10 ng/ml) Tsc2 fl/fl and Tsc2 fl/fl Lyz2 -Cre BMDM treated with solvent or 100 nM rapamycin for four days. Right: Quantification of cluster formation. Clusters in three pictures per condition (4x magnification) were counted (n= 3). ( d ) Flow cytometry scatterplot of CSF1-deprived BMDM on day 7. ( e,f ) Cell cycle analysis of ( e ) BMDM by 7-AAD and EdU staining and of ( f ) peritoneal macrophages by PI staining from Tsc2 fl/fl and Tsc2 fl/fl Lyz2 -Cre mice (n= 5). ( g ) IHC of Ki-67 in lung sections of 3 month-old Tsc2 fl/fl and Tsc2 fl/fl Lyz2 -Cre mice. ( h ) Proliferation analysis of BMDM stimulated with 40 ng/ml CSF1 and treated with solvent or 100 nM rapamycin (n=4). Shown are means ± SE ( b , f,h ) or SD ( c ). *p < 0.001 (Student's t test) ( c , f ). Data are representative of three ( a , d , g,c ) or two ( e ) or cumulative from two independent experiments ( b,f,h ) Scale bar, 100 µm ( c ), 50 μm ( g ).

Article Snippet: Primary antibodies were TSC2 (D93F12) 1:1000, p-Akt S473 (D9E) 1:1000, 4E-BP1 (53H11) 1:1000, p-S6 S240/244 (rabbit polyclonal) 1:3000, S6 (54D2) 1:1000, p-Akt T308 (D25E6) 1:1000, pan-Akt (40D4) 1:1000, β-tubulin (9F3) 1:1000 , p-RB Ser807/811 (D20B12) 1:1000, PDCD4 (D29C6) 1:1000, cleaved caspase 3 Asp175 (5A1E or rabbit polyclonal) 1:500, survivin (71G4B7) 1:1000 and p-S6K1 T389 (108D2) 1:1000, p-TSC2 S939 (rabbit polyclonal) 1:1000 obtained from Cell Signaling Technology.

Techniques: Western Blot, Derivative Assay, Solvent, Control, Expressing, Flow Cytometry, Cell Cycle Assay, Staining

Journal: Nature immunology

Article Title: Chronic signaling via the metabolic checkpoint kinase mTORC1 induces macrophage granuloma formation and marks sarcoidosis progression

doi: 10.1038/ni.3655

Figure Lengend Snippet: ( a ) Scatterplot of global gene-expression profiles of CSF1-deprived Tsc2 fl/fl and Tsc2 fl/fl Lyz2 -Cre BMDM. Blue dots, 426 genes significantly higher expressed in Tsc2 fl/fl BMDM; red dots, 401 genes significantly higher expressed in Tsc2 fl/fl Lyz2 -Cre BMDM. ( b ) Gene-set enrichment analysis (GSEA) of hallmark gene sets (H.all) from the Molecular Signatures Database of the Broad Institute, showing the most significantly enriched gene sets in Tsc2 fl/fl and Tsc2 fl/fl Lyz2 -Cre BMDM and their normalized enrichment scores (NES) as well as their false discovery rates (FDR). ( c,d ) GSEA plot of the E2F targets (c) and Tnfa signaling via NFKB (d) gene signature in Tsc2 fl/fl Lyz2 -Cre BMDM relative to Tsc2 fl/fl BMDM from the analysis in (b) with members of the gene set presented in the ranked list of genes ('bar code' below) and the signal-to-noise ranking metric (bar at bottom). ( e ) GSEA of the 'KEGG_apoptosis' gene signature in Tsc2 fl/fl Lyz2 -Cre BMDM relative to Tsc2 fl/fl BMDM. ( f ) The most significantly enriched transcription factor target gene sets (C3.TFT) from the Molecular Signatures Database in unstimulated Tsc2 fl/fl and Tsc2 fl/fl Lyz2 -Cre BMDM derived by GSEA. NES, normalized enrichment score; FDR, false discovery rate. Data are from one experiment with four biological replicates per genotype obtained from two independent experiments.

Article Snippet: Primary antibodies were TSC2 (D93F12) 1:1000, p-Akt S473 (D9E) 1:1000, 4E-BP1 (53H11) 1:1000, p-S6 S240/244 (rabbit polyclonal) 1:3000, S6 (54D2) 1:1000, p-Akt T308 (D25E6) 1:1000, pan-Akt (40D4) 1:1000, β-tubulin (9F3) 1:1000 , p-RB Ser807/811 (D20B12) 1:1000, PDCD4 (D29C6) 1:1000, cleaved caspase 3 Asp175 (5A1E or rabbit polyclonal) 1:500, survivin (71G4B7) 1:1000 and p-S6K1 T389 (108D2) 1:1000, p-TSC2 S939 (rabbit polyclonal) 1:1000 obtained from Cell Signaling Technology.

Techniques: Gene Expression, Derivative Assay

Journal: Nature immunology

Article Title: Chronic signaling via the metabolic checkpoint kinase mTORC1 induces macrophage granuloma formation and marks sarcoidosis progression

doi: 10.1038/ni.3655

Figure Lengend Snippet: ( a,b,c ) BMDM were treated with 100 nM rapamycin, solvent control and 10 ng/ml CSF1 as indicated for 18 h. Whole cell lysates were analyzed by immunoblotting with the indicated antibodies. ( d ) IHC of CDK4 in lung sections of 3 month-old mice. ( e ) Left, analysis of CSF1-induced (40 ng/ml) proliferation of BMDM treated with solvent or the indicated amounts of the CKD4 inhibitor PD-0332991 (n= 4). Right, cell cycle analysis of CSF1-stimulated BMDM treated with solvent or 1 µM PD-0332991 for 18 h (n= 3). ( f,g ) Tsc2 fl/fl Lyz2 -Cre bone marrow was injected into irradiated wild-type recipient mice. After ten days mice were treated daily with PD-0332991 (n=10) or solvent control (n=10) daily for two month. ( f ) Lung (left panel) and liver (right panel) sections were evaluated by IHC of Mac-2. ( g ) Area of Mac-2 positive cells compared to total lung area of the treated mice. Two random images per animal were evaluated. ( h ) BMDM were treated with 1 µM PD-0332991 or solvent control and stimulated with 10 ng/ml CSF1 as indicated for 18 h. Whole cell lysates were analyzed by immunoblotting with the indicated antibodies. Shown are means ± SE ( e ) or SD ( g ). *p < 0.01, **p < 0.001 (Student's t test). Data are representative of three ( a , b , c , d ), one ( f,g ) or two ( h ) independent, or cumulative of two ( e ) experiments. Scale bar, 40 μm ( d ), 100 μm ( f ).

Article Snippet: Primary antibodies were TSC2 (D93F12) 1:1000, p-Akt S473 (D9E) 1:1000, 4E-BP1 (53H11) 1:1000, p-S6 S240/244 (rabbit polyclonal) 1:3000, S6 (54D2) 1:1000, p-Akt T308 (D25E6) 1:1000, pan-Akt (40D4) 1:1000, β-tubulin (9F3) 1:1000 , p-RB Ser807/811 (D20B12) 1:1000, PDCD4 (D29C6) 1:1000, cleaved caspase 3 Asp175 (5A1E or rabbit polyclonal) 1:500, survivin (71G4B7) 1:1000 and p-S6K1 T389 (108D2) 1:1000, p-TSC2 S939 (rabbit polyclonal) 1:1000 obtained from Cell Signaling Technology.

Techniques: Solvent, Control, Western Blot, Cell Cycle Assay, Injection, Irradiation

Journal: Nature immunology

Article Title: Chronic signaling via the metabolic checkpoint kinase mTORC1 induces macrophage granuloma formation and marks sarcoidosis progression

doi: 10.1038/ni.3655

Figure Lengend Snippet: ( a ) Basal extracellular acidification rate (ECAR) and oxygen consumption rate (OCR) of BMDM reported in units of mpH/min and pmol/min, respectively (n=8). ( b ) Uptake of 2-NBDG in BMDM treated with solvent control or 100 nM rapamycin for 18 h; analyzed by flow cytometry (n=4). ( c ) Uptake of 2-NBDG in peritoneal macrophages from Tsc2 fl/fl (n=4) and Tsc2 fl/fl Lyz2 -Cre (n=5) mice. ( d ) Total amount of glucose in lung tissue of mice (n=10). ( e ) Mitotracker Green staining analyzed by flow cytometry of BMDM treated with solvent control or 100 nM rapamycin for 18 h (n=4). ( f ) Immunofluorescence for F-ATPase β of BMDM deprived of CSF1. ( g ) ECAR of Tsc2 fl/fl Lyz2 -Cre BMDM that were treated for 30 min with solvent or 1 µM PD-0332991 (n=8). ( h ) ECAR of Tsc2 fl/fl that were treated overnight with solvent or 10 ng/ml CSF1 and at the indicated time point with 1 µM PD-0332991 (n=8). ( i ) Images of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) activity on frozen lung sections of mice in situ . Sections were additionally stained for Mac-2, p-S6, and DAPI. ( j ) Fraction (in %) of p-S6-positive Mac-2 macrophages in lungs of mice with high activities of GAPDH, lactate dehydrogenase (LDH), isocitrate dehydrogenase (IDH) and succinate dehydrogenase (SDH) (n=3-4). ( k ) Analysis of CSF1-induced (40 ng/ml) proliferation of BMDM treated with solvent or the indicated amounts of 2-DG (n= 4). ( l ) Cell cycle analysis of CSF1-stimulated BMDM treated with solvent or 1 mM 2-DG for 18 h (n= 3). ( m ) BMDM were treated with 1 mM 2-DG or solvent control and then stimulated with 10 ng/ml CSF1 for 18 h. Whole cell lysates were analyzed by immunoblotting with the indicated antibodies. Shown are means ± SD ( a , g , h , j ) or SE ( b , c , e , k , l ) or boxplots with means, 25 th to 75 th percentile and minimum to maximum bars ( d ). *p<0.05, **p<0.001, ***p<0.001 (Student's t test). Data are representative of one ( d ) or two ( a,f-j,m ) independent experiments or cumulative of 2 experiments ( b,c,e,k,l) . Scale bar, 10 μm ( f ), 100 μm ( i ).

Article Snippet: Primary antibodies were TSC2 (D93F12) 1:1000, p-Akt S473 (D9E) 1:1000, 4E-BP1 (53H11) 1:1000, p-S6 S240/244 (rabbit polyclonal) 1:3000, S6 (54D2) 1:1000, p-Akt T308 (D25E6) 1:1000, pan-Akt (40D4) 1:1000, β-tubulin (9F3) 1:1000 , p-RB Ser807/811 (D20B12) 1:1000, PDCD4 (D29C6) 1:1000, cleaved caspase 3 Asp175 (5A1E or rabbit polyclonal) 1:500, survivin (71G4B7) 1:1000 and p-S6K1 T389 (108D2) 1:1000, p-TSC2 S939 (rabbit polyclonal) 1:1000 obtained from Cell Signaling Technology.

Techniques: Solvent, Control, Flow Cytometry, Staining, Immunofluorescence, Activity Assay, In Situ, Cell Cycle Assay, Western Blot

Journal: Nature immunology

Article Title: Chronic signaling via the metabolic checkpoint kinase mTORC1 induces macrophage granuloma formation and marks sarcoidosis progression

doi: 10.1038/ni.3655

Figure Lengend Snippet: ( a ) Sections of three sarcoidosis patients stained with p-S6 by IHC. ( b ) Immunofluorescence for Mac-2 and p-S6 in a sarcoidosis granuloma. ( c ) GSEA analysis of the ‘mTORC1 signaling’, ‘E2F targets’, and ´Glycolysis´ gene signatures in progressive relative to self-limiting sarcoidosis from the data of Lockstone et al . ( d ) An unsupervised cluster analysis of the microarray data of Tsc2 fl/fl and Tsc2 fl/fl Lyz2 -Cre BMDM performed with the genes that were differentially expressed in progressive relative to self-limiting sarcoidosis patients. ( e ) IHC for p-S6 and Ki-67 of granulomas from sarcoidosis patients. ( f ) Immunofluorescence for Mac-2 and Ki-67 in a sarcoidosis granuloma. ( g ) Relationship between p-S6 and Ki-67 expression in granulomas of 27 human sarcoidosis biopsies. Ki-67 high, > 5 % Ki-67 positive cells in the granuloma; Ki-67 low, < 5 % Ki-67 positive cells in the granulomas. The relationship was investigated using fisher’s exact test. Data are representative of 27 human sarcoidosis patients ( a , b , e , f ) or from 8 self-limiting and 7 progressive sarcoidosis patients ( c , d ). Scale bar, 200 μm ( a ), 20 μm ( b ), 100 μm ( e,f ).

Article Snippet: Primary antibodies were TSC2 (D93F12) 1:1000, p-Akt S473 (D9E) 1:1000, 4E-BP1 (53H11) 1:1000, p-S6 S240/244 (rabbit polyclonal) 1:3000, S6 (54D2) 1:1000, p-Akt T308 (D25E6) 1:1000, pan-Akt (40D4) 1:1000, β-tubulin (9F3) 1:1000 , p-RB Ser807/811 (D20B12) 1:1000, PDCD4 (D29C6) 1:1000, cleaved caspase 3 Asp175 (5A1E or rabbit polyclonal) 1:500, survivin (71G4B7) 1:1000 and p-S6K1 T389 (108D2) 1:1000, p-TSC2 S939 (rabbit polyclonal) 1:1000 obtained from Cell Signaling Technology.

Techniques: Staining, Immunofluorescence, Microarray, Expressing

Journal: Nature immunology

Article Title: Chronic signaling via the metabolic checkpoint kinase mTORC1 induces macrophage granuloma formation and marks sarcoidosis progression

doi: 10.1038/ni.3655

Figure Lengend Snippet: ( a ) 2 month-old and ( b ) 6 month-old Tsc2 fl/fl Lyz2 -Cre mice were treated with placebo or everolimus for three weeks and lung sections were evaluated by H&E staining. ( c ) Paw and tail images of a 6 month-old Tsc2 fl/fl Lyz2 -Cre mouse at day 0 and 14 of everolimus treatment. ( d ) IHC for Mac-2 of paw sections of 6 month-old Tsc2 fl/fl Lyz2 -Cre mice treated with placebo or everolimus. ( e ) IHC for p-S6 and cleaved caspase 3 of paw sections of 6 month-old Tsc2 fl/fl Lyz2 -Cre mice treated with placebo or everolimus for two days. Representative histological images are from one mouse; each analysis was performed on four ( a , b , c ) or three ( d , e ) mice per genotype. Scale bar, 40 μm ( a ), 100 μm ( b ), 1 mm ( d ), 50 μm ( e ).

Article Snippet: Primary antibodies were TSC2 (D93F12) 1:1000, p-Akt S473 (D9E) 1:1000, 4E-BP1 (53H11) 1:1000, p-S6 S240/244 (rabbit polyclonal) 1:3000, S6 (54D2) 1:1000, p-Akt T308 (D25E6) 1:1000, pan-Akt (40D4) 1:1000, β-tubulin (9F3) 1:1000 , p-RB Ser807/811 (D20B12) 1:1000, PDCD4 (D29C6) 1:1000, cleaved caspase 3 Asp175 (5A1E or rabbit polyclonal) 1:500, survivin (71G4B7) 1:1000 and p-S6K1 T389 (108D2) 1:1000, p-TSC2 S939 (rabbit polyclonal) 1:1000 obtained from Cell Signaling Technology.

Techniques: Staining

Journal: Stem cells (Dayton, Ohio)

Article Title: LGR5 Promotes Breast Cancer Progression and Maintains Stem-Like Cells Through Activation of Wnt/β-Catenin Signaling.

doi: 10.1002/stem.2083

Figure Lengend Snippet: Figure 3. LGR5 promotes cell migration, tumor metastasis, and activation of the Wnt/b-catenin pathway to induce epithelial- mesenchymal transition. (A): Transwell invasion assay demonstrating that LGR5 promotes cell invasion. Representative images of invaded cells are shown in the left panel, and the results are summarized in the right panel. The results are expressed as the mean 6 SD of three independent experiments (**, p < .01, independent Student’s t test). (B): Metastatic nodules (arrows) on the lung surface. The number of nodules was quantified on lungs of nude mice (n 5 6 per group) 8 weeks after tail vein injection of LGR5- and empty vector- transfected MCF-7 cells and sh-LGR5- and sh-ctr-transfected MDAMB231 cells (**, p < .01, independent Student’s t test). (C): Represen- tative images of hematoxylin and eosin stained sections derived from metastatic nodules at the lung surface. Original magnification, 3100. (D): Representative IF images showing expression of E-cadherin, vimentin, and b-catenin in LGR5-MCF7 cells compared with Vec- MCF7 cells (upper). sh-LGR5- and sh-ctr-transfected MDAMB231 cells (bottom). Nuclei were counterstained with DAPI. Original magnifi- cation, 3400. (E): Relative expressions of E-cadherin, b-catenin, vimentin, fibronectin, snail, and slug were compared between Vec- MCF7 and LGR5-MCF7 or shc-MDAMB231 and shLGR5-MDAMB231 cells by Western blotting (left). The Western blot image in the right panel illustrates that the Wnt inhibitor Dkk1 could effectively decrease b-catenin, snail, C-myc, and cyclinD1 expression induced by LGR5 in MCF-7 cells, whereas Wnt3a could reactivate expression of these genes in shLGR5-MDAMB231 cells.

Article Snippet: Cells were incubated overnight at 48C with the following primary antibodies: LGR5 (Abcam, 1:100); E-cadherin, vimentin, and b-catenin (Cell Signaling Technology, Danvers, MA, 1:100); cytokeratin14 and cytokeratin18 (Bioss People’s Republic of China; 1:100).

Techniques: Migration, Activation Assay, Transwell Invasion Assay, Injection, Plasmid Preparation, Transfection, Staining, Derivative Assay, Expressing, Western Blot

Journal: Stem cells (Dayton, Ohio)

Article Title: LGR5 Promotes Breast Cancer Progression and Maintains Stem-Like Cells Through Activation of Wnt/β-Catenin Signaling.

doi: 10.1002/stem.2083

Figure Lengend Snippet: Figure 5. LGR5 enhances Wnt/b-catenin pathway activity to induce stemness. (A): Cellular morphology is shown at the second day after passaging the LGR5high cells (0 hour, upper) and culturing in the absence of other factors (control) or the presence of either Wnt3a-conditioned medium (Wnt3a) or Dickkopf1 (Dkk1) (96 hours, lower). (B): Cells treated with R-spondin1 and Wnt3a generated sig- nificantly more spheres, and treatment with DKK1 significantly reduced the number of spheres. The data represent the mean 6 SD of three independent experiments, *, p < .05; **, p < .01. (C): The spheroid formed from LGR5high cells expressed different levels of b-catenin in the control medium or when treated with Wnt3a or DKK1. Nuclei were stained with DAPI. Original magnification: 3400. (D): Breast cancer specimens were analyzed by immunohistochemical staining, and the representative LGR5, b-catenin, c-myc, and cyclinD1 expression are shown. Scale bar 5 50 mm. (E): Analysis of immunohistochemistry tissue microarray data showing linear regres- sions and significant Pearson correlations of LGR5 with b-catenin (n 5 129), LGR5 with C-myc (n 5 123), and LGR5 with cyclinD1 (n 5 109) in breast cancer.

Article Snippet: Cells were incubated overnight at 48C with the following primary antibodies: LGR5 (Abcam, 1:100); E-cadherin, vimentin, and b-catenin (Cell Signaling Technology, Danvers, MA, 1:100); cytokeratin14 and cytokeratin18 (Bioss People’s Republic of China; 1:100).

Techniques: Activity Assay, Passaging, Control, Generated, Staining, Immunohistochemical staining, Expressing, Immunohistochemistry, Microarray

Journal: Journal of Toxicologic Pathology

Article Title: Characteristic Upregulation of Glucose-Regulated Protein 78 in an Early Lesion Negative for Hitherto Established Cytochemical Markers in Rat Hepatocarcinogenesis

doi: 10.1293/tox.22.281

Figure Lengend Snippet: The relative expression of GRP78 mRNA in GST-P-negative HAF induced by DEN→clofibrate was slightly increased compared with the adjacent normal tissue. The vertical axis represents the ratio of GRP78 mRNA expression versus GAPDH mRNA expression in each region. The white bars represent GRP78 mRNA expression in the normal tissue, and the left hatched and black bars represent GST-P-positive and GST-P-negative HAF, respectively.

Article Snippet: That for GRP78 was accomplished in accordance with the protocol provided in the CSA II Kit (Dako Japan, Tokyo), with a minor modification consisting of retrieval of antigens using Target Retrieval Solution (Dako Japan), using a primary goat anti-GRP78 antibody (1:1000, room temperature, 15 min, Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA).

Techniques: Expressing

Journal: Journal of Toxicologic Pathology

Article Title: Characteristic Upregulation of Glucose-Regulated Protein 78 in an Early Lesion Negative for Hitherto Established Cytochemical Markers in Rat Hepatocarcinogenesis

doi: 10.1293/tox.22.281

Figure Lengend Snippet: Immunohistochemistry for GRP78 and GST-P in HAF induced by DEN→clofibrate. (a) GST-P-negative lesion: 1, HE staining; 2, GRP78; 3, GST-P. (b) GST-P positive lesion: 1, HE staining; 2, GRP78; 3, GST-P. Lens × 4.

Article Snippet: That for GRP78 was accomplished in accordance with the protocol provided in the CSA II Kit (Dako Japan, Tokyo), with a minor modification consisting of retrieval of antigens using Target Retrieval Solution (Dako Japan), using a primary goat anti-GRP78 antibody (1:1000, room temperature, 15 min, Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA).

Techniques: Immunohistochemistry, Staining

Journal: Journal of Toxicologic Pathology

Article Title: Characteristic Upregulation of Glucose-Regulated Protein 78 in an Early Lesion Negative for Hitherto Established Cytochemical Markers in Rat Hepatocarcinogenesis

doi: 10.1293/tox.22.281

Figure Lengend Snippet: Immunohistochemistry for GRP78 in GST-P-negative HCAs induced by DEN→clofibrate. (a) HE staining. (b) GST-P. (c), (d) GRP78. (a)-(c) lens × 4; (d) lens × 10.

Article Snippet: That for GRP78 was accomplished in accordance with the protocol provided in the CSA II Kit (Dako Japan, Tokyo), with a minor modification consisting of retrieval of antigens using Target Retrieval Solution (Dako Japan), using a primary goat anti-GRP78 antibody (1:1000, room temperature, 15 min, Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA).

Techniques: Immunohistochemistry, Staining

Journal: BMC Biotechnology

Article Title: Comparative gene expression profiling of mouse ovaries upon stimulation with natural equine chorionic gonadotropin (N-eCG) and tethered recombinant-eCG (R-eCG)

doi: 10.1186/s12896-020-00653-8

Figure Lengend Snippet: Genes that were down-regulated (fold-change) in R-eCG-treated ovaries

Article Snippet: The PMSG ELISA kit was purchased from DRG International, Inc. (Mountain side, NJ, USA), Centriplus Centrifugal Filter Devices from Amicon Bio separations (Merck, Billerica, MA, USA), and an anti- myc antibody and antibodies against HSD17β1, ADAMTS1, Edn2, and OVGP1 were purchased from Santa Cruz Biotechnology (Dallas, TX, USA).

Techniques:

Journal: BMC Biotechnology

Article Title: Comparative gene expression profiling of mouse ovaries upon stimulation with natural equine chorionic gonadotropin (N-eCG) and tethered recombinant-eCG (R-eCG)

doi: 10.1186/s12896-020-00653-8

Figure Lengend Snippet: Localization of HSD17β1, ADAMTS1, EDN2, and OVGP1. The ovaries were induced to superovulate with 10 IU of either N-eCG or R-eCGβ/α, followed by 10 IU of hCG after 48 h. Representative immunohistochemical analyses for HSD17β1, ADAMTS1, EDN2, and OVGP1 were conducted with antisera, and a goat anti-rabbit IgG antibody (secondary antibody). According to the microarray and qRT-PCR results, HSD17β1 was up-regulated in the R-eCG-treated ovaries, while the other three proteins (ADAMTS1, EDN2, and OVGP1) were up-regulated in the N-eCG-treated ovaries. Immunohistochemistry was performed with a Vectastain ABC kit. Scale bar = 200 μm

Article Snippet: The PMSG ELISA kit was purchased from DRG International, Inc. (Mountain side, NJ, USA), Centriplus Centrifugal Filter Devices from Amicon Bio separations (Merck, Billerica, MA, USA), and an anti- myc antibody and antibodies against HSD17β1, ADAMTS1, Edn2, and OVGP1 were purchased from Santa Cruz Biotechnology (Dallas, TX, USA).

Techniques: Immunohistochemical staining, Microarray, Quantitative RT-PCR, Immunohistochemistry

Journal: International journal of cancer

Article Title: The intracellular juxtamembrane domain of discoidin domain receptor 2 (DDR2) is essential for receptor activation and DDR2-mediated cancer progression.

doi: 10.1002/ijc.28901

Figure Lengend Snippet: Figure 1. The IJM region is necessary for collagen-induced DDR2 activation. (a) Overall topology and alignment of the transmembrane (TM) domain and the intracellular juxtamembrane region (IJM) of DDR1a and DDR2. The IJM was divided into three regions: JM1, JM2 and JM3. (b) Schematic diagram of various DDR2 constructs used in our study. (c) HEK293T cells transiently transfected with plasmids encoding the full-length DDR2 and F-DJM1-JM2 mutant were stimulated by Type I collagen for 60 min. Tyrosine phosphorylation of the F-DJM1-JM2 mutant was inhibited compared to that of the full-length DDR2. (d) HEK293T cells were transfected with plasmids encoding full-length DDR2, F- DJM1 and F-DJM2 and were stimulated by collagen. The F-DJM2 mutant showed a significant decrease in tyrosine phosphorylation. **p < 0.01, Student’s t-test.

Article Snippet: The antibodies used in our study were as follows: mouse anti-myc (sc-40; Santa Cruz Biotechnology, Santa Cruz, CA), rabbit anti-HA (sc-805; Santa Cruz Biotechnology), rabbit anti-DDR1 (sc-532; Santa Cruz Biotechnol- ogy), goat anti-DDR2 (sc-7555; Santa Cruz Biotechnology), anti-human DDR2 (AF2538; R&D Systems, Minneapolis, MN), anti-phosphotyrosine (clone 4G10; Upstate Biotechnology, Lake Placid, NY) and peroxidase- and fluoresceinconjugated goat anti-mouse IgG (Jackson ImmunoResearch Laboratories, West Grove, PA).

Techniques: Activation Assay, Construct, Transfection, Mutagenesis, Phospho-proteomics

Journal: International journal of cancer

Article Title: The intracellular juxtamembrane domain of discoidin domain receptor 2 (DDR2) is essential for receptor activation and DDR2-mediated cancer progression.

doi: 10.1002/ijc.28901

Figure Lengend Snippet: Figure 2. DDR2 dimerizes via the JM2 of the IJM region. (a and b) HEK293T cells were transiently cotransfected with plasmids encoding TM- JM1-JM2-myc and TM-JM1-JM2-HA. Immunoprecipitation and Western blot analysis showed that the cytoplasmic domains of DDR2 bind to each other via the intact TM-JM1-JM2 domain and form homodimers. Asterisks indicate the expected size of TM-JM1-JM2. (c) HEK293T cells were transfected with plasmids encoding F-DJM1 and F-DJM2 mutants. A crosslinking assay showed that dimers of F-DJM1 were not changed compared to full-length DDR2, whereas dimers were significantly decreased for F-DJM2.

Article Snippet: The antibodies used in our study were as follows: mouse anti-myc (sc-40; Santa Cruz Biotechnology, Santa Cruz, CA), rabbit anti-HA (sc-805; Santa Cruz Biotechnology), rabbit anti-DDR1 (sc-532; Santa Cruz Biotechnol- ogy), goat anti-DDR2 (sc-7555; Santa Cruz Biotechnology), anti-human DDR2 (AF2538; R&D Systems, Minneapolis, MN), anti-phosphotyrosine (clone 4G10; Upstate Biotechnology, Lake Placid, NY) and peroxidase- and fluoresceinconjugated goat anti-mouse IgG (Jackson ImmunoResearch Laboratories, West Grove, PA).

Techniques: Immunoprecipitation, Western Blot, Transfection

Journal: International journal of cancer

Article Title: The intracellular juxtamembrane domain of discoidin domain receptor 2 (DDR2) is essential for receptor activation and DDR2-mediated cancer progression.

doi: 10.1002/ijc.28901

Figure Lengend Snippet: Figure 3. JM2 has a dominant-negative effect on DDR2 activation. (a and b) HEK293T cells were cotransfected with plasmids encoding full- length DDR2-myc and TM-JM1-JM2-HA. Immunoprecipitation and Western blotting showed that the full-length DDR2 and TM-JM1-JM2 bind to each other to form heterodimers. The immunoprecipitates obtained with anti-IgG antibodies were used as a negative control. (c) H1299 cells transfected with plasmids encoding full-length DDR2 and TM-JM1-JM2 and HeLa cells were lysed, and the whole cell lysates (W) were separated into the plasma membrane (P) and cytosol (C) fractions. EGFR and a-tubulin were used as positive controls for the plasma mem- brane and cytosol fractions, respectively. Endogenous full-length DDR2 (HeLa cells), forced-expressed full-length DDR2 and TM-JM1-JM2 pro- teins were appropriately localized in the plasma membrane. (d) HEK293T cells were transfected with plasmids encoding a C-terminally myc- tagged full-length DDR2 and TM-JM1-JM2. Only under the permeabilized condition, full-length DDR2-myc and TM-JM1-JM2-myc were visual- ized, indicating that the C-termini of these proteins were located in the cytosol and not extracellular space. Full-length DDR2 was used as a positive control. Bar, 50 mm. (e) HEK293T cells were cotransfected with plasmids encoding full-length DDR2 (500 ng) and an increasing amount of TM-JM1-JM2 (100, 300 and 500 ng) as indicated and then stimulated with collagen. Tyrosine phosphorylation gradually decreased with an increasing amount of TM-JM1-JM2. (f) HeLa cells were transiently transfected with a plasmid encoding TM-JM1-JM2 and were then stimulated. Tyrosine phosphorylation was significantly decreased in endogenous DDR2. **p < 0.01, Student’s t-test. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

Article Snippet: The antibodies used in our study were as follows: mouse anti-myc (sc-40; Santa Cruz Biotechnology, Santa Cruz, CA), rabbit anti-HA (sc-805; Santa Cruz Biotechnology), rabbit anti-DDR1 (sc-532; Santa Cruz Biotechnol- ogy), goat anti-DDR2 (sc-7555; Santa Cruz Biotechnology), anti-human DDR2 (AF2538; R&D Systems, Minneapolis, MN), anti-phosphotyrosine (clone 4G10; Upstate Biotechnology, Lake Placid, NY) and peroxidase- and fluoresceinconjugated goat anti-mouse IgG (Jackson ImmunoResearch Laboratories, West Grove, PA).

Techniques: Dominant Negative Mutation, Activation Assay, Immunoprecipitation, Western Blot, Negative Control, Transfection, Clinical Proteomics, Membrane, Positive Control, Phospho-proteomics, Plasmid Preparation

Journal: International journal of cancer

Article Title: The intracellular juxtamembrane domain of discoidin domain receptor 2 (DDR2) is essential for receptor activation and DDR2-mediated cancer progression.

doi: 10.1002/ijc.28901

Figure Lengend Snippet: Figure 4. JM2 regulates the collagen-binding affinity of DDR2. (a and b) HEK293T cells transiently transfected with various DDR2 constructs were harvested, and protein expression was verified by Western blot- ting (a). Collagen-binding affinities were reduced in F-DJM2, F-DJM1- JM2 and extra mutants but not the F-DJM1 mutant in a dose- dependent manner (b). Nontransfected (NC) and TM-JM1-JM2 samples were used as negative controls. **p< 0.01, Student’s t-test.

Article Snippet: The antibodies used in our study were as follows: mouse anti-myc (sc-40; Santa Cruz Biotechnology, Santa Cruz, CA), rabbit anti-HA (sc-805; Santa Cruz Biotechnology), rabbit anti-DDR1 (sc-532; Santa Cruz Biotechnol- ogy), goat anti-DDR2 (sc-7555; Santa Cruz Biotechnology), anti-human DDR2 (AF2538; R&D Systems, Minneapolis, MN), anti-phosphotyrosine (clone 4G10; Upstate Biotechnology, Lake Placid, NY) and peroxidase- and fluoresceinconjugated goat anti-mouse IgG (Jackson ImmunoResearch Laboratories, West Grove, PA).

Techniques: Binding Assay, Transfection, Construct, Expressing, Western Blot, Mutagenesis

Journal: International journal of cancer

Article Title: The intracellular juxtamembrane domain of discoidin domain receptor 2 (DDR2) is essential for receptor activation and DDR2-mediated cancer progression.

doi: 10.1002/ijc.28901

Figure Lengend Snippet: Figure 5. Colony formation and proliferation of tumor cells are suppressed by overexpression of JM2. (a and b) Formalin-fixed tissue micro- array slides were used in immunohistochemistry experiments. DDR2 was overexpressed in bladder, testis, lung, kidney, prostate and stom- ach cancers. Bar, 50 mm. (c) Stable TM-JM1-JM2–expressing H1299 cells were stimulated by collagen and then harvested. Immunoprecipitation and Western blot analysis showed that tyrosine phosphorylation of DDR2 was decreased by TM-JM1-JM2 overexpres- sion (labeled JM1/2), but phosphorylation of DDR1 was unaffected. (d) A colony-forming assay of H1299 cells showed that the number and projected area of colonies were decreased by TM-JM1-JM2 overexpression. Bar, 100 mm. (e) Proliferation of H1299 cells was assessed by cell counting (left) and an MTT assay (right). Cell proliferation was inhibited by TM-JM1-JM2 overexpression. **p < 0.01, Student’s t-test; control, nontransfected cells; Mock, empty vector stably transfected cells; JM1/2, TM-JM1-JM2 stably transfected cells. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

Article Snippet: The antibodies used in our study were as follows: mouse anti-myc (sc-40; Santa Cruz Biotechnology, Santa Cruz, CA), rabbit anti-HA (sc-805; Santa Cruz Biotechnology), rabbit anti-DDR1 (sc-532; Santa Cruz Biotechnol- ogy), goat anti-DDR2 (sc-7555; Santa Cruz Biotechnology), anti-human DDR2 (AF2538; R&D Systems, Minneapolis, MN), anti-phosphotyrosine (clone 4G10; Upstate Biotechnology, Lake Placid, NY) and peroxidase- and fluoresceinconjugated goat anti-mouse IgG (Jackson ImmunoResearch Laboratories, West Grove, PA).

Techniques: Over Expression, Microarray, Immunohistochemistry, Expressing, Immunoprecipitation, Western Blot, Phospho-proteomics, Labeling, Cell Counting, MTT Assay, Control, Plasmid Preparation, Stable Transfection, Transfection

Journal: Nature Communications

Article Title: DOT1L safeguards cartilage homeostasis and protects against osteoarthritis

doi: 10.1038/ncomms15889

Figure Lengend Snippet: ( a ) Immunohistochemistry showing reduced methylated H3K79 levels (H3K79me2) that reflect loss of DOT1L activity in damaged areas from osteoarthritic patients (OA) as compared to their corresponding preserved areas and to cartilage from non-OA patients. Images are representative of images from four different patients. Scale bar, 400 μm. ( b ) Heat maps of differential mRNA expression determined by quantitative PCR in chondrocytes treated with DOT1L inhibitor EPZ-5676 (EPZ) or vehicle (V) from passage 0 (P0) until P2, and from preserved versus damaged areas in OA cartilage. The colour code represents the mean expression level of six and four independent patient samples respectively. ( c ) Immunoblot analysis showing decreased methylated H3K79 levels in mouse articular chondrocytes after intra-articular injection of EPZ into C57Bl/6 wild-type mouse knees. The image is representative of one experiment with protein extracts pooled from two or three mice per condition. Unprocessed original scans of blots are shown in . ( d , e ) C57/Bl6 wild-type mouse knees were injected with EPZ (5 mg kg –1 ) or vehicle and killed after 2 or 4 weeks. Knees were sectioned and stained with Hematoxylin-Safranin O ( d ). Scale bar, 200 μm. Cartilage damage was scored (see Methods section) and is shown in ( e ). One experiment was performed with n =10 and 5. Representative images from the 4 week evaluation are shown. * P <0.05 (two-tailed t -test). Error bars indicate mean±s.e.m.

Article Snippet: Antibody binding to the column was performed using 75 μg of either a mock antibody (donkey anti-goat IgG) as a control or DOT1L antibody (R&D Systems, MAB6546, clone 653613).

Techniques: Immunohistochemistry, Methylation, Activity Assay, Expressing, Real-time Polymerase Chain Reaction, Western Blot, Injection, Staining, Two Tailed Test

Journal: Nature Communications

Article Title: DOT1L safeguards cartilage homeostasis and protects against osteoarthritis

doi: 10.1038/ncomms15889

Figure Lengend Snippet: ( a ) KEGG pathway enrichment analysis of microarray data obtained from human articular chondrocytes treated with EPZ-5676 or vehicle. Nominal P values by EASE modified Fisher Exact test using the DAVID analysis tool (see Methods section) are shown. n =5 independent patient-derived cell cultures. ( b ) Co-immunoprecipitation (Co-IP) using an anti-DOT1L antibody showing interaction between DOT1L and β -catenin in human articular chondrocytes, that is increased upon Wnt activation by LiCl and disrupted upon DOT1L inhibition. The image is representative of three experiments. ( c ) TOP/FOP reporter assay in human articular chondrocytes after Wnt stimulation by LiCl and DOT1L inhibition by EPZ. Activity is compared to untreated cells (dotted line). n =3 biologically independent experiments. *** P <0.001 by one-way ANOVA. ( d , e ) LEF1 , TCF1 and c-MYC expression measured by quantitative PCR in chondrocytes treated with EPZ-5676 and LiCl ( d ) or in LiCl-treated chondrocytes transfected with siRNA directed against DOT1L or scrambled siRNA (siDOT1L or siSCR, respectively) ( e ). Data are from one experiment with three technical replicates. ( f ) Immunohistochemistry demonstrating increased TCF1 levels in the articular cartilage of C57/Bl6 wild-type mice after injection of EPZ-5676. The images are representative of three different animals. Scale bar, 200 μm.

Article Snippet: Antibody binding to the column was performed using 75 μg of either a mock antibody (donkey anti-goat IgG) as a control or DOT1L antibody (R&D Systems, MAB6546, clone 653613).

Techniques: Microarray, Modification, Derivative Assay, Immunoprecipitation, Co-Immunoprecipitation Assay, Activation Assay, Inhibition, Reporter Assay, Activity Assay, Expressing, Real-time Polymerase Chain Reaction, Transfection, Immunohistochemistry, Injection

Journal: Nature Communications

Article Title: DOT1L safeguards cartilage homeostasis and protects against osteoarthritis

doi: 10.1038/ncomms15889

Figure Lengend Snippet: All experiments were performed in healthy human articular chondrocytes: treated as indicated with DOT1L inhibitor EPZ-5676, Wnt activator LiCl, SIRT1 antagonist EX527 or SIRT1 agonist SRT1720; or transfected with DOT1L or scrambled siRNA. All data are presented as mean±s.e.m. ( a ) Chromatin immunoprecipitation-quantitative PCR (ChIP-qPCR) analysis of DOT1L and methylated H3K79 and ( b ) acetylated H3K9 (H3K9Ac) and methylated H3K4 (H3K4me3) as markers of active transcription on the transcriptional start site (TSS) of Wnt target genes. Data are from two to five experiments. ( c ) Expression levels of TCF1 Wnt target gene measured by quantitative PCR in chondrocytes transfected with indicated specific or scrambled siRNA (siSCR). Data are from one experiment with technical triplicates. ( d ) TCF1 expression measured by quantitative PCR in the presence of SIRT1 agonist and antagonist. Data from two experiments each with technical triplicates. ( e ) Co-IP analysis using the indicated antibodies demonstrating the interaction of DOT1L and SIRT1. The image is a representative image of three biologically independent experiments. ( f ) SIRT1 activity relative to vehicle-treated cells (dotted line). Data are from three biologically independent experiments. * P <0.05, *** P <0.001 by one-way ANOVA. ( g ) ChIP-qPCR analysis of SIRT1, PPARGC1A, GCN5 and EP300 binding on the TCF1 promoter and ( h ) TCF1 expression after siRNA transfection with indicated specific or scrambled siRNA. Data from two biologically independent experiments.

Article Snippet: Antibody binding to the column was performed using 75 μg of either a mock antibody (donkey anti-goat IgG) as a control or DOT1L antibody (R&D Systems, MAB6546, clone 653613).

Techniques: Transfection, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, ChIP-qPCR, Methylation, Expressing, Co-Immunoprecipitation Assay, Activity Assay, Binding Assay

Journal: Nature Communications

Article Title: DOT1L safeguards cartilage homeostasis and protects against osteoarthritis

doi: 10.1038/ncomms15889

Figure Lengend Snippet: ( a – c ) Inactivation of SIRT1 protects against DOT1L inhibitor-induced osteoarthritis: ( a ) C57/Bl6 wild-type mouse knees were injected with DOT1L inhibitor EPZ-5676 (5 mg kg −1 ) and SIRT1 inhibitor EX527 (1.25 mg kg –1 ), or vehicle (V) and killed after 4 weeks. Knees were sectioned and stained with Hematoxylin-Safranin O ( a ). Scale bar, 200 μm. Cartilage damage was scored (see Methods section) and is shown in ( b ). One experiment was performed with n =3 (vehicle), 8 (EPZ) and 10 (EPZ+EX527). * P <0.05 by one-way ANOVA. Error bars indicate mean±s.e.m. ( c ) Immunohistochemistry of TCF1 in the indicated groups. TCF1 levels are increased after EPZ treatment and normalized by additional EX527 treatment. The images are representative of three different animals. Scale bar, 200 μm. ( d , e ) Loss of DOT1L function causes severe growth retardation as demonstrated by skeletal staining ( d ) and histology of the growth plate ( e ) of 4-week-old Dot1l fl/fl ;Col2-Cre −/− (Cre-neg) and Dot1l fl/fl ;Col2-Cre +/− (Dot1l Cart-KO ) mice. ( f , g ) Increased TCF1 levels in Dot1l Cart-KO mice as shown by immunohistochemistry in the indicated mice strains in the articular cartilage ( f ) and growth plate ( g ). The images are representative of three different animals. Scale bar, 200 and 100 μm.

Article Snippet: Antibody binding to the column was performed using 75 μg of either a mock antibody (donkey anti-goat IgG) as a control or DOT1L antibody (R&D Systems, MAB6546, clone 653613).

Techniques: Injection, Staining, Immunohistochemistry

Journal: Nature Communications

Article Title: DOT1L safeguards cartilage homeostasis and protects against osteoarthritis

doi: 10.1038/ncomms15889

Figure Lengend Snippet: Upon Wnt signalling activation, DOT1L-containing complexes bind Wnt target gene chromatin. DOT1L interacts with SIRT1 and inhibits its function, preventing Wnt pathway hyper-activation. When Wnt signalling is activated in the absence of DOT1L function, high SIRT1 activity mediates the recruitment of transcriptional activators to LEF1 and TCF1 genes. High Wnt signalling leads to deleterious downstream effects and loss of cartilage homeostasis.

Article Snippet: Antibody binding to the column was performed using 75 μg of either a mock antibody (donkey anti-goat IgG) as a control or DOT1L antibody (R&D Systems, MAB6546, clone 653613).

Techniques: Activation Assay, Activity Assay